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Posts Tagged ‘antiapoptosis’


Biology, Physiology and Pathophysiology of Heat Shock Proteins

Curation: Larry H. Bernstein, MD, FCAP

 

 

Heat Shock Proteins (HSP)

  1. Exploring the association of molecular chaperones, heat shock proteins, and the heat shock response in physiological/pathological processes

Hsp70 chaperones: Cellular functions and molecular mechanism

M. P. MayerB. Bukau
Cell and Molec Life Sci  Mar 2005; 62:670  http://dx.doi.org:/10.1007/s00018-004-4464-6

Hsp70 proteins are central components of the cellular network of molecular chaperones and folding catalysts. They assist a large variety of protein folding processes in the cell by transient association of their substrate binding domain with short hydrophobic peptide segments within their substrate proteins. The substrate binding and release cycle is driven by the switching of Hsp70 between the low-affinity ATP bound state and the high-affinity ADP bound state. Thus, ATP binding and hydrolysis are essential in vitro and in vivo for the chaperone activity of Hsp70 proteins. This ATPase cycle is controlled by co-chaperones of the family of J-domain proteins, which target Hsp70s to their substrates, and by nucleotide exchange factors, which determine the lifetime of the Hsp70-substrate complex. Additional co-chaperones fine-tune this chaperone cycle. For specific tasks the Hsp70 cycle is coupled to the action of other chaperones, such as Hsp90 and Hsp100.

70-kDa heat shock proteins (Hsp70s) assist a wide range of folding processes, including the folding and assembly of newly synthesized proteins, refolding of misfolded and aggregated proteins, membrane translocation of organellar and secretory proteins, and control of the activity of regulatory proteins [17]. Hsp70s have thus housekeeping functions in the cell in which they are built-in components of folding and signal transduction pathways, and quality control functions in which they proofread the structure of proteins and repair misfolded conformers. All of these activities appear to be based on the property of Hsp70 to interact with hydrophobic peptide segments of proteins in an ATP-controlled fashion. The broad spectrum of cellular functions of Hsp70 proteins is achieved through

  • the amplification and diversification of hsp70genes in evolution, which has generated specialized Hsp70 chaperones,
  • co-chaperones which are selectively recruited by Hsp70 chaperones to fulfill specific cellular functions and
  • cooperation of Hsp70s with other chaperone systems to broaden their activity spectrum. Hsp70 proteins with their co-chaperones and cooperating chaperones thus constitute a complex network of folding machines.

Protein folding processes assisted by Hsp70

The role of Hsp70s in the folding of non-native proteins can be divided into three related activities: prevention of aggregation, promotion of folding to the native state, and solubilization and refolding of aggregated proteins. In the cellular milieu, Hsp70s exert these activities in the quality control of misfolded proteins and the co- and posttranslational folding of newly synthesized proteins. Mechanistically related but less understood is the role of Hsp70s in the disassembly of protein complexes such as clathrin coats, viral capsids and the nucleoprotein complex, which initiates the replication of bacteriophage λ DNA. A more complex folding situation exists for the Hsp70-dependent control of regulatory proteins since several steps in the folding and activation process of these substrates are assisted by multiple chaperones.

Hsp70 proteins together with their co-chaperones of the J-domain protein (JDP) family prevent the aggregation of non-native proteins through association with hydrophobic patches of substrate molecules, which shields them from intermolecular interactions (‘holder’ activity). Some JDPs such as Escherichia coli DnaJ and Saccharomyces cerevisiae Ydj1 can prevent aggregation by themselves through ATP-independent transient and rapid association with the substrates. Only members of the Hsp70 family with general chaperone functions have such general holder activity.

Hsp70 chaperone systems assist non-native folding intermediates to fold to the native state (‘folder’ activity). The mechanism by which Hsp70-chaperones assist the folding of non-native substrates is still unclear. Hsp70-dependent protein folding in vitro occurs typically on the time scale of minutes or longer. Substrates cycle between chaperone-bound and free states until the ensemble of molecules has reached the native state. There are at least two alternative modes of action. In the first mechanism Hsp70s play a rather passive role. Through repetitive substrate binding and release cycles they keep the free concentration of the substrate sufficiently low to prevent aggregation, while allowing free molecules to fold to the native state (‘kinetic partitioning’). In the second mechanism, the binding and release cycles induce local unfolding in the substrate, e.g. the untangling of a misfolded β-sheet, which helps to overcome kinetic barriers for folding to the native state (‘local unfolding’) [8–11]. The energy of ATP may be used to induce such conformational changes or alternatively to drive the ATPase cycle in the right direction.

Hsp70 in cellular physiology and pathophysiology

Two Hsp70 functions are especially interesting, de novo folding of nascent polypeptides and interaction with signal transduction proteins, and therefore some aspects of these functions shall be discussed below in more detail. Hsp70 chaperones were estimated to assist the de novo folding of 10–20% of all bacterial proteins whereby the dependence on Hsp70 for efficient folding correlated with the size of the protein [12]. Since the average protein size in eukaryotic cells is increased (52 kDa in humans) as compared to bacteria (35 kDa in E. coli) [25], it is to be expected that an even larger percentage of eukaryotic proteins will be in need of Hsp70 during de novo folding. This reliance on Hsp70 chaperones increases even more under stress conditions. Interestingly, mutated proteins [for example mutant p53, cystis fibrosis transmembrane regulator (CFTR) variant ΔF508, mutant superoxid dismutase (SOD) 1] seem to require more attention by the Hsp70 chaperones than the corresponding wild-type protein [2629]. As a consequence of this interaction the function of the mutant protein can be preserved. Thereby Hsp70 functions as a capacitor, buffering destabilizing mutations [30], a function demonstrated earlier for Hsp90 [3132]. Such mutations are only uncovered when the overall need for Hsp70 action exceeds the chaperone capacity of the Hsp70 proteins, for example during stress conditions [30], at certain stages in development or during aging, when the magnitude of stress-induced increase in Hsp70 levels declines [3334]. Alternatively, the mutant protein can be targeted by Hsp70 and its co-chaperones to degradation as shown e.g. for CFTRΔF508 and some of the SOD1 mutant proteins [35,36]. Deleterious mutant proteins may then only accumulate when Hsp70 proteins are overwhelmed by other, stress-denatured proteins. Both mechanisms may contribute to pathological processes such as oncogenesis (mutant p53) and neurodegenerative diseases, including amyotrophic, lateral sclerosis (SOD1 mutations), Parkinsonism (α-synuclein mutations), Huntington’s chorea (huntingtin with polyglutamin expansions) and spinocerebellar ataxias (proteins with polyglutamin expansions).

De novo folding is not necessarily accelerated by Hsp70 chaperones. In some cases folding is delayed for different reasons. First, folding of certain proteins can only proceed productively after synthesis of the polypeptide is completed as shown, e.g. for the reovirus lollipop-shaped protein sigma 1 [37]. Second, proteins destined for posttranslational insertion into organellar membranes are prevented from aggregation and transported to the translocation pore [38]. Third, in the case of the caspase-activated DNase (CAD), the active protein is dangerous for the cell and therefore can only complete folding in the presence of its specific inhibitor (ICAD). Hsp70 binds CAD cotranslationally and mediates folding only to an intermediate state. Folding is completed after addition of ICAD, which is assembled into a complex with CAD in an Hsp70-dependent manner [39]. Similar folding pathways may exist also for other potentially dangerous proteins.

As mentioned above Hsp70 interacts with key regulators of many signal transduction pathways controlling cell homeostasis, proliferation, differentiation and cell death. The interaction of Hsp70 with these regulatory proteins continues in activation cycles that also involve Hsp90 and a number of co-chaperones. The regulatory proteins, called clients, are thereby kept in an inactive state from which they are rapidly activated by the appropriate signals. Hsp70 and Hsp90 thus repress regulators in the absence of the upstream signal and guarantee full activation after the signal transduction pathway is switched on [6]. Hsp70 can be titrated away from these clients by other misfolded proteins that may arise from internal or external stresses. Consequently, through Hsp70 disturbances of the cellular system induced by environmental, developmental or pathological processes act on these signal transduction pathways.

In this way stress response and apoptosis are linked to each other. Hsp70 inhibits apoptosis acting on the caspase-dependent pathway at several steps both upstream and downstream of caspase activation and on the caspase-independent pathway. Overproduction of Hsp70 leads to increased resistance against apoptosis-inducing agents such as tumor necrosis factor-α(TNFα), staurosporin and doxorubicin, while downregulation of Hsp70 levels by antisense technology leads to increased sensitivity towards these agents [1840]. This observation relates to many pathological processes, such as oncogenesis, neurodegeneration and senescence. In many tumor cells increased Hsp70 levels are observed and correlate with increased malignancy and resistance to therapy. Downregulation of the Hsp70 levels in cancer cells induce differentiation and cell death [41]. Neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, Huntington’s corea and spinocerebellar ataxias are characterized by excessive apoptosis. In several different model systems overexpression of Hsp70 or one of its co-chaperones could overcome the neurodegenerative symptoms induced by expression of a disease-related gene (huntingtin, α-synuclein or ataxin) [20,42]. Senescence in cell culture as well as aging in vivo is correlated with a continuous decline in the ability to mount a stress response [3443]. Age-related symptoms and diseases reflect this decreased ability to cope with cellular stresses. Interestingly, centenarians seem to be an exception to the rule, as they show a significant induction of Hsp70 production after heat shock challenge [44].

ATPase domain and ATPase cycle

Substrate binding

The coupling mechanism: nucleotide-controlled opening and closing of the substrate binding cavity

The targeting activity of co-chaperones

J-domain proteins

Bag proteins

Hip, Hop and CHIP

Perspectives

The Hsp70 protein family and their co-chaperones constitute a complex network of folding machines which is utilized by cells in many ways. Despite considerable progress in the elucidation of the mechanistic basis of these folding machines, important aspects remain to be solved. With respect to the Hsp70 proteins it is still unclear whether their activity to assist protein folding relies on the ability to induce conformational changes in the bound substrates, how the coupling mechanism allows ATP to control substrate binding and to what extent sequence variations within the family translate into variations of the mechanism. With respect to the action of co-chaperones we lack a molecular understanding of the coupling function of JDPs and of how co-chaperones target their Hsp70 partner proteins to substrates. Furthermore, it can be expected that more cellular processes will be discovered that depend on the chaperone activity of Hsp70 chaperones.

 

  1. The biochemistry and ultrastructure of molecular chaperones

Structure and Mechanism of the Hsp90 Molecular Chaperone Machinery

Laurence H. Pearl and Chrisostomos Prodromou
Ann Rev of Biochem July 2006;75:271-294
http://dx.doi.org:/10.1146/annurev.biochem.75.103004.142738

Heat shock protein 90 (Hsp90) is a molecular chaperone essential for activating many signaling proteins in the eukaryotic cell. Biochemical and structural analysis of Hsp90 has revealed a complex mechanism of ATPase-coupled conformational changes and interactions with cochaperone proteins, which facilitate activation of Hsp90’s diverse “clientele.” Despite recent progress, key aspects of the ATPase-coupled mechanism of Hsp90 remain controversial, and the nature of the changes, engendered by Hsp90 in client proteins, is largely unknown. Here, we discuss present knowledge of Hsp90 structure and function gleaned from crystallographic studies of individual domains and recent progress in obtaining a structure for the ATP-bound conformation of the intact dimeric chaperone. Additionally, we describe the roles of the plethora of cochaperones with which Hsp90 cooperates and growing insights into their biochemical mechanisms, which come from crystal structures of Hsp90 cochaperone complexes.

 

  1. Properties of heat shock proteins (HSPs) and heat shock factor (HSF)

Heat shock factors: integrators of cell stress, development and lifespan

Malin Åkerfelt,*‡ Richard I. Morimoto,§ and Lea Sistonen*‡
Nat Rev Mol Cell Biol. 2010 Aug; 11(8): 545–555.  doi:  10.1038/nrm2938

Heat shock factors (HSFs) are essential for all organisms to survive exposures to acute stress. They are best known as inducible transcriptional regulators of genes encoding molecular chaperones and other stress proteins. Four members of the HSF family are also important for normal development and lifespan-enhancing pathways, and the repertoire of HSF targets has thus expanded well beyond the heat shock genes. These unexpected observations have uncovered complex layers of post-translational regulation of HSFs that integrate the metabolic state of the cell with stress biology, and in doing so control fundamental aspects of the health of the proteome and ageing.

In the early 1960s, Ritossa made the seminal discovery of temperature-induced puffs in polytene chromosomes of Drosophila melanogaster larvae salivary glands1. A decade later, it was shown that the puffing pattern corresponded to a robust activation of genes encoding the heat shock proteins (HSPs), which function as molecular chaperones2. The heat shock response is a highly conserved mechanism in all organisms from yeast to humans that is induced by extreme proteotoxic insults such as heat, oxidative stress, heavy metals, toxins and bacterial infections. The conservation among different eukaryotes suggests that the heat shock response is essential for survival in a stressful environment.

The heat shock response is mediated at the transcriptional level by cis-acting sequences called heat shock elements (HSEs; BOX 1) that are present in multiple copies upstream of the HSP genes3. The first evidence for a specific transcriptional regulator, the heat shock factor (HSF) that can bind to the HSEs and induce HSP gene expression, was obtained through DNA–protein interaction studies on nuclei isolated from D. melanogaster cells4,5. Subsequent studies showed that, in contrast to a single HSF in invertebrates, multiple HSFs are expressed in plants and vertebrates68. The mammalian HSF family consists of four members: HSF1,HSF2, HSF3 and HSF4. Distinct HSFs possess unique and overlapping functions (FIG. 1), exhibit tissue-specific patterns of expression and have multiple post-translational modifications (PTMs) and interacting protein partners7,9,10. Functional crosstalk between HSF family members and PTMs facilitates the fine-tuning of HSF-mediated gene regulation. The identification of many targets has further extended the impact of HSFs beyond the heat shock response. Here, we present the recent discoveries of novel target genes and physiological functions of HSFs, which have changed the view that HSFs act solely in the heat shock response. Based on the current knowledge of small-molecule activators and inhibitors of HSFs, we also highlight the potential for pharmacologic modulation of HSF-mediated gene regulation.

Box 1

The heat shock element

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3402356/bin/nihms281610u1.jpg

Heat shock factors (HSFs) act through a regulatory upstream promoter element, called the heat shock element (HSE). In the DNA-bound form of a HSF, each DNA-binding domain (DBD) recognizes the HSE in the major groove of the double helix6. The HSE was originally identified using S1 mapping of transcripts of the Drosophila melanogaster heat shock protein (HSP) genes3 (see the figure; part a). Residues –47 to –66 are necessary for heat inducibility. HSEs in HSP gene promoters are highly conserved and consist of inverted repeats of the pentameric sequence nGAAn132. The type of HSEs that can be found in the proximal promoter regions of HSP genes is composed of at least three contiguous inverted repeats: nTTCnnGAAnnTTCn132134. The promoters of HSF target genes can also contain more than one HSE, thereby allowing the simultaneous binding of multiple HSFs. The binding of an HSF to an HSE occurs in a cooperative manner, whereby binding of an HSF trimer facilitates binding of the next one135. More recently, Trinklein and colleagues used chromatin immunoprecipitation to enrich sequences bound by HSF1 in heat-shocked human cells to define the HSE consensus sequence. They confirmed the original finding of Xiao and Lis, who identified guanines as the most conserved nucleotides in HSEs87,133 (see the figure; part b). Moreover, in a pair of inverted repeats, a TTC triplet 5′ of a GAA triplet is separated by a pyrimidine–purine dinucleotide, whereas the two nucleotides separating a GAA triplet 5′ from a TTC triplet is unconstrained87. The discovery of novel HSF target genes that are not involved in the heat shock response has rendered it possible that there may be HSEs in many genes other than the HSP genes. Although there are variations in these HSEs, the spacing and position of the guanines are invariable7. Therefore, both the nucleotides and the exact spacing of the repeated units are considered as key determinants for recognition by HSFs and transcriptional activation. Part b of the figure is modified, with permission, from REF. 87 © (2004) The American Society for Cell Biology.

Figure 1     http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3402356/bin/nihms281610f1.gif

The mammalian HSF machinery

HSFs as stress integrators

A hallmark of stressed cells and organisms is the increased synthesis of HSPs, which function as molecular chaperones to prevent protein misfolding and aggregation to maintain protein homeostasis, also called proteostasis11. The transcriptional activation of HSP genes is mediated by HSFs (FIG. 2a), of which HSF1 is the master regulator in vertebrates. Hsf1-knockout mouse and cell models have revealed that HSF1 is a prerequisite for the transactivation of HSP genes, maintenance of cellular integrity during stress and development of thermotolerance1215. HSF1 is constitutively expressed in most tissues and cell types16, where it is kept inactive in the absence of stress stimuli. Thus, the DNA-binding and transactivation capacity of HSF1 are coordinately regulated through multiple PTMs, protein–protein interactions and subcellular localization. HSF1 also has an intrinsic stress-sensing capacity, as both D. melanogaster and mammalian HSF1 can be converted from a monomer to a homotrimer in vitro in response to thermal or oxidative stress1719.

Figure 2    http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3402356/bin/nihms281610f2.gif

Members of the mammalian HSF family

Functional domains

HSFs, like other transcription factors, are composed of functional domains. These have been most thoroughly characterized for HSF1 and are schematically presented in FIG. 2b. The DNA-binding domain (DBD) is the best preserved domain in evolution and belongs to the family of winged helix-turn-helix DBDs2022. The DBD forms a compact globular structure, except for a flexible wing or loop that is located between β-strands 3 and 4 (REF. 6). This loop generates a protein– protein interface between adjacent subunits of the HSF trimer that enhances high-affinity binding to DNA by cooperativity between different HSFs23. The DBD can also mediate interactions with other factors to modulate the transactivating capacity of HSFs24. Consequently, the DBD is considered as the signature domain of HSFs for target-gene recognition.

The trimerization of HSFs is mediated by arrays of hydrophobic heptad repeats (HR-A and HR-B) that form a coiled coil, which is characteristic for many Leu zippers6,25 (FIG. 2b). The trimeric assembly is unusual, as Leu zippers typically facilitate the formation of homodimers or heterodimers. Suppression of spontaneous HSF trimerization is mediated by yet another hydrophobic repeat, HR-C2628. Human HSF4 lacks the HR-C, which could explain its constitutive trimerization and DNA-binding activity29. Positioned at the extreme carboxyl terminus of HSFs is the transactivation domain, which is shared among all HSFs6except for yeast Hsf, which has transactivation domains in both the amino and C termini, and HSF4A, which completely lacks a transactivation domain2931. In HSF1, the transactivation domain is composed of two modules — AD1 and AD2, which are rich in hydrophobic and acidic residues (FIG. 3a) — that together ensures a rapid and prolonged response to stress32,33. The transactivation domain was originally proposed to provide stress inducibility to HSF1 (REFS 34,35), but it soon became evident that an intact regulatory domain, located between the HR-A and HR-B and the transactivation domain, is essential for the responsiveness to stress stimuli32,33,36,37. Because several amino acids that are known targets for different PTMs reside in the regulatory domain33,3842, the structure and function of this domain are under intensive investigation.

Figure 3    http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3402356/bin/nihms281610f3.gif

HSF1 undergoes multiple PTMs on activation

Regulation of the HSF1 activation–attenuation cycle

The conversion of the inactive monomeric HSF1 to high-affinity DNA-binding trimers is the initial step in the multistep activation process and is a common feature of all eukaryotic HSFs43,44 (FIG. 3b). There is compelling evidence for HSF1 interacting with multiple HSPs at different phases of its activation cycle. For example, monomeric HSF1 interacts weakly with HSP90 and, on stress, HSF1 dissociates from the complex, allowing HSF1 trimerization45,46 (FIG. 3b). Trimeric HSF1 can be kept inactive when its regulatory domain is bound by a multi-chaperone complex of HSP90, co-chaperone p23 (also known as PTGES3) and immunophilin FK506-binding protein 5 (FKBP52; also known as FKBP4)4651. Elevated levels of both HSP90 and HSP70 negatively regulate HSF1 and prevent trimer formation on heat shock52. Activated HSF1 trimers also interact with HSP70 and the co-chaperone HSP40 (also known as DNAJB1), but instead of suppressing the DNA-binding activity of HSF1, this interaction inhibits its transactivation capacity5254. Although the inhibitory mechanism is still unknown, the negative feedback from the end products of HSF1-dependent transcription (the HSPs) provides an important control step in adjusting the duration and intensity of HSF1 activation according to the levels of chaperones and presumably the levels of nascent and misfolded peptides.

A ribonucleoprotein complex containing eukaryotic elongation factor 1A (eEF1A) and a non-coding RNA, heat shock RNA-1 (HSR-1), has been reported to possess a thermosensing capacity. According to the proposed model, HSR-1 undergoes a conformational change in response to heat stress and together with eEF1A facilitates trimerization of HSF1 (REF. 55). How this activation mode relates to the other regulatory mechanisms associated with HSFs remains to be elucidated.

Throughout the activation–attenuation cycle, HSF1 undergoes extensive PTMs, including acetylation, phosphorylation and sumoylation (FIG. 3). HSF1 is also a phosphoprotein under non-stress conditions, and the results from mass spectrometry (MS) analyses combined with phosphopeptide mapping experiments indicate that at least 12 Ser residues are phosphorylated41,5659. Among these sites, stress-inducible phosphorylation of Ser230 and Ser326 in the regulatory domain contributes to the transactivation function of HSF1 (REFS 38,41). Phosphorylation-mediated sumoylation on a single Lys residue in the regulatory domain occurs rapidly and transiently on exposure to heat shock; Ser303 needs to be phosphorylated before a small ubiquitin-related modifier (SUMO) can be conjugated to Lys298 (REF. 39). The extended consensus sequence ΨKxExxSP has been named the phosphorylation-dependent sumoylation motif (PDSM; FIG. 3)40. The PDSM was initially discovered in HSF1 and subsequently found in many other proteins, especially transcriptional regulators such as HSF4, GATA1, myocyte-specific enhancer factor 2A (MEF2A) and SP3, which are substrates for both SUMO conjugation and Pro-directed kinases40,6062.

Recently, Mohideen and colleagues showed that a conserved basic patch on the surface of the SUMO-conjugating enzyme ubiquitin carrier protein 9 (UBC9; also known as UBE2I) discriminates between the phosphorylated and non-phosphorylated PDSM of HSF1 (REF. 63). Future studies will be directed at elucidating the molecular mechanisms for dynamic phosphorylation and UBC9-dependent SUMO conjugation in response to stress stimuli and establishing the roles of kinases, phosphatases and desumoylating enzymes in the heat shock response. The kinetics of phosphorylation-dependent sumoylation of HSF1 correlates inversely with the severity of heat stress, and, as the transactivation capacity of HSF1 is impaired by sumoylation and this PTM is removed when maximal HSF1 activity is required40, sumoylation could modulate HSF1 activity under moderate stress conditions. The mechanisms by which SUMO modification represses the transactivating capacity of HSF1, and the functional relationship of this PTM with other modifications that HSF1 is subjected to, will be investigated with endogenous substrate proteins.

Phosphorylation and sumoylation of HSF1 occur rapidly on heat shock, whereas the kinetics of acetylation are delayed and coincide with the attenuation phase of the HSF1 activation cycle. Stress-inducible acetylation of HSF1 is regulated by the balance of acetylation by p300–CBP (CREB-binding protein) and deacetylation by the NAD+-dependent sirtuin, SIRT1. Increased expression and activity of SIRT1 enhances and prolongs the DNA-binding activity of HSF1 at the human HSP70.1promoter, whereas downregulation of SIRT1 enhances the acetylation of HSF1 and the attenuation of DNA-binding without affecting the formation of HSF1 trimers42. This finding led to the discovery of a novel regulatory mechanism of HSF1 activity, whereby SIRT1 maintains HSF1 in a state that is competent for DNA binding by counteracting acetylation (FIG. 3). In the light of current knowledge, the attenuation phase of the HSF1 cycle is regulated by a dual mechanism: a dependency on the levels of HSPs that feed back directly by weak interactions with HSF1, and a parallel step that involves the SIRT1-dependent control of the DNA-binding activity of HSF1. Because SIRT1 has been implicated in caloric restriction and ageing, the age-dependent loss of SIRT1 and impaired HSF1 activity correlate with an impairment of the heat shock response and proteostasis in senescent cells, connecting the heat shock response to nutrition and ageing (see below).

HSF dynamics on the HSP70 promoter

For decades, the binding of HSF to the HSP70.1 gene has served as a model system for inducible transcription in eukaryotes. In D. melanogaster, HSF is constitutively nuclear and low levels of HSF are associated with the HSP70promoter before heat shock6466. The uninduced HSP70 promoter is primed for transcription by a transcriptionally engaged paused RNA polymerase II (RNAP II)67,68. RNAP II pausing is greatly enhanced by nucleosome formation in vitro, implying that chromatin remodelling is crucial for the release of paused RNAP II69. It has been proposed that distinct hydrophobic residues in the transactivation domain of human HSF1 can stimulate RNAP II release and directly interact withBRG1, the ATPase subunit of the chromatin remodelling complex SWI/SNF70,71. Upon heat shock, RNAP II is released from its paused state, leading to the synthesis of a full-length transcript. Rapid disruption of nucleosomes occurs across the entire HSP70 gene, at a rate that is faster than RNAP II-mediated transcription72. The nucleosome displacement occurs simultaneously with HSF recruitment to the promoter in D. melanogaster. Downregulation of HSF abrogates the loss of nucleosomes, indicating that HSF provides a signal for chromatin rearrangement, which is required for HSP70 nucleosome displacement. Within seconds of heat shock, the amount of HSF at the promoter increases drastically and HSF translocates from the nucleoplasm to several native loci, including HSP genes. Interestingly, the levels of HSF occupying the HSP70 promoter reach saturation soon after just one minute65,73.

HSF recruits the co-activating mediator complex to the heat shock loci, which acts as a bridge to transmit activating signals from transcription factors to the basal transcription machinery. The mediator complex is recruited by a direct interaction with HSF: the transactivation domain of D. melanogaster HSF binds to TRAP80(also known as MED17), a subunit of the mediator complex74. HSF probably has other macromolecular contacts with the preinitiation complex as it binds to TATA-binding protein (TBP) and the general transcription factor TFIIB in vitro75,76. In contrast to the rapid recruitment and elongation of RNAP II on heat shock, activated HSF exchanges very slowly at the HSP70 promoter. HSF stays stably bound to DNA in vivo and no turnover or disassembly of transcription activator is required for successive rounds of HSP70 transcription65,68.

Functional interplay between HSFs

Although HSF1 is the principal regulator of the heat shock response, HSF2 also binds to the promoters of HSP genes. In light of our current knowledge, HSF2 strictly depends on HSF1 for its stress-related functions as it is recruited to HSP gene promoters only in the presence of HSF1 and this cooperation requires an intact HSF1 DBD77. Nevertheless, HSF2 modulates, both positively and negatively, the HSF1-mediated inducible expression of HSP genes, indicating that HSF2 can actively participate in the transcriptional regulation of the heat shock response. Coincident with the stress-induced transcription of HSP genes, HSF1 and HSF2 colocalize and accumulate rapidly on stress into nuclear stress bodies (NSBs; BOX 2), where they bind to a subclass of satellite III repeats, predominantly in the human chromosome 9q12 (REFS 7880). Consequently, large and stable non-coding satellite III transcripts are synthesized in an HSF1-dependent manner in NSBs81,82. The function of these transcripts and their relationship with other HSF1 targets, and the heat shock response in general, remain to be elucidated.

 

Box 2

Nuclear stress bodies  

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3402356/bin/nihms281610u2.jpg

The cell nucleus is highly compartmentalized and dynamic. Many nuclear factors are diffusely distributed throughout the nucleoplasm, but they can also accumulate in distinct subnuclear compartments, such as nucleoli, speckles, Cajal bodies and promyelocytic leukaemia (PML) bodies136. Nuclear stress bodies (NSBs) are different from any other known nuclear bodies137,138. Although NSBs were initially thought to contain aggregates of denatured proteins and be markers of heat-shocked cells, their formation can be elicited by various stresses, such as heavy metals and proteasome inhibitors137. NSBs are large structures, 0.3–3 μm in diameter, and are usually located close to the nucleoli or nuclear envelope137,138. NSBs consist of two populations: small, brightly stained bodies and large, clustered and ring-like structures137.

NSBs appear transiently and are the main site of heat shock factor 1 (HSF1) and HSF2 accumulation in stressed human cells80. HSF1 and HSF2 form a physically interacting complex and colocalize into small and barely detectable NSBs after only five minutes of heat shock, but the intensity and size of NSBs increase after hours of continuous heat shock. HSF1 and HSF2 colocalize in HeLa cells that have been exposed to heat shock for one hour at 42°C (see the figure; confocal microscopy image with HSF1–green fluorescent protein in green and endogenous HSF2 in red). NSBs form on specific chromosomal loci, mainly on q12 of human chromosome 9, where HSFs bind to a subclass of satellite III repeats78,79,83. Stress-inducible and HSF1-dependent transcription of satellite III repeats has been shown to produce non-coding RNA molecules, called satellite III transcripts81,82. The 9q12 locus consists of pericentromeric heterochromatin, and the satellite III repeats provide scaffolds for docking components, such as splicing factors and other RNA-processing proteins139143.

HSF2 also modulates the heat shock response through the formation of heterotrimers with HSF1 in the NSBs when bound to the satellite III repeats83 (FIG. 4). Studies on the functional significance of heterotrimerization indicate that HSF1 depletion prevents localization of HSF2 to NSBs and abolishes the stress-induced synthesis of satellite III transcripts. By contrast, increased expression of HSF2 leads to its own activation and the localization of both HSF1 and HSF2 to NSBs, where transcription is spontaneously induced in the absence of stress stimuli. These results suggest that HSF2 can incorporate HSF1 into a transcriptionally competent heterotrimer83. It is possible that the amounts of HSF2 available for heterotrimerization with HSF1 influence stress-inducible transcription, and that HSF1–HSF2 heterotrimers regulate transcription in a temporal manner. During the acute phase of heat shock, HSF1 is activated and HSF1–HSF2 heterotrimers are formed, whereas upon prolonged exposures to heat stress the levels of HSF2 are diminished, thereby limiting heterotrimerization83. Intriguingly, in specific developmental processes such as corticogenesis and spermatogenesis, the expression of HSF2 increases spatiotemporarily, leading to its spontaneous activation. Therefore, it has been proposed that HSF-mediated transactivation can be modulated by the levels of HSF2 to provide a switch that integrates the responses to stress and developmental stimuli83 (FIG. 4). Functional relationships between different HSFs are emerging, and the synergy of DNA-binding activities among HSF family members offers an efficient way to control gene expression in a cell- and stimulus-specific manner to orchestrate the differential upstream signalling and target-gene networks.

Figure 4   http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3402356/bin/nihms281610f4.gif

 

Interactions between different HSFs provide distinct functional modes in transcriptional regulation

A new member of the mammalian HSF family, mouse HSF3, was recently identified10. Avian HSF3 was shown to be activated at higher temperatures and with different kinetics than HSF1 (REF. 84), whereas in mice, heat shock induces the nuclear translocation of HSF3 and activation of stress-responsive genes other than HSP genes10. Future experiments will determine whether HSF3 is capable of interacting with other HSFs, potentially through heterocomplex formation. HSF4 has not been implicated in the heat shock response, but it competes with HSF1 for common target genes in mouse lens epithelial cells85, which will be discussed below. It is important to elucidate whether the formation of homotrimers or hetero trimers between different family members is a common theme in HSF-mediated transcriptional regulation.

 

HSFs as developmental regulators

Evidence is accumulating that HSFs are highly versatile transcription factors that, in addition to protecting cells against proteotoxic stress, are vital for many physioogical functions, especially during development. The initial observations using deletion experiments of the D. melanogaster Hsf gene revealed defective oogenesis and larvae development86. These effects were not caused by obvious changes in HSP gene expression patterns, which is consistent with the subsequent studies showing that basal expression of HSP genes during mouse embryogenesis is not affected by the lack of HSF1 (REF. 13). These results are further supported by genome-wide gene expression studies revealing that numerous genes, not classified as HSP genes or molecular chaperones, are under HSF1-dependent control87,88.

Although mice lacking HSF1 can survive to adulthood, they exhibit multiple defects, such as increased prenatal lethality, growth retardation and female infertility13. Fertilized oocytes do not develop past the zygotic stage when HSF1-deficient female mice are mated with wild-type male mice, indicating that HSF1 is a maternal factor that is essential for early post-fertilization development89. Recently, it was shown that HSF1 is abundantly expressed in maturing oocytes, where it regulates specifically Hsp90α transcription90. The HSF1-deficient oocytes are devoid of HSP90α and exhibit a blockage of meiotic maturation, including delayed G2–M transition or germinal vesicle breakdown and defective asymmetrical division90. Moreover, intra-ovarian HSF1-depleted oocytes contain dysfunctional mitochondria and are sensitive to oxidative stress, leading to reduced survival91. The complex phenotype of Hsf1-knockout mice also demonstrates the involvement of HSF1 in placenta formation, placode development and the immune system15,85,92,93, further strengthening the evidence for a protective function of HSF1 in development and survival.

Both HSF1 and HSF2 are key regulators in the developing brain and in maintaining proteostasis in the central nervous system. Disruption of Hsf1 results in enlarged ventricles, accompanied by astrogliosis, neurodegeneration, progressive myelin loss and accumulation of ubiquitylated proteins in specific regions of the postnatal brain under non-stressed conditions94,95. The expression of HSP25 (also known as HSPB1) and α-crystallin B chain (CRYAB), which are known to protect cells against stress-induced protein damage and cell death, is dramatically decreased in brains lacking HSF1 (REF. 13). In contrast to HSF1, HSF2 is already at peak levels during early brain development in mice and is predominantly expressed in the proliferative neuronal progenitors of the ventricular zone and post-mitotic neurons of the cortical plate9699. HSF2-deficient mice have enlarged ventricles and defects in cortical lamination owing to abnormal neuronal migration9799. Incorrect positioning of superficial neurons during cortex formation in HSF2-deficient embryos is caused by decreased expression of the cyclin-dependent kinase 5 (CDK5) activator p35, which is a crucial regulator of the cortical migration signalling pathway100,101. The p35 gene was identified as the first direct target of HSF2 in cortex development99. As correct cortical migration requires the coordination of multiple signalling molecules, it is likely that HSF2, either directly or indirectly, also regulates other components of the same pathway.

 

Cooperativity of HSFs in development

In adult mice, HSF2 is most abundantly expressed in certain cell types of testes, specifically pachytene spermatocytes and round spermatids102. The cell-specific expression of HSF2 in testes is regulated by a microRNA, miR-18, that directly binds to the 3′ untranslated region (UTR) of HSF2 (J.K. Björk, A. Sandqvist, A.N. Elsing, N. Kotaja and L.S., unpublished observations). Targeting of HSF2 in spermatogenesis reveals the first physiological role for miR-18, which belongs to the oncomir-1 cluster associated mainly with tumour progression103. In accordance with the expression pattern during the maturation of male germ cells, HSF2-null male mice display several abnormal features in spermatogenesis, ranging from smaller testis size and increased apoptosis at the pachytene stage to a reduced amount of sperm and abnormal sperm head shape97,98,104. A genome-wide search for HSF2 target promoters in mouse testis revealed the occupancy of HSF2 on the sex chromosomal multi-copy genes spermiogenesis specific transcript on the Y 2 (Ssty2), Sycp3-like Y-linked (Sly) and Sycp3-like X-linked (Slx), which are important for sperm quality104. Compared with the Hsf2-knockout phenotype, disruption of both Hsf1 and Hsf2 results in a more pronounced phenotype, including larger vacuolar structures, more widely spread apoptosis and a complete lack of mature spermatozoa and male sterility105. The hypo thesis that the activities of HSF1 and HSF2 are intertwined and essential for spermatogenesis is further supported by our results that HSF1 and HSF2 synergistically regulate the sex chromosomal multi-copy genes in post-meiotic round spermatids (M.Å., A. Vihervaara, E.S. Christians, E. Henriksson and L.S., unpublished observations). Given that the sex chromatin mostly remains silent after meiosis, HSF1 and HSF2 are currently the only known transcriptional regulators during post-meiotic repression. These results, together with the earlier findings that HSF2 can also form heterotrimers with HSF1 in testes83, strongly suggest that HSF1 and HSF2 act in a heterocomplex and fine-tune transcription of their common target genes during the maturation of male germ cells.

HSF1 and HSF4 are required for the maintenance of sensory organs, especially when the organs are exposed to environmental stimuli for the first time after birth85,88. During the early postnatal period, Hsf1-knockout mice display severe atrophy of the olfactory epithelium, increased accumulation of mucus and death of olfactory sensory neurons88. Although lens development in HSF4-deficient mouse embryos is normal, severe abnormalities, including inclusion-like structures in lens fibre cells, appear soon after birth and the mice develop cataracts85,106,107. Intriguingly, inherited severe cataracts occurring in Chinese and Danish families have been associated with a mutation in the DBD of HSF4 (REF. 108). In addition to the established target genes, Hsp25Hsp70 and Hsp90, several new targets for HSF1 and HSF4, such as crystallin γF (Crygf), fibroblast growth factor 7 (Fgf7) and leukaemia inhibitory factor (Lif) have been found to be crucial for sensory organs85,88. Furthermore, binding of either HSF1 or HSF4 to the Fgf7 promoter shows opposite effects on gene expression, suggesting competitive functions between the two family members85. In addition to the proximal promoters, HSF1, HSF2 and HSF4 bind to other genomic regions (that is, introns and distal parts of protein-coding genes in mouse lens), and there is also evidence for either synergistic interplay or competition between distinct HSFs occupying the target-gene promoters109. It is possible that the different HSFs are able to compensate for each other to some extent. Thus, the identification of novel functions and target genes for HSFs has been a considerable step forward in understanding their regulatory mechanisms in development.

 

HSFs and lifespan

The lifespan of an organism is directly linked to the health of its tissues, which is a consequence of the stability of the proteome and functionality of its molecular machineries. During its lifetime, an organism constantly encounters environmental and physiological stress and requires an efficient surveillance of protein quality to prevent the accumulation of protein damage and the disruption of proteostasis. Proteotoxic insults contribute to cellular ageing, and numerous pathophysiological conditions, associated with impaired protein quality control, increase prominently with age11. From studies on the molecular basis of ageing, in which a wide range of different model systems and experimental strategies have been used, the insulin and insulin-like growth factor 1 receptor (IGF1R) signalling pathway, which involves the phosphoinositide 3-kinase (PI3K) and AKT kinases and the Forkhead box protein O (FOXO) transcription factors (such as DAF-16 in Caenorhabditis elegans), has emerged as a key process. The downregulation of HSF reduces the lifespan and accelerates the formation of protein aggregates in C. elegans carrying mutations in different components of the IGF1R-mediated pathway. Conversely, inhibition of IGF1R signalling results in HSF activation and promotes longevity by maintaining proteostasis110,111. These results have prompted many laboratories that use other model organisms to investigate the functional relationship between HSFs and the IGF1R signalling pathway.

The impact of HSFs on the lifespan of whole organisms is further emphasized by a recent study, in which proteome stability was examined during C. elegansageing112. The age-dependent misfolding and downregulation of distinct metastable proteins, which display temperature-sensitive missense mutations, was examined in different tissues. Widespread failure in proteostasis occurred rapidly at an early stage of adulthood, coinciding with the severely impaired heat shock response and unfolded protein response112. The age-dependent collapse of proteostasis could be restored by overexpression of HSF and DAF-16, strengthening the evidence for the unique roles of these stress-responsive transcription factors to prevent global instability of the proteome.

Limited food intake or caloric restriction is another process that is associated with an enhancement of lifespan. In addition to promoting longevity, caloric restriction slows down the progression of age-related diseases such as cancer, cardiovascular diseases and metabolic disorders, stimulates metabolic and motor activities, and increases resistance to environmental stress stimuli113. To this end, the dynamic regulation of HSF1 by the NAD+-dependent protein deacetylase SIRT1, a mammalian orthologue of the yeast transcriptional regulator Sir2, which is activated by caloric restriction and stress, is of particular interest. Indeed, SIRT1 directly deacetylates HSF1 and keeps it in a state that is competent for DNA binding. During ageing, the DNA-binding activity of HSF1 and the amount of SIRT1 are reduced. Consequently, a decrease in SIRT1 levels was shown to inhibit HSF1 DNA-binding activity in a cell-based model of ageing and senescence42. Furthermore, an age-related decrease in the HSF1 DNA-binding activity is reversed in cells exposed to caloric restriction114. These results indicate that HSF1 and SIRT1 function together to protect cells from stress insults, thereby promoting survival and extending lifespan. Impaired proteostasis during ageing may at least partly reflect the compromised HSF1 activity due to lowered SIRT1 expression.

 

Impact of HSFs in disease

The heat shock response is thought to be initiated by the presence of misfolded and damaged proteins, and is thus a cell-autonomous response. When exposed to heat, cells in culture, unicellular organisms, and cells in a multicellular organism can all trigger a heat shock response autonomously115117. However, it has been proposed that multicellular organisms sense stress differently to isolated cells. For example, the stress response is not properly induced even if damaged proteins are accumulated in neurodegenerative diseases like Huntington’s disease and Parkinson’s disease, suggesting that there is an additional control of the heat shock response at the organismal level118. Uncoordinated activation of the heat shock response in cells in a multicellular organism could cause severe disturbances of interactions between cells and tissues. In C. elegans, a pair of thermosensory neurons called AFDs, which sense and respond to temperature, regulate the heat shock response in somatic tissues by controlling HSF activity119,120. Moreover, the heat shock response in C. elegans is influenced by the metabolic state of the organism and is reduced under conditions that are unfavourable for growth and reproduction121. Neuronal control may therefore allow organisms to coordinate the stress response of individual cells with the varying metabolic requirements in different tissues and developmental stages. These observations are probably relevant to diseases of protein misfolding that are highly tissue-specific despite the often ubiquitous expression of damaged proteins and the heat shock response.

Elevated levels of HSF1 have been detected in several types of human cancer, such as breast cancer and prostate cancer122,123. Mice deficient in HSF1 exhibit a lower incidence of tumours and increased survival than their wild-type counterparts in a classical chemical skin carcinogenesis model and in a genetic model expressing an oncogenic mutation of p53. Similar results have been obtained in human cancer cells lines, in which HSF1 was depleted using an RNA interference strategy124. HSF1 expression is likely to be crucial for non-oncogene addiction and the stress phenotype of cancer cells, which are attributes given to many cancer cells owing to their high intrinsic level of proteotoxic and oxidative stress, frequent spontaneous DNA damage and aneuploidy125. Each of these features may disrupt proteostasis, raising the need for efficient chaperone and proteasome activities. Accordingly, HSF1 would be essential for the survival of cancer cells that experience constant stress and develop non-oncogene addiction.

 

HSFs as therapeutic targets

Given the unique role of HSF1 in stress biology and proteostasis, enhanced activity of this principal regulator during development and early adulthood is important for the stability of the proteome and the health of the cell. However, HSF1 is a potent modifier of tumorigenesis and, therefore, a potential target for cancer therapeutics125. In addition to modulating the expression of HSF1, the various PTMs of HSF1 that regulate its activity should be considered from a clinical perspective. As many human, age-related pathologies are associated with stress and misfolded proteins, several HSF-based therapeutic strategies have been proposed126. In many academic and industrial laboratories, small molecule regulators of HSF1 are actively being searched for (see Supplementary information S1 (table)). For example, celastrol, which has antioxidant properties and is a natural compound derived from the Celastreace family of plants, activates HSF1 and induces HSP expression with similar kinetics to heat shock, and could therefore be a potential candidate molecule for treating neurodegenerative diseases127,128. In a yeast-based screen, a small-molecule activator of human HSF1 was found and named HSF1A129. HSF1A, which is structurally distinct from the other known activators, activates HSF1 and enhances chaperone expression, thereby counteracting protein misfolding and cell death in polyQ-expressing neuronal precursor cells129. Triptolide, also from the Celastreace family of plants, is a potent inhibitor of the transactivating capacity of HSF1 and has been shown to have beneficial effects in treatments of pancreatic cancer xenografts130,131. These examples of small-molecule regulators of HSF1 are promising candidates for drug discovery and development. However, the existence of multiple mammalian HSFs and their functional interplay should also be taken into consideration when planning future HSF-targeted therapies.

 

Concluding remarks and future perspectives

HSFs were originally identified as specific heat shock-inducible transcriptional regulators of HSP genes, but now there is unambiguous evidence for a wide variety of HSF target genes that extends beyond the molecular chaperones. The known functions governed by HSFs span from the heat shock response to development, metabolism, lifespan and disease, thereby integrating pathways that were earlier strictly divided into either cellular stress responses or normal physiology.

Although the extensive efforts from many laboratories focusing on HSF biology have provided a richness of understanding of the complex regulatory mechanisms of the HSF family of transcription factors, several key questions remain. For example, what are the initial molecular events (that is, what is the ‘thermometer’) leading to the multistep activation of HSFs? The chromatin-based interaction between HSFs and the basic transcription machinery needs further investigation before the exact interaction partners at the chromatin level can be established. The activation and attenuation mechanisms of HSFs require additional mechanistic insights, and the roles of the multiple signal transduction pathways involved in post-translational regulation of HSFs are only now being discovered and are clearly more complex than anticipated. Although still lacking sufficient evidence, the PTMs probably serve as rheostats to allow distinct forms of HSF-mediated regulation in different tissues during development. Further emphasis should therefore be placed on understanding the PTMs of HSFs during development, ageing and different protein folding diseases. Likewise, the subcellular distribution of HSF molecules, including the mechanism by which HSFs shuttle between the cytoplasm and the nucleus, remains enigmatic, as do the movements of HSF molecules in different nuclear compartments such as NSBs.

Most studies on the impact of HSFs in lifespan and disease have been conducted with model organisms such as D. melanogaster and C. elegans, which express a single HSF. The existence of multiple members of the HSF family in mammals warrants further investigation of their specific and overlapping functions, including their extended repertoire of target genes. The existence of multiple HSFs in higher eukaryotes with different expression patterns suggests that they may have functions that are triggered by distinct stimuli, leading to activation of specific target genes. The impact of the HSF family in the adaptation to diverse biological environments is still poorly understood, and future studies are likely to broaden the prevailing view of HSFs being solely stress-inducible factors. To this end, the crosstalk between distinct HSFs that has only recently been uncovered raises obvious questions about the stoichiometry between the components in different complexes residing in different cellular compartments, and the mechanisms by which the factors interact with each other. Interaction between distinct HSF family members could generate new opportunities in designing therapeutics for protein-folding diseases, metabolic disorders and cancer.

 

  1. Role in the etiology of cancer

Expression of heat shock proteins and heat shock protein messenger ribonucleic acid in human prostate carcinoma in vitro and in tumors in vivo

Dan Tang,1 Md Abdul Khaleque,2 Ellen L. Jones,1 Jimmy R. Theriault,2 Cheng Li,3 Wing Hung Wong,3 Mary Ann Stevenson,2 and Stuart K. Calderwood1,2,4
Cell Stress Chaperones. 2005 Mar; 10(1): 46–58. doi:  10.1379/CSC-44R.1

Heat shock proteins (HSPs) are thought to play a role in the development of cancer and to modulate tumor response to cytotoxic therapy. In this study, we have examined the expression of hsf and HSP genes in normal human prostate epithelial cells and a range of prostate carcinoma cell lines derived from human tumors. We have observed elevated expressions of HSF1, HSP60, and HSP70 in the aggressively malignant cell lines PC-3, DU-145, and CA-HPV-10. Elevated HSP expression in cancer cell lines appeared to be regulated at the post–messenger ribonucleic acid (mRNA) levels, as indicated by gene chip microarray studies, which indicated little difference in heat shock factor (HSF) or HSP mRNA expression between the normal and malignant prostate cell lines. When we compared the expression patterns of constitutive HSP genes between PC-3 prostate carcinoma cells growing as monolayers in vitro and as tumor xenografts growing in nude mice in vivo, we found a marked reduction in expression of a wide spectrum of the HSPs in PC-3 tumors. This decreased HSP expression pattern in tumors may underlie the increased sensitivity to heat shock of PC-3 tumors. However, the induction by heat shock of HSP genes was not markedly altered by growth in the tumor microenvironment, and HSP40, HSP70, and HSP110 were expressed abundantly after stress in each growth condition. Our experiments indicate therefore that HSF and HSP levels are elevated in the more highly malignant prostate carcinoma cells and also show the dominant nature of the heat shock–induced gene expression, leading to abundant HSP induction in vitro or in vivo.

Heat shock proteins (HSPs) were first discovered as a cohort of proteins that is induced en masse by heat shock and other chemical and physical stresses in a wide range of species (Lindquist and Craig 1988Georgopolis and Welch 1993). The HSPs (Table 1) have been subsequently characterized as molecular chaperones, proteins that have in common the property of modifying the structures and interactions of other proteins (Lindquist and Craig 1988Beckmann et al 1990;Gething and Sambrook 1992Georgopolis and Welch 1993Netzer and Hartl 1998). Molecular chaperone function dictates that the HSP often interact in a stoichiometric, one-on-one manner with their substrates, necessitating high intracellular concentrations of the proteins (Lindquist and Craig 1988Georgopolis and Welch 1993). As molecules that shift the balance from denatured, aggregated protein conformation toward ordered, functional conformation, HSPs are particularly in demand when the protein structure is disrupted by heat shock, oxidative stress, or other protein-damaging events (Lindquist and Craig 1988;Gething and Sambrook 1992Georgopolis and Welch 1993). The HSP27, HSP40,HSP70, and HSP110 genes have therefore evolved a highly efficient mechanism for mass synthesis during stress, with powerful transcriptional activation, efficient messenger ribonucleic acid (mRNA) stabilization, and selective mRNA translation (Voellmy 1994). HSP27, HSP70, HSP90, and HSP110 increase to become the dominantly expressed proteins after stress (Hickey and Weber 1982Landry et al 1982Li and Werb 1982Subjeck et al 1982Henics et al 1999) (Zhao et al 2002). Heat shock factor (HSF) proteins have been shown to interact with the promoters of many HSP genes and ensure prompt transcriptional activation in stress and equally precipitous switch off after recovery (Sorger and Pelham 1988Wu 1995). The hsf gene family includes HSF1 (hsf1), the molecular coordinator of the heat shock response, as well as 2 less well-characterized genes, hsf2 and hsf4(Rabindran et al 1991Schuetz et al 1991) (Nakai et al 1997). In addition to the class of HSPs induced by heat, cells also contain a large number of constitutively expressed HSP homologs, which are also listed in Table 1. The constitutive HSPs are found in a variety of multiprotein complexes containing both HSPs and cofactors (Buchner 1999). These include HSP10-HSP60 complexes that mediate protein folding and HSP70- and HSP90-containing complexes that are involved in both generic protein-folding pathways and in specific association with regulatory proteins within the cell (Netzer and Hartl 1998). HSP90 plays a particularly versatile role in cell regulation, forming complexes with a large number of cellular kinases, transcription factors, and other molecules (Buchner 1999Grammatikakis et al 2002).

 

Table 1     http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1074571/bin/i1466-1268-10-1-46-t01.jpg

 

Heat shock protein family genes studied by microchip array analysis

Many tumor types contain high concentrations of HSP of the HSP28, HSP70, and HSP90 families compared with adjacent normal tissues (Ciocca et al 1993Yano et al 1999Cornford et al 2000Strik et al 2000Ricaniadis et al 2001Ciocca and Vargas-Roig 2002). We have concentrated here on HSP gene expression in prostate carcinoma. The progression of prostatic epithelial cells to the fully malignant, metastatic phenotype is a complex process and involves the expression of oncogenes as well as escape from androgen-dependent growth and survival (Cornford et al 2000). There is a molecular link between HSP expression and tumor progression in prostate cancer in that HSP56, HSP70, and HSP90 regulate the function of the androgen receptor (AR) (Froesch et al 1998Grossmann et al 2001). Escape from AR dependence during tumorigenesis may involve altered HSP-AR interactions (Grossmann et al 2001). The role of HSPs in tumor development may also be related to their function in the development of tolerance to stress (Li and Hahn 1981). Thermotolerance is induced in cells preconditioned by mild stress coordinately with the expression of high HSP levels (Landry et al 1982Li and Werb 1982Subjeck et al 1982). Elevated HSP expression appears to be a factor in tumor pathogenesis, and, among other mechanisms, this may involve the ability of individual HSPs to block the pathways of apoptosis and permit malignant cells to arise despite the triggering of apoptotic signals during transformation (Volloch and Sherman 1999). De novo HSP expression may also afford protection of cancer cells from treatments such as chemotherapy and hyperthermia by thwarting the proapoptotic influence of these modalities (Gabai et al 1998Hansen et al 1999Blagosklonny 2001Asea et al 2001Van Molle et al 2002). The mechanisms underlying HSP induction in tumor cells are not known but may reflect the genetic alterations accompanying malignancy or the disordered state of the tumor microenvironment, which would be expected to lead to cellular stress.

Here, we have examined expression of hsf and HSP genes in immortalized normal human prostate epithelial cells and a range of prostate carcinoma cells obtained from human tumors at the mRNA and protein levels. Our aim was to determine whether hsf-HSP expression profiles are conserved in cells that express varying degrees of malignancy, under resting conditions and after heat and ionizing radiation. In addition, we have compared HSP expression profiles of a metastatic human prostate carcinoma cell line growing either in monolayer culture or as a tumor xenograft in nude mice. These studies were prompted by findings in our laboratory that prostate carcinoma cells are considerably more sensitive to heat-induced apoptosis in vivo growing as tumors compared with similar cells growing in tissue culture in vitro. Our studies show that, although the hsf-HSP expression profiles are similar in normal and malignant prostate-derived cells at the mRNA level, expression at the protein level was very different. HSF1 and HSP protein expression was highest in the 3 aggressively metastatic prostate cancer cell lines (PC-3, DU-145, and CA-HPV-10). Although the gene expression patterns of constitutive HSP differ enormously in PC-3 cells in vitro and in xenografts in vivo, stress induction of HSP genes is not markedly altered by exposure to the tumor microenvironment, indicating the hierarchical rank of the stress response that permits it to override other forms of regulation. ……

The experiments described here are largely supportive of the notion that HSP gene expression and HSF activity and expression are increased in more advanced stages of cancer (Fig 4). The most striking finding in the study was the elevation of HSF1 and HSP levels in aggressively malignant prostate carcinoma cell lines (Fig 4). It is significant that these changes in HSF and HSP levels would not have been predicted from microarray studies of HSF (Fig 3) and HSP (Fig 1) mRNA levels. The increased HSF levels observed in the metastatic prostate carcinoma cell lines in particular appear to be due to altered regulation of either mRNA translation or protein turnover (or both) (Figs 3 and ​and4).4). Although we do not at this stage know the mechanisms involved, 1 candidate could be differential activity of the proteosome in the metastatic cell lines: both HSF1 and HSF2 are targets for proteosomal degradation (Mathew et al 1998). Despite these differences in HSP expression between cells of varying degrees of malignancy under growth conditions, stress caused a major shift in HSP gene expression and activation of HSP40-1, HSP70-1A, HSP70-1B, HSP70-6 (HSP70B), DNA-J2–like, and HSP105 in all cells (Fig 2). Even in LnCap cells with minimal HSF1 and HSF2 expression, heat-inducible HSP70 protein expression was observed (Fig 4). Interestingly, we observed minimal induction of the HSP70B gene in LnCap cells: because the HSP70B promoter is known to be almost exclusively induced by stress through the HSE in its promoter, the findings may suggest that a mechanism for HSP70 induction alternative to HSF1 activation may be operative in LnCap cells (Schiller et al 1988). Increased HSP expression in cancer patients has been shown to signal a poor response to treatment by a number of modalities, suggesting that HSP expression is involved with development of resistance to treatment in addition to being involved in the mechanisms of malignant progression (Ciocca et al 1993Cornford et al 2000Yamamoto et al 2001Ciocca and Vargas-Roig 2002;Mese et al 2002). In addition, subpopulations of LnCap-derived cells, selected for enhanced capacity to metastasize, have been shown to express elevated levels of HSF1, HSP70, and HSP27 compared with nonselected controls (Hoang et al 2000). This may be highly significant because our studies indicate minimal levels of HSF1 and HSP in the poorly metastatic parent LnCap cells (Figs 1 and ​and4).4). Previous studies have also indicated that elevated HSP70 expression occurs at an early stage in cellular immortalization from embryonic stem cells (Ravagnan et al 2001). We had to use immortalized prostatic epithelial cells for our normal controls and may have missed a very early change in HSP expression during the immortalization process.

As indicated by the kinetic studies (Figs 5–7), HSPs are activated at a number of regulatory levels by stress in addition to transcriptional activation, and these may include stress-induced mRNA stabilization, differential translation, and protein stabilization (Hickey and Weber 1982Zhao et al 2002). HSF1 activity and HSP expression appear to be subject to differential regulation by a number of pathways at normal temperatures but are largely independent of such regulation when exposed to heat shock, which overrides constitutive regulation and permits prompt induction of this emergency response.

Growth of PC-3 cells in vivo as tumor xenografts was accompanied by a marked decrease in constitutive HSP expression (Figs 8 and ​and11).11). Decreased HSP expression was part of a global switch in gene expression that accompanies the switch of PC-3 cells from growth as monolayers in tissue culture to growth as tumors in vivo (D. Tang and S.K. Calderwood, in preparation). Many reports indicate changes in a wide range of cellular properties as cells grow as tumors, and these properties may reflect the remodeling of gene expression patterns. These changes may reflect adaptation to the chemical nature of the tumor microenvironment and the alterations in cell-cell interaction in growth as a tumor in vivo. Our studies also indicate the remarkable sturdiness of the heat shock response that remains intact in the PC-3 cells growing in vivo despite the global rearrangements in other gene expressions mentioned above (Figs 10 and ​and1111).

The elevation in HSF1 and HSP levels in cancer shown in our studies and in those of others and its association with a poor prognosis and inferior response to therapy suggests the strategy of targeting HSP in cancer therapy. Treatment with HSP70 antisense oligonucleotides, for instance, can cause tumor cell apoptosis on its own and can synergize with heat shock in cell killing (Jones et al 2004). Indeed, it has been shown that antagonizing heat-inducible HSP expression with quercitin, a bioflavonoid drug that inhibits HSF1 activation, or by using antisense oligonucleotides directed against HSP70 mRNA further sensitizes PC-3 cells to heat-induced apoptosis in vitro and leads to tumor regression in vivo (Asea et al 2001Lepchammer et al 2002Jones et al 2004) (A. Asea et al, personal communication). The strategy of targeting HSP expression or function in cancer cells may thus be indicated. Such a strategy might prove particularly effective because constitutive HSP expression is reduced in tumors, and this might be related to increased killing of PC-3 tumor cells by heat (Fig 12).

 

  1. Molecular chaperones in aging

Aging and molecular chaperones

Csaba So˝ti*, Pe´ter Csermely
Exper Geront 2003; 38:1037–1040  http://195.111.72.71/docs/pcs/03exger.pdf

Chaperone function plays a key role in sequestering damaged proteins and in repairing proteotoxic damage. Chaperones are induced by environmental stress and are called as stress or heat shock proteins. Here, we summarize the current knowledge about protein damage in aged organisms, about changes in proteolytic degradation, chaperone expression and function in the aging process, as well as the involvement of chaperones in longevity and cellular senescence. The role of chaperones in aging diseases, such as in Alzheimer’s disease, Parkinson’s disease, Huntington’s disease and in other neurodegenerative diseases as well as in atherosclerosis and in cancer is discussed. We also describe how the balance between chaperone requirement and availability becomes disturbed in aged organisms, or in other words, how chaperone overload develops. The consequences of chaperone overload are also outlined together with several new research strategies to assess the functional status of chaperones in the aging process.

Molecular chaperones Chaperones are ubiquitous, highly conserved proteins (Hartl, 1996), either assisting in the folding of newly synthesized or damaged proteins in an ATP-dependent active process or working in an ATP-independent passive mode sequestering damaged proteins for future refolding or digestion. Environmental stress leads to proteotoxic damage. Damaged, misfolded proteins bind to chaperones, and liberate the heat shock factor (HSF) from its chaperone complexes. HSF is activated and transcription of chaperone genes takes place (Morimoto, 2002). Most chaperones, therefore, are also called stress or (after the archetype of experimental stress) heat shock proteins (Hsp-s).

Aging proteins—proteins of aging organisms During the life-span of a stable protein, various posttranslational modifications occur including backbone and side chain oxidation, glycation, etc. In aging organisms, the disturbed cellular homeostasis leads to an increased rate of protein modification: in an 80-year old human, half of all proteins may become oxidized (Stadtman and Berlett, 1998). Susceptibility to various proteotoxic damages is mainly increased due to dysfunction of mitochondrial oxidation of starving yeast cells (Aguilaniu et al., 2001). In prokaryotes, translational errors result in folding defects and subsequent protein oxidation (Dukan et al., 2000), which predominantly takes place in growth arrested cells (Ballesteros et al., 2001). Additionally, damaged signalling networks loose their original stringency, and irregular protein phosphorylation occurs (e.g.: the Parkinson disease-related a-synuclein also becomes phosphorylated, leading to misfolding and aggregation; Neumann et al., 2002).

Aging protein degradation Irreversibly damaged proteins are recognized by chaperones, and targeted for degradation. Proteasome level and function decreases with aging, and some oxidized, aggregated proteins exert a direct inhibition on proteasome activity. Chaperones also aid in lysosomal degradation. The proteolytic changes are comprehensively reviewed by Szweda et al. (2002). Due to the degradation defects, damaged proteins accumulate in the cells of aged organisms, and by aggregation may cause a variety of protein folding diseases (reviewed by So˝ti and Csermely, 2002a).

Aging chaperones I: defects in chaperone induction Damaged proteins compete with the HSF in binding to the Hsp90-based cytosolic chaperone complex, which may contribute to the generally observed constitutively elevated chaperone levels in aged organisms (Zou et al., 1998; So˝ti and Csermely, 2002b). On the contrary, the majority of the reports showed that stress-induced synthesis of chaperones is impaired in aged animals. While HSF activation does not change, DNA binding activity may be reduced during aging (Heydari et al., 2000). A number of signaling events use an overlapping network of chaperones not only to establish the activation-competent state of different transcription factors (e.g. steroid receptors), but also as important elements in the attenuation of respective responses. HSF transcriptional activity is also negatively influenced by higher levels of chaperones (Morimoto, 2002). Differential changes of these proteins in various organisms and tissues may lead to different extents of (dys)regulation. More importantly, the cross-talk between different signalling pathways through a shared pool of chaperones may have severe consequences during aging when the cellular conformational homeostasis is deranged (see below).

Aging chaperones II: defects in chaperone function   Direct studies on chaperone function in aged organisms are largely restricted to a-crystallin having a decreased activity in aged human lenses (Cherian and Abraham, 1995; Cherian-Shaw et al., 1999). In a recent study, an initial test of passive chaperone function of whole cytosols was assessed showing a decreased chaperone capacity in aged rats compared to those of young counterparts (Nardai et al., 2002). What can be the mechanism behind these deleterious changes in chaperone function? Chaperones may also be prone to oxidative damage, as GroEL is preferentially oxidized in growth-arrested E. coli (Dukan and Nystro¨m, 1999). Macario and Conway de Macario (2002) raised the idea of ‘sick chaperones’ in aged organisms in a recent review. Indeed, chaperones are interacting with a plethora of other proteins (Csermely, 2001a), which requires rather extensive binding surfaces. These exposed areas may make chaperones a preferential target for proteotoxic damage: chaperones may behave as ‘suicide proteins’ during aging, sacrificing themselves instead of ‘normal’ proteins. The high abundance of chaperones (which may constitute more than 5% of cellular proteins), and their increased constitutive expression in aged organisms makes them a good candidate for this ‘altruistic courtesy.’ It may be especially true for mitochondrial Hsp60, the role of which would deserve extensive studies.

Aging chaperones III: defects in capacity, the chaperone overload Another possible reason of decreased chaperone function is chaperone overload (Csermely, 2001b). In aging organisms, the balance between misfolded proteins and available free chaperones is grossly disturbed: increased protein damage, protein degradation defects increase the amount of misfolded proteins, while chaperone damage, inadequate synthesis of molecular chaperones and irreparable folding defects (due to posttranslational changes) decrease the amount of available free chaperones. Chaperone overload occurs, where the need for chaperones may greatly exceed the available chaperone capacity (Fig. 1). Under these conditions, the competition for available chaperones becomes fierce and the abundance of damaged proteins may disrupt the folding assistance to other chaperone targets, such as: (1) newly synthesized proteins; (2) ‘constantly damaged’ (mutant) proteins; and (3) constituents of the cytoarchitecture (Csermely, 2001a). This may cause defects in signal transduction, protein transport, immune recognition, cellular organization as well as the appearance of previously buffered, hidden mutations in the phenotype of the cell (Csermely, 2001b). Chaperone overload may significantly decrease the robustness of cellular networks, as well as shift their function towards a more stochastic behavior. As a result of this, aging cells become more disorganized, their adaptation is impaired.

Fig. 1. Chaperone overload: a shift in the balance between misfolded proteins and available free chaperones in aging organisms. The accumulation of chaperone substrates along with an impaired chaperone function may exhaust the folding assistance to specific chaperone targets and leads to deterioration in vital processes. Chaperone overload may significantly decrease the robustness of cellular networks, and compromise the adaptative responses. See text for details.

Senescent cells and chaperones The involvement of chaperones in aging at the cellular level is recently reviewed (So˝ti et al., 2003). Non-dividingsenescent-peripheral cells tend to have increased chaperone levels (Verbeke et al., 2001), and cannot preserve the induction of several chaperones (Liu et al., 1989), similarly to cells from aged animals. Activation and binding of HSF to the heat shock element is decreased in aged cells (Choi et al., 1990). Interestingly, cellular senescence seems to unmask a proteasomal activity leading to the degradation of HSF (Bonelli et al., 2001). Chaperone induction per se seems to counteract senescence. Repeated mild heat shock (a kind of hormesis) has been reported to delay fibroblast aging (Verbeke et al., 2001), though it does not seem to extend replicative lifespan. A major chaperone, Hsp90 is required for the correct function of telomerase, an important enzyme to extend the life-span of cells (Holt et al., 1999). Mortalin (mtHsp70/Grp75), a member of the Hsp70 family, produces opposing phenotypic effects related to its localization. In normal cells, it is pancytoplasmically distributed, and its expression causes senescence. Its upregulation and perinuclear distribution, however, is connected to transformation, probably via p53 inactivation. Mortalin also induces life-span extension in human fibroblasts or in C. elegans harboring extra copies of the orthologous gene (Kaul et al., 2002).

Aging organisms and chaperones: age-related diseases Unbalanced chaperone requirement and chaperone capacity in aged organisms helps the accumulation of aggregated proteins, which often cause folding diseases, mostly of the nervous system, due to the very limited proliferation potential of neurons. Over expression of chaperones often delays the onset or diminishes the symptoms of the disease (So˝ti and Csermely, 2002b). Other aging diseases, such as atherosclerosis and cancer are also related to chaperone action. Here space limitation precludes a detailed description of these rapidly developing fields, however, numerous recent reviews were published on these subjects, where the interested readers may find a good summary and several hints for further readings (Ferreira and Carlos, 2002; Neckers, 2002; Sarto et al., 2000; Wick and Xu, 1999).

 

Chaperones and Longevity

Increased chaperone induction leads to increased longevity (Tatar et al., 1997). Moreover, a close correlation exists between stress resistance and longevity in several long-lived C. elegans and Drosophila mutants (Lithgow and Kirkwood, 1996). As the other side of the same coin, damaged HSF has been found as an important gene to cause accelerated aging in C. elegans (Garigan et al., 2002). Caloric restriction, the only effective experimental manipulation known to retard aging in rodents and primates (Ramsey et al., 2000), restores age-impaired chaperone induction, while reversing the age-induced changes in constitutive Hsp levels (see So˝ti and Csermely, 2002a,b). These examples confirm the hypothesis that a better adaptation capacity to various stresses greatly increases the chances to reach longevity. 10. Conclusions and perspectives Aging can be defined as a multicausal process leading to a gradual decay of self-defensive mechanisms, and an exponential accumulation of damage at the molecular, cellular and organismal level. The protein oxidation, damage, misfolding and aggregation together with the simultaneously impaired function and induction of chaperones in aged organisms disturb the balance between chaperone requirement and availability. There are several important aspects for future investigation of this field: † the measurement of active chaperone function (i.e. chaperone-assisted refolding of damaged proteins) in cellular extracts does not have a well-established method yet; † we have no methods to measure free chaperone levels; † among the consequences of chaperone overload, changes in signal transduction, protein transport, immune recognition and cellular organization have not been systematically measured and/or related to the protein folding homeostasis of aging organisms and cells.

 

  1. Extracellular HSPs in inflammation and immunity

Cutting Edge: Heat Shock Protein (HSP) 60 Activates the Innate Immune Response: CD14 Is an Essential Receptor for HSP60 Activation of Mononuclear Cells1

Amir Kol,* Andrew H. Lichtman,† Robert W. Finberg,‡ Peter Libby,*† and Evelyn A. Kurt-Jones2‡
J  Immunol 2000; 164: 13–17.  https://www.researchgate.net/profile/Robert_Finberg/publication/12696457_Cutting_Edge_Heat_Shock_Protein_(HSP)_60_Activates_the_Innate_Immune_Response_CD14_Is_an_Essential_Receptor_for_HSP60_Activation_of_Mononuclear_Cells/links/53ee00460cf23733e80b21c0.pdf

Heat shock proteins (HSP), highly conserved across species, are generally viewed as intracellular proteins thought to serve protective functions against infection and cellular stress. Recently, we have reported the surprising finding that human and chlamydial HSP60, both present in human atheroma, can activate vascular cells and macrophages. However, the transmembrane signaling pathways by which extracellular HSP60 may activate cells remains unclear. CD14, the monocyte receptor for LPS, binds numerous microbial products and can mediate activation of monocytes/macrophages and endothelial cells, thus promoting the innate immune response. We show here that human HSP60 activates human PBMC and monocyte-derived macrophages through CD14 signaling and p38 mitogen-activated protein kinase, sharing this pathway with bacterial LPS. These findings provide further insight into the molecular mechanisms by which extracellular HSP may participate in atherosclerosis and other inflammatory disorders by activating the innate immune system.

There is increasing interest in the role of nontraditional mediators of inflammation in atherosclerosis (1). Recent studies from our laboratory have shown that chlamydial and human heat shock protein 60 (HSP60)3 colocalize in human atheroma (2), and either HSP60 induces adhesion molecule and cytokine production by human vascular cells and macrophages, in a pattern similar to that induced by Escherichia coli LPS (3, 4). These results suggested that HSP60 and LPS might share similar signaling mechanisms. CD14 is the major high-affinity receptor for bacterial LPS on the cell membrane of mononuclear cells and macrophages (5, 6). In addition to LPS, CD14 functions as a signaling receptor for other microbial products, including peptidoglycan from Gram-positive bacteria and mycobacterial lipoarabinomann (7, 8). CD14 is considered a pattern recognition receptor for microbial Ags and, with Toll-like receptor (TLR) proteins, an important mediator of innate immune responses to infection (9–14). We have examined the role of CD14 in the response of human monocytes and macrophages to HSP60.  …..

HSP may play a central role in the innate immune response to microbial infections. Because both microbes and stressed or injured host cells produce abundant HSP (36), and dying cells likely release these proteins, it is conceivable that HSP furnish signals that inform the innate immune system of the presence of infection and cell damage. The findings reported here, that human HSP60 induces IL-6 production by mononuclear cells and macrophages via the CD14, supports this hypothesis, suggesting that human HSP60 may act together with LPS or other microbial products to provoke innate immune responses.

Inflammation and immunity can contribute to the pathogenesis and complications of atherosclerosis (37). Moreover, the search for novel risk factors for atherosclerosis has revived the concept that microbial products might substantially contribute to the inflammatory reaction in the atheromatous vessel wall (38, 39). We have shown that chlamydial HSP60 colocalizes with human HSP60 in the macrophages of human atheroma (2). Therefore, bacterial and human HSP60, released from dying or injured cells during atherogenesis (40) or myocardial injury (41), may further promote local inflammation and possibly activate the innate immune system. Previous reports that immunization with mycobacterial HSP65 enhances atheroma formation in rabbits (42), have suggested an important role for HSPs in atherogenesis, particularly because the high degree of homology between HSPs of the same m.w. among different species might stimulate autoimmunity (43).

In conclusion, our findings, that CD14 mediates cellular activation induced by human HSP60 provide further insight into the molecular mechanisms by which HSP may activate the innate immune system and participate in atherogenesis and other inflammatory disorders.

DAMPs, PAMPs and alarmins: all we need to know about danger

Marco E. Bianchi1
J. Leukoc. Biol. 81: 1–5; 2007.   http://aerozon.ru/documents/publications/37_Bianche.pdf

Multicellular animals detect pathogens via a set of receptors that recognize pathogen associated molecular patterns (PAMPs). However, pathogens are not the only causative agents of tissue and cell damage: trauma is another one. Evidence is accumulating that trauma and its associated tissue damage are recognized at the cell level via receptor-mediated detection of intracellular proteins released by the dead cells. The term “alarmin” is proposed to categorize such endogenous molecules that signal tissue and cell damage. Intriguingly, effector cells of innate and adaptive immunity can secrete alarmins via nonclassical pathways and often do so when they are activated by PAMPs or other alarmins. Endogenous alarmins and exogenous PAMPs therefore convey a similar message and elicit similar responses; they can be considered subgroups of a larger set, the damage associated molecular patterns (DAMPs).

Multicellular animals must distinguish whether their cells are alive or dead and detect when microorganisms intrude, and have evolved surveillance/defense/repair mechanisms to this end. How these mechanisms are activated and orchestrated is still incompletely understood, and I will argue that that these themes define a unitary field of investigation, of both basic and medical interest.

A complete system for the detection, containment, and repair of damage caused to cells in the organism requires warning signals, cells to respond to them via receptors and signaling pathways, and outputs in the form of physiological responses. Classically, a subset of this system has been recognized and studied in a coherent form: pathogen-associated molecular patterns (PAMPs) are a diverse set of microbial molecules which share a number of different recognizable biochemical features (entire molecules or, more often, part of molecules or polymeric assemblages) that alert the organism to intruding pathogens [1]. Such exogenous PAMPs are recognized by cells of the innate and acquired immunity system, primarily through toll-like receptors (TLRs), which activate several signaling pathways, among which NF-kB is the most distinctive. As a result, some cells are activated to destroy the pathogen and/or pathogen-infected cells, and an immunological response is triggered in order to produce and select specific T cell receptors and antibodies that are best suited to recognize the pathogen on a future occasion. Most of the responses triggered by PAMPs fall into the general categories of inflammation and immunity.

However, pathogens are not the only causative agents of tissue and cell damage: trauma is another one. Tissues can be ripped, squashed, or wounded by mechanical forces, like falling rocks or simply the impact of one’s own body hitting the ground. Animals can be wounded by predators. In addition, tissues can be damaged by excessive heat (burns), cold, chemical insults (strong acids or bases, or a number of different cytotoxic poisons), radiation, or the withdrawal of oxygen and/or nutrients. Finally, humans can also be damaged by specially designed drugs, such as chemotherapeutics, that are meant to kill their tumor cells with preference over their healthy cells. Very likely, we would not be here to discuss these issues if evolution had not incorporated in our genetic program ways to deal with these damages, which are not caused by pathogens but are nonetheless real and common enough. Tellingly, inflammation is also activated by these types of insults. A frequently quoted reason for the similarity of the responses evoked by pathogens and trauma is that pathogens can easily breach wounds, and infection often follows trauma; thus, it is generally effective to respond to trauma as if pathogens were present. In my opinion, an additional reason is that pathogens and trauma both cause tissue and cell damage and thus trigger similar responses.

None of these considerations is new; however, a new awareness of the close relationship between trauma- and pathogenevoked responses emerged from the EMBO Workshop on Innate Danger Signals and HMGB1, which was held in February 2006 in Milano (Italy); many of the findings presented at the meeting are published in this issue of the Journal of Leukocyte Biology. At the end of the meeting, Joost Oppenheim proposed the term “alarmin” to differentiate the endogenous molecules that signal tissue and cell damage. Together, alarmins and PAMPs therefore constitute the larger family of damage-associated molecular patterns, or DAMPs.

Extranuclear expression of HMGB1 has been involved in a number of pathogenic conditions: sepsis [44], arthritis [45, 46], atherosclerosis [10], systemic lupus erythematosus (SLE) [47], cancer [48] and hepatitis [49, this issue]. Uric acid has been known to be the aethiologic agent for gout since the 19th century. S100s may be involved in arthritis [31, this issue] and psoriasis [50]. However, although it is clear that excessive alarmin expression might lead to acute and chronic diseases, the molecular mechanisms underlying these effects are still largely unexplored.

The short list of alarmins presented above is certainly both provisional and incomplete and serves only as an introduction to the alarmin concept and to the papers published in this issue of JLB. Other molecules may be added to the list, including cathelicidins, defensins and eosinophil-derived neurotoxin (EDN) [51], galectins [52], thymosins [53], nucleolin [54], and annexins [55; and 56, this issue]; more will emerge with time. Eventually, the concept will have to be revised and adjusted to the growing information. Indeed, I have previously argued that any misplaced protein in the cell can signal damage [57], and Polly Matzinger has proposed that any hydrophobic surface (“Hyppo”, or Hydrophobic protein part) might act as a DAMP [58]. As most concepts in biology, the alarmin category serves for our understanding and does not correspond to a blueprint or a plan in the construction of organisms. Biology proceeds via evolution, and evolution is a tinkerer or bricoleur, finding new functions for old molecules. In this, the reuse of cellular components as signals for alerting cells to respond to damage and danger, is a prime example.

 

  1. Role of heat shock and the heat shock response in immunity and cancer

 

Heat Shock Proteins: Conditional Mediators of Inflammation in Tumor Immunity

Stuart K. Calderwood,1,* Ayesha Murshid,1 and Jianlin Gong1
Front Immunol. 2012; 3: 75.  doi:  10.3389/fimmu.2012.00075

Heat shock protein (HSP)-based anticancer vaccines have undergone successful preclinical testing and are now entering clinical trial. Questions still remain, however regarding the immunological properties of HSPs. It is now accepted that many of the HSPs participate in tumor immunity, at least in part by chaperoning tumor antigenic peptides, introducing them into antigen presenting cells such as dendritic cells (DC) that display the antigens on MHC class I molecules on the cell surface and stimulate cytotoxic lymphocytes (CTL). However, in order for activated CD8+ T cells to function as effective CTL and kill tumor cells, additional signals must be induced to obtain a sturdy CTL response. These include the expression of co-stimulatory molecules on the DC surface and inflammatory events that can induce immunogenic cytokine cascades. That such events occur is indicated by the ability of Hsp70 vaccines to induce antitumor immunity and overcome tolerance to tumor antigens such as mucin1. Secondary activation of CTL can be induced by inflammatory signaling through Toll-like receptors and/or by interaction of antigen-activated T helper cells with the APC. We will discuss the role of the inflammatory properties of HSPs in tumor immunity and the potential role of HSPs in activating T helper cells and DC licensing.

Heat shock protein, vaccine, inflammation, antigen presentation

Heat shock proteins (HSP) were first discovered as a group of polypeptides whose level of expression increases to dominate the cellular proteome after stress (Lindquist and Craig, 1988). These increases in HSPs synthesis correlate with a marked resistance to potentially toxic stresses such as heat shock (Li and Werb,1982). The finding that such proteins have extracellular immune functions suggested that, as highly abundant intracellular proteins they could be prime candidates as danger signals to the immune response (Srivastava and Amato,2001). There are several human HSP gene families with known immune significance and their classification is reviewed in Kampinga et al. (2009). These include the HSPA (Hsp70) family, which includes the HPA1A and HSPA1B genes encoding the two major stress-inducible Hsp70s, that together are often referred to as Hsp72. When referring to Hsp70 in this chapter, we generally refer to the products of these two genes. The Hsp70 family also includes two other members with immune function – HSPA8 and HSPA5 genes, whose protein products are known as Hsc70 the major constitutive Hsp70 family member and Grp78, a key ER-resident protein. In addition two more Hsp70 related genes have immune significance and these include HSPH2 (Hsp110) and HSPH4 the ER-resident class H protein Grp170. The Hsp90 family also has major functions in tumor immunity and these include HSPC2 and HSPC3, which encode the major cytoplasmic proteins Hsp90a and Hsp90b, and HSPC4 that encodes ER chaperone Grp94. In addition, the product of the HSPD1 gene, the mitochondrial chaperone Hsp60 has some immunological functions. Mice have been shown to encode orthologs of each of these genes (Kampinga et al., 2009).

It has been suggested that many of the HSPs have the property of damage associated molecular patterns (DAMPs), inducers of sterile inflammation and innate immunity (Kono and Rock, 2008). The additional discovery that intracellular HSPs function as molecular chaperones and can bind to a wide spectrum of intracellular polypeptides further indicated that they could play a broad role in the immune response and might mediate both innate immunity due to their status as DAMPs and adaptive immunity by chaperoning antigens.

Heat shock proteins are currently employed as vaccines in cancer immunotherapy (Tamura et al., 1997; Murshid et al., 2011a). The rationale behind the approach is that if HSPs can be extracted from tumor tissue bound to the polypeptides which they chaperone during normal metabolism, they may retain antigenic peptides specific to the tumor (Noessner et al., 2002; Srivastava, 2002; Wang et al., 2003; Enomoto et al., 2006; Gong et al., 2010). Indeed, vaccines based on Hsp70, Hsp90, Grp94, Hsp110, and Grp170 polypeptide complexes have been used successfully to immunize mice to a range of tumor types and Hsp70 and Grp94 vaccines have undergone recent clinical trials (rev: Murshid et al., 2011a). These effects of the HSP vaccines on tumor immunity appear to be mediated largely to the associated, co-isolated tumor polypeptides, although in the case of Grp94 this question is still controversial and tumor regression was observed in mice treated with the chaperone devoid of its peptide binding domain (Udono and Srivastava, 1993; Srivastava, 2002; Nicchitta, 2003; Chandawarkar et al., 2004; Nicchitta et al.,2004). Use of such HSP vaccines is potentially a powerful approach to tumor immunotherapy as the majority of the antigenic repertoire of most individual tumor cells is unknown (Srivastava and Old, 1988; Srivastava, 1996). Individual cancer cells are likely to take a lone path in accumulating a spectrum of random mutations. Although some mutations are functional, permitting cells to become transformed and to progress into a highly malignant state, many such changes are likely to be passenger mutations not required to drive tumor growth (Srivastava and Old, 1988; Srivastava, 1996). Some of these individual mutant sequences will be novel antigenic epitopes and together with the few known shared tumor antigens comprise an “antigenic fingerprint” for each individual tumor (Srivastava,1996). Accumulation of mutations in cancer appears to be related to, and may drive the increases in HSPs observed in many tumors (Kamal et al., 2003; Whitesell and Lindquist, 2005; Trepel et al., 2010). As the mutant conformations of tumor proteins are “locked in” due to the covalent nature of the alterations, cancer cells appear to be under permanent proteotoxic stress and rich in HSP expression (Ciocca and Calderwood, 2005). For tumor immunology these conditions may offer a therapeutic opportunity as individual HSPs, whose expression is expanded in cancer will chaperone a cross-section of the “antigenic fingerprint” of the individual tumors (Murshid et al., 2011a). This approach was first utilized by Srivastava (20002006) and led to the development of immunotherapy using HSP–peptide complexes.

In addition to using HSP–peptide complexes extracted from tumors, in cases where tumor antigens are known, these can be directly loaded onto purified or recombinant HSPs and the complex used as a vaccine. This procedure has been carried out successfully in the case of the “large HSPs,” Hsp110 and Grp170 (Manjili et al., 20022003). A variant of this approach employs the molecular engineering of tumor antigens in order to produce molecular chaperone-fusion genes which encode products in which the HSP is fused covalently to the antigen. The fusion proteins are then employed as vaccines. This approach was pioneered by Young et al. who showed that a fusion between mycobacterial Hsp70 and ovalbumin could induced cytotoxic lymphocytes (CTL) in mice with the capacity to kill Ova-expressing cancer cells (Suzue et al., 1997). The vaccines could be used effectively without adjuvant and adjuvant properties were ascribed to the molecular chaperone component of the fusion protein. Subsequent studies have confirmed the utility of the approach in targeting common tumor antigens such as the melanoma antigen Mage3 (Wang et al., 2009).

HSPs and Immunosurveillance in Cancer

The question next arises as to the role of endogenous HSPs, with or without bound antigens in immunosurveillance of cancer cells. Although the immune system can recognize tumor antigens and generate a CTL response, most cancers evade immune cell killing by a range of strategies (van der Bruggen et al., 1991; Pardoll,2003). These include the down-regulation of surface MHC class I molecules by individual tumor cells and release of immunosuppressive IL-10 by tumors (Moller and Hammerling, 1992; Chouaib et al., 2002). Tumors in vivo also appear to attract a range of hematopoietic cells with immunosuppressive action including regulatory CD4+CD25+FoxP3+ T cells (Treg), M2 macrophages, myeloid-derived suppressor cells (MDSC) and some classes of natural killer cells (Pekarek et al.,1995; Terabe et al., 2005; Mantovani et al., 2008; Marigo et al., 2008). The tumor milieu also contain a small fraction of cells of mesenchymal origin identified by surface fibroblast activation protein-a (FAP cells) that suppress antitumor immune responses (Kraman et al., 2010). Endogenous tumor HSPs may also participate in immune suppression. Although the majority of the HSPs function as intracellular molecular chaperones, a fraction of these proteins can be released from cells even under unstressed conditions and may participate in immune functions (rev: Murshid and Calderwood, 2012). Intracellular Hsp70 can be actively secreted from tumor cells in either free form or packaged into lipid-bounded structures called exosomes (Mambula and Calderwood, 2006b; Chalmin et al., 2010). In addition Hsp70 and Hsp90 can also be found associated with the surfaces of tumor cells where they can function as molecular chaperones or as recognition structures for immune cells (Sidera et al., 2008; Qin et al., 2010; Multhoff and Hightower, 2011). As Hsp70 was shown in a number of earlier studies to be pro-inflammatory due to its interaction with pattern recognition receptors such as Toll-like receptors 2 and 4 (TLR2 and TLR4), these findings might suggest, as mentioned above, that Hsp70 released by tumors could be pro-inflammatory and possess the properties of DAMPs (Asea et al., 20002002; Vabulas et al., 2002). However, subsequent studies indicated that a portion of the TLR4 activation detected in the earlier reports, involving exposure of monocytes, macrophages, or dendritic cells (DC) to HSPs in vitro may be due to trace contamination with bacterial pathogen associated molecular patterns (PAMPs), potent TLR activators (Tsan and Gao,2004). In spite of these drawbacks, an overwhelming amount of evidence now seems to indicate the interaction of Hsp70 and other HSPs with TLRs (particularly TLR4) in vivo – in a wide range of physiological and pathological conditions, leading to acute inflammation in many conditions (Chase et al., 2007; Wheeler et al., 2009; see Appendix for a full list of references). Thus both TLR2 and TLR4 appear to be important components of inflammatory responses to Hsp70 under many pathophysiological conditions. In cancer therapy it has been shown that autoimmunity can be triggered in mice through necrotic killing of melanocytes engineered to overexpress Hsp70; such treatment led to the concomitant immune destruction of B16 melanoma tumors that share patterns of antigen expression with the killed melanocytes (Sanchez-Perez et al., 2006). Hsp70 appears to play an adjuvant role in this form of therapy through its interaction with TLR4 and induction of the cytokine TNF-a (Sanchez-Perez et al., 2006). However, despite these findings it has also been shown that depletion of Hsp70 in cancer cells can, in the absence of other treatments lead to tumor regression by inducing antitumor immunity (Rerole et al., 2011). This effect appears to be due to the secretion by cancer cells of immunosuppressive exosomes containing Hsp70 that activate MDSC and lead to local immunosuppression (Chalmin et al., 2010). Under normal circumstances therefore, release of endogenous Hsp70 into the extracellular microenvironment may be a component of the tumor defenses against immunosurveillance. Extracellular Hsp60 has also been shown be immunomodulatory and can increase levels of FoxP3 Treg in vitro and suppress T cell-mediated immunity (de Kleer et al., 2010; Aalberse et al., 2011).

The pro-inflammatory properties of extracellular HSPs may be more evident underin vivo situations particularly in the context of tissue damage (Sanchez-Perez et al.,2006). For instance when elevated temperatures were used to boost Hsp70 release from Lewis Lung carcinoma cells in vivo, antitumor immunity was activated along with release of chemokines CCL2, CCL5, and CCL10, in a TLR4-dependent manner, leading to attraction of DC and T cells into the tumor (Chen et al., 2009). Thus under resting conditions, the tumor milieu appears to be a specialized immunosuppressive environment, rich in inhibitory cells such as Treg, MDSC, and M2 macrophages and inaccessible to “exhausted” CD8+ T cells that often fail to penetrate the tumor microcirculation. However, under inflammatory conditions involving necrotic cell killing of tumor cells, extracellular HSPs may be able to amplify the anticancer immune response, intracellular HSPs may be released to further increase such a response and CTL may triggered to penetrate the tumor milieu, inducing antigen-specific cancer cell killing (Evans et al., 2001; Mambula and Calderwood, 2006a; Sanchez-Perez et al., 2006; Chen et al., 2009).

 

HSP-Based Anticancer Vaccines

It is apparent that a number of HSP types, conjugated to peptide complexes (HSP.PC) from cancer cells form effective bases for immunotherapy approaches with unique properties, as mentioned above (Calderwood et al., 2008; Murshid et al., 2011a). The immunogenicity of most HSP.PC appears to involve the ability of the HSPs to sample the tumor “antigenic fingerprint,” deliver the antigens to antigen presenting cells (APC) such as DC and stimulate activation of CTL (Tamura et al., 1997; Singh-Jasuja et al., 2000b; Wang et al., 2003; Murshid et al.,2010). A number of studies show that HSPs can chaperone tumor antigens and deliver them to the appropriate destination – MHC class I molecules on the DC surface (Singh-Jasuja et al., 2000a,b; Srivastava and Amato, 2001; Delneste et al.,2002; Enomoto et al., 2006; Gong et al., 2009). In addition, Hsp70 has been shown to chaperone viral antigenic peptides and increase cross priming of human CTL under ex vivo conditions (Tischer et al., 2011). However, it is still far from clear how the process of HSP-mediated cross priming unfolds. For instance, the CD8+ expressing DC subpopulation in lymph nodes is regarded as the primary cross-presenting APC (Heath and Carbone, 2009). It is not however currently known whether the CD8+ DC subset or other peripheral or lymph-node resident, DC interact with HSP vaccines to induce cross presentation. HSPs appear to be able to enter APC, such as mouse bone marrow derived DC (BMDC) and human DC in a receptor-mediated manner (Basu et al., 2001; Delneste et al., 2002; Gong et al.,2009; Murshid et al., 2010). However, no unique endocytosing HSP receptor has emerged and HSP–antigen complexes appear instead to be taken up by proteins with “scavenger” function such as LOX-1, SRECI, and CD91 that can each take up a wide range of extracellular ligands (Basu et al., 2001; Delneste et al., 2002; Theriault et al., 2006; Murshid et al., 2010). A pathway for Hsp90–peptide (Hsp90.PC) uptake has been characterized in mouse BMDC by scavenger receptor SRECI (Murshid et al., 2010). SRECI is able to mediate the whole process of Hsp90.PC endocytosis, trafficking through the cytoplasm to the sites of antigen processing and presentation of antigens to CD8+ T lymphocytes on MHC class I molecules (Murshid et al., 2010). This process is known as antigen cross presentation (Kurts et al., 2010). It is not currently clear what the relative contribution to antigen cross presentation of the various HSP receptors might be under in vivo conditions. It may be that each receptor class contributes to an individual aspect of CTL activation by HSP peptide complexes although a definitive understanding may await studies in mice deficient in each receptor class.

 

HSPs and CTL Programming

It is evident that that HSPs can mediate antigen cross presentation and activate CD8+ T lymphocytes. However, presentation of tumor antigens by DC is not sufficient for CTL programming and, in the absence of co-stimulatory molecules and innate immunity, the “helpless” CD8+ cells will cease to proliferate abundantly and will most likely undergo apoptosis (Schurich et al., 2009; Kurts et al., 2010). One mechanism for enhancing CTL programming involves activation of the TLR pathways that lead to synthesis of co-stimulatory molecules (Rudd et al.,2009; Yamamoto and Takeda, 2010). The co-stimulatory molecules, including CD80 and CD86 then become expressed on the DC cell surface and amplify the signals induced by binding of the T cell receptor on CD8+ T cells to MHC class I peptide complexes on the presenting DC (Parra et al., 1995; Rudd et al., 2009). This process is important in pathogen infection in which microbially derived antigens are encountered in the presence of inflammatory PAMPs that can activate innate immune transcriptional networks. Originally it had been thought that HSPs could provide analogous stimulation through their suspected activity as DAMPs and their inbuilt ability to trigger innate immunity through TLR2 and TLR4 on DC (Asea et al., 20002002; Vabulas et al., 2002). (The potential role of HSPs as DAMPs has been the subject of a recent review: van Eden et al., 2012). Subsequent studies on the capacity of HSPs to bind TLRs do not indicate avid binding of Hsp70 to either TLR2 or TLR4 when expressed in cells deficient in HSP receptors in vitro (Theriault et al., 2006). In vivo however, TLR signaling is essential for Hsp70 vaccine-induced tumor cell killing. Studies of tumor-bearing mice treated with an Hsp70 vaccine in vivo indicated that vaccine function is depleted by knockout of the TLR signaling intermediate Myd88 and completely abrogated by double knockout of TLR2 and TLR4 (Gong et al., 2009). These findings were somewhat complicated by the fact that TLR4 is involved in upstream regulation of the expression of Hsp70 receptor SRECI, but do strongly implicate a role for these receptors in amplifying immune signaling by Hsp70 vaccines and Hsp70-based immunotherapy (Sanchez-Perez et al., 2006; Gong et al., 2009). It is still not clear to what degree HSPs are capable of providing a sturdy DC maturing signal through TLR2/TLR4. The potency of HSP anticancer vaccines could potentially be improved by addition of PAMPs such as CpG DNA shown to activate TLR9, or double stranded RNA that can activate TLR3 (Murshid et al., 2011a). As mentioned, one contradictory factor in the earlier studies was that, although TLR2 and TLR4 are required for a sturdy Hsp70 vaccine-mediated immune response, direct binding of Hsp70 to these receptors was not observed (Theriault et al., 2006; Gong et al., 2009; Murshid et al., 2012). A rationale for these findings might be that HSPs can activate TLR signaling indirectly through primary binding to established HSP receptors such as LOX-1 and SRECI which secondarily recruit and activate the TLRs (Murshid et al., 2011b). Both of these scavenger receptors bind to TLR2 upon stimulation and activate TLR2-based signaling (Jeannin et al., 2005; A. Murshid and SK Calderwood, in preparation). In addition, we have found that Hsp90–SRECI complexes move to the lipid raft compartment of the cell, an environment highly enriched in TLR2 and TLR4 (Triantafilou et al., 2002; Murshid et al., 2010).

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3342006/bin/fimmu-03-00075-g001.jpg

Heat shock protein–peptide complexes extracted from tumor cells interact with endocytosing receptors (HSP-R) such as SRECI or signaling receptors (TLR) such as TLR4 on DC. SREC1 mediates uptake and intracellular processing of antigens and the presentation of resulting peptides on surface MHC class I and MHC class II proteins. MHC class II receptor–peptide complexes then bind to T cell receptors on CD4+ cells. One consequence of binding is interaction of CD40 ligand on the MHC class II cell with CD40 on the DC leading to the licensing interaction that results in enhanced expression of co-stimulatory proteins on the DC cell surface. The licensed DC may then interact with CD8+ T cells through T cell interaction with MHC class I peptide complexes. This effect will be enhanced by simultaneous interaction of CD80 or Cd86 co-stimulatory complexes on the DC with CD28 on the CD8+ cells, leading to effective CD8+ CTL that can lyse tumor cells. T cell programming can also be amplified by signals emanating from activated TLR that can boost levels of CD80 and CD86 as well as inflammatory cytokines (not shown).

 

Hsp70, Cell Damage, and Inflammation

The question of whether Hsp70 acts as DAMP and could by itself induce an inflammatory response in cancer patients in vivo is still open. However, some recent studies by Vile et al. using a gene therapy approach may shed some light on the inflammatory role of Hsp70 in tumor therapy. In this approach, as mentioned above, normal murine tissues were engineered to express high Hsp70 levels then subjected to treatments that lead to necrotic killing. The aim was to stimulate an autoimmune response that could lead to bystander immune killing of tumor cells that share the antigenic repertoire as the killed normal cells (Sanchez-Perez et al.,2006). In the initial studies, normal melanocytes were preloaded with Hsp70 plasmids and then necrotic cell death was triggered (Daniels et al., 2004). This treatment led to T cell-mediated immune killing of syngeneic B16 melanoma cells transplanted at a distant site in the mouse, presumably in response to antigens shared by the killed normal melanocytes and melanoma cell (Daniels et al., 2004). This effect only occurred when melanocytes were induced to undergo necrosis and Hsp70 levels were elevated, indicating a role for high levels of Hsp70 in the tumor specific immune response. Interestingly, these conditions did not lead to a prolonged autoimmune response, an effect mediated by the induction of a delayed Treg response (Srivastava, 2003; Daniels et al., 2004). It is notable that some early studies of chaperone-based tumor vaccines in animal models demonstrated a primary CTL response to tumors in response to treatment followed by delayed activation of a Treg reaction, and that chaperone levels must be carefully titrated for effective induction of tumor immunity (Udono and Srivastava, 1993; Liu et al.,2009). The role of Hsp70 in autoimmune rejection of tumors was also investigated in prostate cancer (Kottke et al., 2007). Ablation of normal prostate cells by necrotic killing with fusogenic viruses in the absence of Hsp70 elevation led to the induction of the cytokines IL-10 and TGF-b in the mouse prostate and a Treg response. However, when Hsp70 levels were elevated in these cells, IL-10, TGF-b, and IL-6 were induced simultaneously, the IL-6 component leading to further induction of IL-17, a profound Th17 response and tumor rejection (Kottke et al.,2007). Thus elevated levels of Hsp70, presumably released from cells undergoing necrosis can influence the local cytokine patterns and lead to an inflammatory statein vivo. Interestingly, these results seem to be tissue specific as inflammatory killing of pancreatic cells even in the presence of elevated Hsp70 did not provoke IL-6 release, a Th17 response or tumor rejection and the Treg response dominated under these conditions (Kottke et al., 2009). Thus the role of Hsp70 in tissue inflammation and tumor rejection seems to require elevated concentrations of extracellular chaperones, significant levels of necrotic cell killing, and tissue specific cytokine release.

Conclusion

  • Earlier studies investigating HSP vaccines considered such structures to be the “Swiss penknives” of immunology able to deliver antigens directly to APC and confer a maturing signal that could render DC able to effectively program CTL (Srivastava and Amato, 2001; Noessner et al., 2002). It is well established now that Hsp70, Hsp90, Hsp110, and GRP170 can chaperone tumor antigens and activate antigen cross presentation (Murshid et al., 2011a). In addition, HSPs were thought to be DAMPs with ability to strongly activate TLR signaling and innate immunity (Asea et al., 2000). However, although there is compelling evidence to indicate that Hsp70, for instance can interact with TLR4 under a number of pathological situations (see Appendix, Sanchez-Perez et al., 2006), it remains unclear whether free Hsp70 binds directly to the Toll-like receptor and induces innate immunity in the absence of other treatments in vitro(Tsan and Gao, 2004).
  • Elevated levels of extracellular HSPs appear to have the capacity to amplify the effects of inflammatory signals emanating from necrotic cells in vivoin a TLR4-dependent manner (Daniels et al., 2004; Sanchez-Perez et al., 2006; Kottke et al., 2007). In the presence of cell injury and death, elevated levels of Hsp70 appear to increase the production of inflammatory signals that involve cytokines such as IL-6 and IL-17 and lead to a specific T cell-mediated immune response to tumor cells sharing antigens with the dying cells (Kottke et al., 2007). The mechanisms involved in these processes are not clear although one possibility is that HSPs can induce the engulfment of necrotic cells. Hsp70 has been shown to increase bystander engulfment of a variety of structures (Wang et al., 2006a,b). In addition, tumor cells treated with elevated temperatures release inflammatory chemokines in an Hsp70 and TLR4-dependent mechanisms and this effect may be significant in CTL programming and tumor cell killing (Chen et al., 2009). Our studies indicate that CTL induction by Hsp70 vaccines in vivo has an absolute requirement for TLR2 and TLR4 suggesting that at least in vivo HSPs can trigger innate immunity through TLR signaling (Gong et al., 2009).
  • HSPs appear also to be able to direct antigen presentation through the class II pathway in DC and may stimulate T helper cells (Gong et al., 2009). It may thus be possible that HSPs participate in DC licensing and reinforce CTL programming during exposure to HSP vaccines. Future studies will address these questions.
  • A further interesting consideration is whether HSPs released from untreated tumor cells enhance or depress tumor immunity. One initial study shows that Hsp70 released from tumor cells in exosomes can strongly decrease tumor immunity through effects on MDSC (Chalmin et al., 2010). Further studies will be required to make a definitive statement on these questions.

 

  1. Protein aggregation disorders and HSP expression

Chaperone suppression of aggregation and altered subcellular proteasome localization imply protein misfolding in SCA1

Christopher J. Cummings1,5, Michael A. Mancini3, Barbara Antalffy4, Donald B. DeFranco7, Harry T. Orr8 & Huda Y. Zoghbi1,2,6
Nature Genetics 19, 148 – 154 (1998) http://dx.doi.org:/10.1038/502

Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disorder caused by expansion of a polyglutamine tract in ataxin-1. In affected neurons of SCA1 patients and transgenic mice, mutant ataxin-1 accumulates in a single, ubiquitin-positive nuclear inclusion. In this study, we show that these inclusions stain positively for the 20S proteasome and the molecular chaperone HDJ-2/HSDJ. Similarly, HeLa cells transfected with mutant ataxin-1 develop nuclear aggregates which colocalize with the 20S proteasome and endogenous HDJ-2/HSDJ. Overexpression of wild-type HDJ-2/HSDJ in HeLa cells decreases the frequency of ataxin-1 aggregation. These data suggest that protein misfolding is responsible for the nuclear aggregates seen in SCA1, and that overexpression of a DnaJ chaperone promotes the recognition of a misfolded polyglutamine repeat protein, allowing its refolding and/or ubiquitin-dependent degradation.

Effects of heat shock, heat shock protein 40 (HDJ-2), and proteasome inhibition on protein aggregation in cellular models of Huntington’s disease

Andreas Wyttenbach, Jenny Carmichael, Jina Swartz, Robert A. Furlong, Yolanda Narain, Julia Rankin, and David C. Rubinsztein*
https://www.researchgate.net/profile/David_Rubinsztein/publication/24447892_Effects_of_heat_shock_heat_shock_protein_40_(HDJ2)_and_proteasome_inhibition_on_protein_aggregation_in_cellular_models_of_Huntington’s_disease/links/00b7d528b80aab69bb000000.pdf

Huntington’s disease (HD), spinocerebellar ataxias types 1 and 3 (SCA1, SCA3), and spinobulbar muscular atrophy (SBMA) are caused by CAGypolyglutamine expansion mutations. A feature of these diseases is ubiquitinated intraneuronal inclusions derived from the mutant proteins, which colocalize with heat shock proteins (HSPs) in SCA1 and SBMA and proteasomal components in SCA1, SCA3, and SBMA. Previous studies suggested that HSPs might protect against inclusion formation, because overexpression of HDJ-2yHSDJ (a human HSP40 homologue) reduced ataxin-1 (SCA1) and androgen receptor (SBMA) aggregate formation in HeLa cells. We investigated these phenomena by transiently transfecting part of huntingtin exon 1 in COS-7, PC12, and SH-SY5Y cells. Inclusion formation was not seen with constructs expressing 23 glutamines but was repeat length and time dependent for mutant constructs with 43–74 repeats. HSP70, HSP40, the 20S proteasome and ubiquitin colocalized with inclusions. Treatment with heat shock and lactacystin, a proteasome inhibitor, increased the proportion of mutant huntingtin exon 1-expressing cells with inclusions. Thus, inclusion formation may be enhanced in polyglutamine diseases, if the pathological process results in proteasome inhibition or a heat-shock response. Overexpression of HDJ-2yHSDJ did not modify inclusion formation in PC12 and SH-SY5Y cells but increased inclusion formation in COS-7 cells. To our knowledge, this is the first report of an HSP increasing aggregation of an abnormally folded protein in mammalian cells and expands the current understanding of the roles of HDJ-2yHSDJ in protein folding.

 

  1. Hsp70 in blood cell differentiation.

 

Apoptosis Versus Cell Differentiation -Role of Heat Shock Proteins HSP90, HSP70 and HSP27

David Lanneau, Aurelie de Thonel, Sebastien Maurel, Celine Didelot, and Carmen Garrido
Prion. 2007 Jan-Mar; 1(1): 53–60.  http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2633709/

Heat shock proteins HSP27, HSP70 and HSP90 are molecular chaperones whose expression is increased after many different types of stress. They have a protective function helping the cell to cope with lethal conditions. The cytoprotective function of HSPs is largely explained by their anti-apoptotic function. HSPs have been shown to interact with different key apoptotic proteins. As a result, HSPs can block essentially all apoptotic pathways, most of them involving the activation of cystein proteases called caspases. Apoptosis and differentiation are physiological processes that share many common features, for instance, chromatin condensation and the activation of caspases are frequently observed. It is, therefore, not surprising that many recent reports imply HSPs in the differentiation process. This review will comment on the role of HSP90, HSP70 and HSP27 in apoptosis and cell differentiation. HSPs may determine de fate of the cells by orchestrating the decision of apoptosis versus differentiation.

Key Words: apoptosis, differentiation, heat shock proteins, chaperones, cancer cells, anticancer drugs

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Introduction

Stress or heat shock proteins (HSPs) were first discovered in 19621 as a set of highly conserved proteins whose expression was induced by different kinds of stress. It has subsequently been shown that most HSPs have strong cytoprotective effects and behave as molecular chaperones for other cellular proteins. HSPs are also induced at specific stages of development, differentiation and during oncogenesis.2 Mammalian HSPs have been classified into five families according to their molecular size: HSP100, HSP90, HSP70, HSP60 and the small HSPs. Each family of HSPs is composed of members expressed either constitutively or regulated inducibly, and/or targeted to different sub-cellular compartments. The most studied HSPs are HSP90, the inducible HSP70 (also called HSP72) and the small heat shock protein HSP27.

HSP90 is a constitutively abundant chaperone that makes up 1–2% of cytosolic proteins. It is an ATP-dependent chaperone that accounts for the maturation and functional stability of a plethora of proteins termed HSP90 client proteins. In mammals, HSP90 comprises 2 homologue proteins (HSP90α and HSP90β) encoded by separated but highly conserved genes that arose through duplication during evolution.3 Most studies do not differentiate between the two isoforms because for a long time they have been considered as having the same function in the cells. However, recent data and notably out-of-function experiments indicate that at least some functions of the beta isoform are not overlapped by HSP90α’s functions.4 HSP70, like HSP90, binds ATP and undergoes a conformational change upon ATP binding, needed to facilitate the refolding of denatured proteins. The chaperone function of HSP70 is to assist the folding of newly synthesized polypeptides or misfolded proteins, the assembly of multi-protein complexes and the transport of proteins across cellular membranes.5,6 HSP90 and HSP70 chaperone activity is regulated by co-chaperones like Hip, CHIP or Bag-1 that increase or decrease their affinity for substrates through the stabilization of the ADP or ATP bound state. In contrast to HSP90 and HSP70, HSP27 is an ATP-independent chaperone, its main chaperone function being protection against protein aggregation.7 HSP27 can form oligomers of more than 1000 Kda. The chaperone role of HSP27 seems modulated by its state of oligomerization, the multimer being the chaperone competent state.8 This oligomerization is a very dynamic process modulated by the phosphorylation of the protein that favors the formation of small oligomers. Cell-cell contact and methylglyoxal can also modulate the oligomerization of the protein.9

It is now well accepted that HSPs are important modulators of the apoptotic pathway. Apoptosis, or programmed cell death, is a type of death essential during embryogenesis and, latter on in the organism, to assure cell homeostasis. Apoptosis is also a very frequent type of cell death observed after treatment with cytotoxic drugs.10 Mainly, two pathways of apoptosis can be distinguished, although cross-talk between the two signal transducing cascades exists (Fig. 1). The extrinsic pathway is triggered through plasma membrane proteins of the tumor necrosis factor (TNF) receptor family known as death receptors, and leads to the direct activation of the proteases called caspases, starting with the receptor-proximal caspase-8. The intrinsic pathway involves intracellular stress signals that provoke the permeabilization of the outer mitochondrial membrane, resulting in the release of pro-apoptotic molecules normally confined to the inter-membrane space. Such proteins translocate from mitochondria to the cytosol in a reaction that is controlled by Bcl-2 and Bcl-2-related proteins.11 One of them is the cytochrome c, which interacts with cytosolic apoptosis protease-activating factor-1 (Apaf-1) and pro-caspase-9 to form the apoptosome, the caspase-3 activation complex.12Apoptosis inducing factor (AIF) and the Dnase, EndoG, are other mitochondria intermembrane proteins released upon an apoptotic stimulus. They translocate to the nucleus and trigger caspase-independent nuclear changes.13,14 Two additional released mitochondrial proteins, Smac/Diablo and Htra2/Omi, activate apoptosis by neutralizing the inhibitory activity of the inhibitory apoptotic proteins (IAPs) that associate with and inhibit caspases15 (Fig. 1).

Figure 1     http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2633709/figure/F1/

Modulation of apoptosis and differentiation by HSP90, HSP70 and HSP27. In apoptosis (upper part), HSP90 can inhibit caspase (casp.) activation by its interaction with Apaf1. HSP90 stabilizes proteins from the survival signaling including RIP, Akt and 

Apoptosis and differentiation are two physiological processes that share different features like chromatin condensation or the need of caspase activity.16 It has been demonstrated in many differentiation models that the activation of caspases is preceded by a mitochondrial membrane depolarization and release of mitochondria apoptogenic molecules.17,18 This suggests that the mitochondrial-caspase dependent apoptotic pathway is a common intermediate for conveying apoptosis and differentiation. Timing, intensity and cellular compartmentalization might determine whether a cell is to die or differentiate. HSPs might be essential to orchestrate this decision. This review will describe the role of HSP90, HSP70 and HSP27 in apoptosis and cell differentiation.

 

HSP27, HSP70 and HSP90 are Anti-Apoptotic Proteins

Overexpression of HSP27, HSP70 or HSP90 prevents apoptosis triggered by various stimuli, including hyperthermia, oxidative stress, staurosporine, ligation of the Fas/Apo-1/CD95 death receptor or anticancer drugs.2,1921 Downregulation or inhibition of HSP27, HSP70 or HSP90 have been shown to be enough to sensitize a cell to apoptosis, proving that endogenous levels of those chaperones seem to be sufficiently high to control apoptosis.2224 It is now known that these chaperones can interact with key proteins of the apoptotic signaling pathways (Fig. 1).

 

HSP90: A survival protein through its client proteins.

HSP90 client proteins include a number of signaling proteins like ligand-dependent transcription factors and signal transducing kinases that play a role in the apoptotic process. Upon binding and hydrolysis of ATP, the conformation of HSP90 changes and the client protein, which is no longer chaperoned, is ubiquitinated and degraded by the proteasome.25

A function for HSP90 in the serine/threonine protein kinase Akt pathway was first suggested by studies using an HSP90 inhibitor that promoted apoptosis in HEK293T and resulted in suppressed Akt activity.26 A direct interaction between Akt and HSP90 was reported later.27 Binding of HSP90 protects Akt from protein phosphatase 2A (PP2A)-mediated dephosphorylation.26 Phosphorylated Akt can then phosphorylate the Bcl-2 family protein Bad and caspase-9 leading to their inactivation and to cell survival.28,29 But Akt has been also shown to phosphorylate IkB kinase, which results in promotion of NFkB-mediated inhibition of apoptosis.30 When the interaction HSP90/Akt was prevented by HSP90 inhibitors, Akt was dephosphorylated and destabilized and the likelihood of apoptosis increased.27 Additional studies showed that another chaperone participates in the Akt-HSP90 complex, namely Cdc37.26 Together this complex protects Akt from proteasome degradation. In human endothelial cells during high glucose exposure, apoptosis can be prevented by HSP90 through augmentation of the protein interaction between eNOS and HSP90 and recruitment of the activated Akt.31 HSP90 has also been shown to interact with and stabilize the receptor interacting protein (RIP). Upon ligation of TNFR-1, RIP-1 is recruited to the receptor and promotes the activation of NFκB and JNK. Degradation of RIP-1 in the absence of HSP90 precludes activation of NFκB mediated by TNFα and sensitizes cells to apoptosis.32 Another route by which HSP90 can affect NFκB survival activity is via the IKK complex.33 The HSP90 inhibitor geldanamycin prevents TNF-induced activation of IKK, highlighting the role of HSP90 in NFκB activation. Some other HSP90 client proteins through which this chaperone could participate in cell survival are p5334 and the transcription factors Her2 and Hif1α.35,36

But the anti-apoptotic role of HSP90 can also be explained by its effect and interaction with proteins not defined as HSP90 client proteins (i.e., whose stability is not regulated by HSP90). HSP90 overexpression in human leukemic U937 cells can prevent the activation of caspases in cytosolic extracts treated with cytochrome c probably because HSP90 can bind to Apaf-1 and inhibit its oligomerization and further recruitment of procaspase-9.37

Unfortunately, most studies do not differentiate between HSP90α and HSP90β. It has recently been demonstrated in multiple myeloma, in which an over expression of HSP90 is necessary for cell survival, that depletion of HSP90β by siRNA is sufficient to induce apoptosis. This effect is strongly increased when also HSP90α is also depleted,23 suggesting different and cooperating anti-apoptotic properties for HSP90α and HSP90β. Confirming this assumption, in mast cells, HSP90β has been shown to associate with the anti-apoptotic protein Bcl-2. Depletion of HSP90β with a siRNA or inhibion of HSP90 with geldanamycin inhibits HSP90β interaction with Bcl-2 and results in cytochrome c release, caspase activation and apoptosis.38

In conclusion, HSP90 anti-apoptotic functions can largely be explained by its chaperone role assuring the stability of different proteins. Recent studies suggest that the two homologue proteins, HSP90α and HSP90β, might have different survival properties. It would be interesting to determine whether HSP90α and HSP90β bind to different client proteins or bind with different affinity.

 

HSP70: A quintessential inhibitor of apoptosis.

HSP70 loss-of-function studies demonstrated the important role of HSP70 in apoptosis. Cells lacking hsp70.1 and hsp70.3, the two genes that code for inductive HSP70, are very sensitive to apoptosis induced by a wide range of lethal stimuli.39Further, the testis specific isoform of HSP70 (hsp70.2) when ablated, results in germ cell apoptosis.40 In cancer cells, depletion of HSP70 results in spontaneous apoptosis.41

HSP70 has been shown to inhibit the apoptotic pathways at different levels (Fig. 1). At the pre-mitochondrial level, HSP70 binds to and blocks c-Jun N-terminal Kinase (JNK1) activity.42,43 Confirming this result, HSP70 deficiency induces JNK activation and caspase-3 activation44 in apoptosis induced by hyperosmolarity. HSP70 also has been shown to bind to non-phosphorylated protein kinase C (PKC) and Akt, stabilizing both proteins.45

At the mitochondrial level, HSP70 inhibits Bax translocation and insertion into the outer mitochondrial membrane. As a consequence, HSP70 prevents mitochondrial membrane permeabilization and release of cytochrome c and AIF.46

At the post-mitochondrial level HSP70 has been demonstrated to bind directly to Apaf-1, thereby preventing the recruitment of procaspase-9 to the apoptosome.47However, these results have been contradicted by a study in which the authors demonstrated that HSP70 do not have any direct effect on caspase activation. They explain these contradictory results by showing that it is a high salt concentration and not HSP70 that inhibits caspase activation.48

HSP70 also prevents cell death in conditions in which caspase activation does not occur.49 Indeed, HSP70 binds to AIF, inhibits AIF nuclear translocation and chromatin condensation.39,50,51 The interaction involves a domain of AIF between aminoacids 150 and 228.52 AIF sequestration by HSP70 has been shown to reduce neonatal hypoxic/ischemic brain injury.53 HSP70 has also been shown to associate with EndoG and to prevent DNA fragmentation54 but since EndoG can form complexes with AIF, its association with HSP70 could involve AIF as a molecular bridge.

HSP70 can also rescue cells from a later phase of apoptosis than any known survival protein, downstream caspase-3 activation.55 During the final phases of apoptosis, chromosomal DNA is digested by the DNase CAD (caspase activated DNase), following activation by caspase-3. The enzymatic activity and proper folding of CAD has been reported to be regulated by HSP70.56

At the death receptors level, HSP70 binds to DR4 and DR5, thereby inhibiting TRAIL-induced assembly and activity of death inducing signaling complex (DISC).57 Finally, HSP70 has been shown to inhibit lysosomal membrane permeabilization thereby preventing cathepsines release, proteases also implicated in apoptosis.58,59

In conclusion, HSP70 is a quintessential regulator of apoptosis that can interfere with all main apoptotic pathways. Interestingly, the ATP binding domain of HSP70 is not always required. For instance, while the ATPase function is needed for the Apaf-150 and AIF binding,51 it is dispensable for JNK60 or GATA-161binding/protection. In this way, in erythroblasts, in which HSP70 blocks apoptosis by protecting GATA-1 from caspase-3 cleavage, a HSP70 mutant that lacks the ATP binding domain of HSP70 is as efficient as wild type HSP70 in assuring the protection of erythroblasts.61

 

HSP27: An inhibitor of caspase activation.

HSP27 depletion reports demonstrate that HSP27 essentially blocks caspase-dependent apoptotic pathways. Small interefence targeting HSP27 induces apoptosis through caspase-3 activation.62,63 This may be consequence of the association of HSP27 with cytochrome c in the cytosol, thereby inhibiting the formation of the caspase-3 activation complex as demonstrated in leukemia and colon cancer cells treated with different apoptotic stimuli.6466 This interaction involves amino-acids 51 and 141 of HSP27 and do not need the phosphorylation of the protein.65 In multiple myeloma cells treated with dexamethasone, HSP27 has also been shown to interact with Smac.67

HSP27 can also interfere with caspase activation upstream of the mitochondria.66This effect seems related to the ability of HSP27 to interact and regulate actin microfilaments dynamics. In L929 murine fibrosarcoma cells exposed to cytochalasin D or staurosporine, overexpressed HSP27 binds to F-actin68preventing the cytoskeletal disruption, Bid intracellular redistribution and cytochrome c release66 (Fig. 1). HSP27 has also important anti-oxidant properties. This is related to its ability to uphold glutathione in its reduced form,69 to decrease reactive oxygen species cell content,19 and to neutralize the toxic effects of oxidized proteins.70 These anti-oxidant properties of HSP27 seem particularly relevant in HSP27 protective effect in neuronal cells.71

HSP27 has been shown to bind to the kinase Akt, an interaction that is necessary for Akt activation in stressed cells. In turn, Akt could phosphorylate HSP27, thus leading to the disruption of HSP27-Akt complexes.72 HSP27 also affects one downstream event elicited by Fas/CD95. The phosphorylated form of HSP27 directly interacts with Daxx.73 In LNCaP tumor cells, HSP27 has been shown to induce cell protection through its interaction with the activators of transcription 3 (Stat3).74 Finally, HSP27 protective effect can also be consequence of its effect favouring the proteasomal degradation of certain proteins under stress conditions. Two of the proteins that HSP27 targets for their ubiquitination/proteasomal degradation are the transcription factor nuclear factor κB (NFκB) inhibitor IκBα and p27kip1. The pronounced degradation of IkBα induced by HSP27 overexpression increases NFκB dependent cell survival75 while that of p27kip1facilitates the passage of cells to the proliferate phases of the cellular cycle. As a consequence HSP27 allows the cells to rapidly resume proliferation after a stress.76

Therefore, HSP27 is able to block apoptosis at different stages because of its interaction with different partners. The capacity of HSP27 to interact with one or another partner seems to be determined by the oligomerization/phosphorylation status of the protein, which, at its turn, might depend on the cellular model/experimental conditions. We have demonstrated in vitro and in vivo that for HSP27 caspase-dependent anti-apoptotic effect, large non-phosphorylated oligomers of HSP27 were the active form of the protein.77 Confirming these results, it has recently been demonstrated that methylglyoxal modification of HSP27 induces large oligomers formation and increases the anti-apoptotic caspase-inhibitory properties of HSP27.78 In contrast, for HSP27 interaction with the F-actin and with Daxx, phosphorylated and small oligomers of HSP27 were necessary73,79 and it is its phosphorylated form that protects against neurotoxicity.80

 

HSP27, HSP70 and HSP90 and Cell Differentiation

Under the prescribed context of HSPs as powerful inhibitors of apoptosis, it is reasonable to assume that an increase or decrease in their expression might modulate the differentiation program. The first evidence of the role of HSPs in cell differentiation comes from their tightly regulated expression at different stages of development and cell differentiation. For instance during the process of endochondrial bone formation, they are differentially expressed in a stage-specific manner.81 In addition, during post-natal development, time at which extensive differentiation takes place, HSPs expression is regulated in neuronal and non-neuronal tissues.82 In hemin-induced differentiation of human K562 erythroleukemic cells, genes coding for HSPs are induced.83

In leukemic cells HSP27 has been described as a pre-differentiation marker84because its induction occurs early during differentiation.8588 HSP27 expression has also been suggested as a differentiation marker for skin keratinocytes89 and for C2C12 muscle cells.90 This role for HSP27 in cell differentiation might be related to the fact that HSP27 expression increases as cells reach the non proliferative/quiescent phases of the cellular cycle (G0/G1).19,76

Subcellular localization is another mechanism whereby HSPs can determine whether a cell is to die or to differentiate. We, and others, have recently demonstrated the essential function of nuclear HSP70 for erythroid differentiation. During red blood cells’ formation, HSP70 and activated caspase-3 accumulate in the nucleus of the erythroblast.91 HSP70 directly associates with GATA-1 protecting this transcription factor required for erythropoiesis from caspase-3 cleavage. As a result, erythroblats continue their differentiation process instead of dying by apoptosis.61 HSP70, during erythropoiesis in TF-1 cells, have been shown to bind to AIF and thereby to block AIF-induced apoptosis, thus allowing the differentiation of erythroblasts to proceed.18

HSP90 has been required for erythroid differentiation of leukemia K562 cells induced by sodium butyrate92 and for DMSO-differentiated HL-60 cells. Regulation of HSP90 isoforms may be a critical event in the differentiation of human embryonic carcinoma cells and may be involved in differentiation into specific cell lineages.93 This effect of HSP90 in cell differentiation is probably because multiple transduction proteins essential for differentiation are client proteins of HSP90 such as Akt,94 RIP32 or Rb.95 Loss of function studies confirm that HSP90 plays a role in cell differentiation and development. In Drosophila melanogaster, point mutations of HSP83 (the drosophila HSP90 gene) are lethal as homozygotes. Heteterozygous mutant combinations produce viable adults with the same developmental defect: sterility.96 In Caenorhabditis elegans, DAF-21, the homologue of HSP90, is necessary for oocyte development.97 In zebrafish, HSP90 is expressed during normal differentiation of triated muscle fibres. Disruption of the activity of the proteins or the genes give rise to failure in proper somatic muscle development.98 In mice, loss-of-function studies demonstrate that while HSP90α loss-of-function phenotype appears to be normal, HSP90β is lethal. HSP90β is essential for trophoblasts differentiation and thereby for placenta development and this function can not be performed by HSP90α.4

HSP90 inhibitors have also been used to study the role of HSP90 in cell differentiation. These inhibitors such as the benzoquinone ansamycin geldanamycin or its derivative the 17-allylamino-17-demethoxygeldanamycin (17-AAG), bind to the ATP-binding “pocket” of HSP90 with higher affinity than natural nucleotides and thereby HSP90 chaperone activity is impaired and its client proteins are degraded. As could be expected by the reported role of HSP90 in cell differentiation, inhibitors of HSP90 block C2C12 myoblasts differentiation.99 In cancer cells and human leukemic blasts, 17-AAG induces a retinoblastoma-dependent G1 block. These G1 arrested cells do not differentiate but instead die by apoptosis.100

However, some reports describe that inhibitors of HSP90 can induce the differentiation process. In acute myeloid leukemia cells, 17-AAG induced apoptosis or differentiation depending on the dose and time of the treatment.101Opposite effects on cell differentiation and apoptosis are also obtained with the HSP90 inhibitor geldanamycin: in PC12 cells it induced apoptosis while in murin neuroblastoma N2A cells it induced differentiation.102 In breast cancer cells, 17-AAG-induced G1 block is accompanied by differentiation followed by apoptosis.103 The HSP90 inhibitor PU3, a synthetic purine that like 17-AAG binds with high affinity to the ATP “pocket” of HSP90, caused breast cancer cells arrest in G1 phase and differentiation.104

These contradictory reports concerning the inhibitors of HSP90 and cell differentiation could be explained if we consider that these drugs, depending on the experimental conditions, can have some side effects more or less independent of HSP90. Another possibility is that these studies do not differentiate between the amount of HSP90α and HSP90β inhibited. It is presently unknown whether HSP90 inhibitors equally block both isoforms, HSP90α and HSP90β. It not known neither whether post-translational modifications of HSP90 (acetylation, phosphorylation.) can affect their affinity for the inhibitors. HSP90α has been reported to be induced by lethal stimuli while the HSP90β can be induced by growth factors or cell differentiating signals.105 Mouse embryos out-of-function studies clearly show the role of HSP90β in the differentiation process and, at least for HSP90β role in embryo cell differentiation, there is not an overlap with HSP90α functions. Therefore, we can hypothesized that it can be the degree of inhibition of HSP90β by the HSP90 inhibitors that would determine whether or not there is a blockade of the differentiation process. This degree of inhibition of the different HSP90 isoforms might be conditioned by their cellular localization and their post-translational modifications. It should be noted, however, that the relative relevance of HSP90β in the differentiation process might depend on the differentiation model studied.

To summarize, we can hypothesize that the role in the differentiation process of a chaperone will be determined by its transient expression, subcellular redistribution and/or post-translational modifications induced at a given stage by a differ- entiation factor. How can HSPs affect the differentiation process? First by their anti-apoptotic role interfering with caspase activity, we and other authors have shown that caspase activity was generally required for cell differentiation.16,17Therefore, HSPs by interfering with caspase activity at a given moment, in a specific cellular compartment, may orchestrate the decision differentiation versus apoptosis. In this way, we have recently shown that HSP70 was a key protein to orchestrate this decision in erythroblasts.61 Second, HSPs may affect the differentiation process by regulating the nuclear/cytosolic shuttling of proteins that take place during differentiation. For instance, c-IAP1 is translocated from the nucleus to the cytosol during differentiation of hematopoietic and epithelial cells, and we have demonstrated that HSP90 is needed for this c-IAP1 nuclear export.106It has also been shown that, during erythroblast differentiation, HSP70 is needed to inhibit AIF nuclear translocation.18 Third, in the case of HSP90, the role in the differentiation process could be through certain of its client proteins, like RIP or Akt, whose stability is assured by the chaperone.

 

Repercussions and Concluding Remarks

The ability of HSPs to modulate the fate of the cells might have important repercussions in pathological situations such as cancer. Apoptosis, differentiation and oncogenesis are very related processes. Defaults in differentiation and/or apoptosis are involved in many cancer cells’ aetiology. HSPs are abnormally constitutively high in most cancer cells and, in clinical tumors, they are associated with poor prognosis. In experimental models, HSP27 and HSP70 have been shown to increase cancer cells’ tumorigenicty and their depletion can induce a spontaneous regression of the tumors.24,107 Several components of tumor cell-associated growth and survival pathways are HSP90 client proteins. These qualities have made HSPs targets for anticancer drug development. Today, although many research groups and pharmaceutical companies look for soluble specific inhibitors of HSP70 and HSP27, only specific soluble inhibitors of HSP90 are available for clinical trials. For some of them (17-AAG) phase II clinical trials are almost finished.108 However, considering the new role of HSP90β in cell differentiation, it seems essential to re-evaluate the functional consequences of HSP90 blockade.

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HSF-1 activates the ubiquitin proteasome system to promote non-apoptotic developmental cell death inC. elegans

A new pathway for non-apoptotic cell death

The results presented here allow us to construct a model for the initiation and execution of LCD in C. elegans (Figure 7). The logic of the LCD pathway may be similar to that of developmental apoptotic pathways. In C. elegans and Drosophila, where the control of specific cell deaths has been primarily examined, cell lineage or fate determinants control the expression of specific transcription factors that then impinge on proteins regulating caspase activation (Fuchs and Steller, 2011). Likewise, LCD is initiated by redundant determinants that require a transcription factor to activate protein degradation genes.

Figure 7.

https://elife-publishing-cdn.s3.amazonaws.com/12821/elife-12821-fig7-v3-480w.jpg

Figure 7. Model for linker cell death.

Green, upstream regulators. Orange, HSF-1. Purple, proteolytic components.    DOI: http://dx.doi.org/10.7554/eLife.12821.016

 

Our data suggest that three partially redundant signals control LCD initiation. The antagonistic Wnt pathways we describe may provide positional information to the linker cell, as the relevant ligands are expressed only near the region where the linker cell dies. The LIN-29 pathway, which controls timing decisions during the L4-adult molt, may ensure that LCD takes place only at the right time. Finally, while the TIR-1/SEK-1 pathway could act constitutively in the linker cell, it may also respond to specific cues from neighboring cells. Indeed, MAPK pathways are often induced by extracellular ligands. We propose that these three pathways, together, trigger activation of HSF-1. Our data support a model in which HSF-1 is present in two forms, HSF-1LC, promoting LCD, and HSF-1HS, protecting cells from stresses, including heat shock. We postulate that the redundant LCD initiation pathways tip the balance in favor of HSF-1LC, allowing this activity to bind to promoters and induce transcription of key LCD effectors, including LET-70/UBE2D2 and other components of the ubiquitin proteasome system (UPS), functioning through E3 ligase complexes consisting of CUL-3, RBX-1, BTBD-2, and SIAH-1.

Importantly, the molecular identification of LCD components and their interactions opens the door to testing the impact of this cell death pathway on vertebrate development. For example, monitoring UBE2D2 expression during development could reveal upregulation in dying cells. Likewise, genetic lesions in pathway components we identified may lead to a block in cell death. Double mutants in apoptotic and LCD genes would allow testing of the combined contributions of these processes.

The proteasome and LCD

As is the case with caspase proteases that mediate apoptosis (Pop and Salvesen, 2009), how the UPS induces LCD is not clear, and remains an exciting area of future work. That loss of BTBD-2, a specific E3 ligase component, causes extensive linker cell survival suggests that a limited set of targets may be required for LCD. Previous work demonstrated that BTBD2, the vertebrate homolog of BTBD-2, interacts with topoisomerase I (Khurana et al., 2010; Xu et al., 2002), raising the possibility that this enzyme may be a relevant target, although other targets may exist.

The UPS has been implicated in a number of cell death processes in which it appears to play a general role in cell dismantling, most notably, perhaps, in intersegmental muscle remodeling during metamorphosis in moths (Haas et al., 1995). However, other studies suggest that the UPS can have specific regulatory functions, as with caspase inhibition by IAP E3 ligases (Ditzel et al., 2008).

During Drosophila sperm development, caspase activity is induced by the UPS to promote sperm individualization, a process that resembles cytoplasm-specific activation of apoptosis (Arama et al., 2007). While C. elegans caspases are dispensible for LCD, it remains possible that they participate in linker cell dismantling or serve as a backup in case the LCD program fails.

Finally, the proteasome contains catalytic domains with target cleavage specificity reminiscent of caspases; however, inactivation of the caspase-like sites does not, alone, result in overt cellular defects (Britton et al., 2009), suggesting that this activity may be needed to degrade only specific substrates. Although the proteasome generally promotes proteolysis to short peptides, site-specific cleavage of proteins by the proteasome has been described (Chen et al., 1999). It is intriguing to speculate, therefore, that caspases and the proteasome may have common, and specific, targets in apoptosis and LCD.

A pro-death developmental function for HSF-1

Our discovery that C. elegans heat-shock factor, HSF-1, promotes cell death is surprising. Heat-shock factors are thought to be protective proteins, orchestrating the response to protein misfolding induced by a variety of stressors, including elevated temperature. Although a role for HSF1 has been proposed in promoting apoptosis of mouse spermatocytes following elevated temperatures (Nakai et al., 2000), it is not clear whether this function is physiological. In this context, HSF1 induces expression of the gene Tdag51 (Hayashida et al., 2006). Both pro- and anti-apoptotic activities have been attributed to Tdag51 (Toyoshima et al., 2004), and which is activated in sperm is not clear. Recently, pathological roles for HSF1 in cancer have been detailed (e.g. Mendillo et al., 2012), but in these capacities HSF1 still supports cell survival.

Developmental functions for HSF1 have been suggested in which HSF1 appears to act through transcriptional targets different from those of the heat-shock response (Jedlicka et al., 1997), although target identity remains obscure. Here, we have shown that HSF-1 has at least partially non-overlapping sets of stress-induced and developmental targets. Indeed, typical stress targets of HSF-1, such as the small heat-shock gene hsp-16.49 as well as genes encoding larger chaperones, likehsp-1, are not expressed during LCD, whereas let-70, a direct transcriptional target for LCD, is not induced by heat shock. Interestingly, the yeast let-70 homologs ubc4 and ubc5 are induced by heat shock (Seufert and Jentsch, 1990), supporting a conserved connection between HSF and UBE2D2-family proteins. However, the distinction between developmental and stress functions is clearly absent in this single-celled organism, raising the possibility that this separation of function may be a metazoan innovation.

What distinguishes the stress-related and developmental forms of HSF-1? One possibility is that whereas the stress response appears to be mediated by HSF-1 trimerization, HSF-1 monomers or dimers might promote LCD roles. Although this model would nicely account for the differential activities in stress responses and LCD of the HSF-1(R145A) transgenic protein, which would be predicted to favor inactivation of a larger proportion of higher order HSF-1 complexes, the identification of conserved tripartite HSEs in the let-70 and rpn-3 regulatory regions argues against this possibility. Alternatively, selective post-translational modification of HSF-1 could account for these differences. In mammals, HSF1 undergoes a variety of modifications including phosphorylation, acetylation, ubiquitination, and sumoylation (Xu et al., 2012), which, depending on the site and modification, stimulate or repress HSF1 activity. In this context, it is of note that p38/MAPK-mediated phosphorylation of HSF1 represses its stress-related activity (Chu et al., 1996), and the LCD regulator SEK-1 encodes a MAPKK. However, no single MAPK has been identified that promotes LCD (E.S.B., M.J.K. unpublished results), suggesting that other mechanisms may be at play.

Our finding that POP-1/TCF does not play a significant role in LCD raises the possibility that Wnt signaling exerts direct control over HSF-1 through interactions with β-catenin. However, we have not been able to demonstrate physical interactions between these proteins to date (M.J.K, unpublished results).

Finally, a recent paper (Labbadia and Morimoto, 2015) demonstrated that in young adult C. elegans, around the time of LCD, global binding of HSF-1 to its stress-induced targets is reduced through changes in chromatin modification. Remarkably, we showed that chromatin regulators play a key role in let-70 induction and LCD (J.A.M., M.J.K and S.S., manuscript in preparation), suggesting, perhaps, that differences in HSF-1 access to different loci may play a role in distinguishing its two functions.

LCD and neurodegeneration

Previous studies from our lab raised the possibility that LCD may be related to degenerative processes that promote vertebrate neuronal death. Nuclear crenellation is evident in dying linker cells and in degenerating cells in polyQ disease (Abraham et al., 2007) and the TIR-1/Sarm adapter protein promotes LCD in C. elegans as well as degeneration of distal axonal segments following axotomy in Drosophila and vertebrates (Osterloh et al., 2012). The studies we present here, implicating the UPS and heat-shock factor in LCD, also support a connection with neurodegeneration. Indeed, protein aggregates found in cells of patients with polyQ diseases are heavily ubiquitylated (Kalchman et al., 1996). Chaperones also colocalize with protein aggregates in brain slices from SCA patients, and HSF1 has been shown to alleviate polyQ aggregation and cellular demise in both polyQ-overexpressing flies and in neuronal precursor cells (Neef et al., 2010). While the failure of proteostatic mechanisms in neurodegenerative diseases is generally thought to be a secondary event in their pathogenesis, it is possible that this failure reflects the involvement of a LCD-like process, in which attempts to engage protective measures instead result in activation of a specific cell death program.

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Metastatic Disease (4.3)

Writer and Curator: Larry H. Bernstein, MD, FCAP 

In the preceding discussions the hematological and nonhematological cancers were elaborated.  These were tumors of blood or solid tumors that are malignant.  Malignant solid tumors have a loss of normal architecture.  Malignant cancers of the blood forming organ also have a disruption of the architecture in the blood forming organs, and they are circulating elements that are either acutely increased in number or chronically increased to very high circulating counts as well as many cells in the marrow.  The diagnosis depends on the type of cell elements and the stage of maturation.  In the case of blood cell cancers, one might consider an intermediate stage that has a long course that is in the case of the myelogenous series, myeloid dysplasia, which includes myelofibrosis, which in either case is not a benign course. In the case of solid tumors, there is an anatomic structure of the cancer site.

The usual structure for a carcinoma is either adjoining cells surrounding a vascular supply, as in the liver, a parenchymal gland, as in pancreas, a tubular structure, as the gastrointestinal tract and lungs (which are embryologically and outpouching of the gut), or a skin surface.  In the case of carcinomas, the cells mature from a basement membrane of small flattened cells that overlie a fibrovascular matrix and an underlying myxoid stroma, perhaps beneath which is a muscular organ, then covered by a flat layer of cells. In the case of all epithelial structures there is an orderly maturation of epithelium from the basal layer to the mature epithelial cells that are elongate, have a brush border, and secrete into the glandular structure.  The cell maturation becomes disrupted and disorderly to different degrees in the development of malignancy from a dysplasia to low grade malignancy, to high grade anaplastic cancer.

The development of a cancer implies the loss of tissue architecture, the replication of cells, the development of a neoplasms circulation (which is the topic of vascular endothelial growth factor (VEGF)), the overgrowth of the circulation so that the tumor has insufficient blood supply, and vascular invasion.  We refer to the Warburg Hypothesis with respect to the malignancy relying on glycolysis in the presence of oxygen (aerobic glycolysis), but it may be questionable to imply that there is sufficient oxygen supply.  In some cases a cancer may occur from a longstanding inflammatory focus.  This has been seen to occur in osteomyelitis and in gastrointestinal fistulas.  The growth of a neoplasm, when it exceeds its blood supply, requires adaptive changes. The most obvious to consider would be a decreased reliance of mitochondrial respiration.  Warburg refer to the increase production of lactic acid as analogous to Pasteur observation of fermentation in yeast (Pasteur effect).  He measured the lactic acid production by various tissues, and the consumption with the oxygen consumption showed that in many tissues approximately two molecules of lactate are prevented from appearing when one molecule of oxygen is consumed – a relationship that Meyerhof had found in muscle. This he expressed as the “Meyerhof quotient”:

Anaerobic glycolysis – aerobic glycolysis/oxygen consumption

Ref: Otto Warburg: Cell Physiologist, Biochemist, and Eccentric
Hans Krebs in collaboration with Roswitha Schmid
Clarendon Press, Oxford, 1981. Pp 19-25.

The special feature of cancer cells was the high rate of glycolysis in the presence of oxygen, whereas muscle can form lactate from carbohydrate in the absence of oxygen. This led to the discovery that all animal tissues are capable of glycolysis both aerobically and anaerobically.  Pasteur had established 60 years earlier that the rates of fermentation are generally hiugh anaerobically, but low aerobically. This led Warburg to the conclusion that cancer cells are distinguished from noncancer cells by their failure to suppress glycolysis in the presence of oxygen. He discovered in 1926 that the link between respiration and fermentation can be severed by a specific inhibitor, ethylcarbylamine. He looked at carbylethylamine as an inhibitor of the ‘Pasteur effect’, and determined that the catalyst was a heavy metal ion. But the proposed mechanism was shown not to be correct by Engelhardt, Lynen, Bucher, Lowry, Racker, and Sols.The activity of the enzyme phospofructokinase is regulated by the concentrations of ATP, ADP and inorganic phosphate (Pi). The “allosteric properties” of PFK could account for the ‘Pasteur effect’.  ATP inactivates PFK, while ADP and Pi activate it. Further, etylcarbylamine was found to be an uncoupler of oxidative phosphorylation (OxPhos), but Warburg was right in postulating that a heavy metal was involved since heavy metals are involved in
OxPhos.  The explanation for this is now that when malignant transformation occurs, the cells’ energy supply is redirected from their normal function to growth. This change was found to be irreversible upon restoration of oxygen supply.

The topic of discussion is metastasis. What does it have to do with malignancy and respiration? Metastasis is the other key feature of cancer cells. What it has to do with respiration would probably tie in with the change in the cells’ energy supply that is directed toward proliferation. As the cell metabolism is reconfigured, there is also a change in the cell signaling with respect to apoptosis and the events regarding autophagy.  This has to extend beyond the mitochondria, mainly because autophagy involves mitophagy, the ER and the entire cytoskeleton.  This means that the cytoplasmic relationship to the intercellular matrix and the fibroblast stroma would have to be affected, as the cell breaks away from its close association with adjacent cells.  Cells can migrate to adjacent lymphatic structures, and either enter the circulation by way of the lymphatics or by invasion of the venous circulation directly. In any case, entry into the circulation allows for transport to distant sites.  With respect to migration to distant sites, we recall the hypothesis of Paget that the cells metastasize directly into the circulating blood, and they may ‘seed’ to favorable organs.

The discussion now turns to the assessment of apoptosis as a means to inhibition of cancer cell lines, which proliferate if unchecked and migrate away from the primary site.  I use a few examples from a symposium volume of the Annals of the New York Academy of Sciences:

Apoptosis: From Signaling Pathways to Therapeutic Tools.
Ed, Mark Diederich
ANYAA9 2003; 1010:1-799
The role of β-glucuronidase in induction of apoptosis by Genistein Combined Polysaccharide (GCP) in xenogenetic mice bearing human mammary cancer cells.
Yuan L, Wagatsuma C, Sun B, Kim Jung-Hwan, Surh Young-Joon
Ann NY Acad Sci 2003;1010: 347-349.
http://dx.doi.org:/10.1196/annals.1299.063

  • GCP inhibits tumor cell growth through multiple mechanisms, including induction of tumor apoptosis
  • The biological activities of genistein (aglycon) are more evident in tumor tissues than in normal tissues.
  • Hiugh doses of genistein administration rarely induces toxicity to normal tissues.
  • Higher levels of β-glucuronidase expression in tumor tissues results in more genistein aglycon, leading to tumor destruction.

Induction of apoptosis in human pancreatic cancer cells by docosahexanoic acid
Merendino N, Molinari R, Loppi B, Pessina G, D’Aquino M, Tomassi G, Velotti F.
Ibid 361-364. http://dx.doi.org:/10.1196/annals.1299.143

Polyunsaturated fatty acids have been indicated to induce anti-proliferative and/or apoptotic effects in various tumor cells. We showed that, at a 200-μM concentration, both alpha-linoleic (18:2 n-6; LA) or docosahexaenoic (22:6 n-3; DHA) acid inhibited cell growth, while only DHA induced apoptosis in the human Paca-44 pancreatic cancer cell line. Investigating the mechanism underlying DHA-induced apoptosis, we showed that DHA induced a rapid and dramatic (>60%) intracellular depletion of reduced glutathione (GSH), without affecting oxidized glutathione (GSSG). Moreover, using two specific inhibitors of carrier-mediated GSH extrusion, cystathionine or methionine, we observed that GSH depletion occurred via an active GSH extrusion, and that inhibition of GSH efflux completely reversed apoptosis. These results provide the first evidence for a possible causative role of GSH depletion in DHA-induced apoptosis.

Opposite phenotypes of cancer and aging arise from alternative regulation of common signaling pathways.
Ukraintseva SV1, Yashin AI.
Ann N Y Acad Sci. 2003 Dec; 1010:489-92.
http://dx.doi.org:/10.1196/annals.1299.089

Phenotypic features of malignant and senescent cells are in many instances opposite. Cancer cells do not “age”; their metabolic, proliferative, and growth characteristics are opposite to those observed with cellular aging (both replicative and functional). In many such characteristics cancer cells resemble embryonic cells. One can say that cancer manifests itself as a local, uncontrolled “rejuvenation” in an organism. Available evidence from human and animal studies suggests that the opposite phenotypic features of aging and cancer arise from the opposite regulation of genes participating in apoptosis/growth arrest or growth signal transduction pathways in cells. This fact may be applicable in the development of new anti-aging treatments. Genes that are contrarily regulated in cancer and aging cells (e.g., proto-oncogenes or tumor suppressors) could be candidate targets for anti-aging interventions. Their “cancer-like” regulation, if strictly controlled, might help to rejuvenate the human organism.

CUGBP2 Plays a Critical Role in Apoptosis of Breast Cancer Cells in Response to Genotoxic Injury
Mukhopadhyay D, Jung J, Murmu N, Houchen CW, Dieckgraefe BK, Anant A
Ibid 504–509. http://dx.doi.org:/10.1196/annals.1299.093

 Posttranscriptional control of gene expression plays a key role in regulating gene expression in cells undergoing apoptosis. Cyclooxygenase-2 (COX-2) is a crucial enzyme in the conversion of arachidonic acid to prostaglandin E2 (PGE2) and is significantly upregulated in many types of adenocarcinomas. COX-2 overexpression leads to increased PGE2 production, resulting in increased cellular proliferation. PGE2 enhances the resistance of cells to ionizing radiation. Accordingly, understanding mechanisms regulating COX-2 expression may lead to important therapeutic advances. Besides transcriptional control, COX-2 expression is significantly regulated by mRNA stability and translation. We have previously demonstrated that RNA binding protein CUGBP2 binds AU-rich sequences to regulate COX-2 mRNA translation. In the current study, we have determined that expression of both COX-2 mRNA and CUGBP2 mRNA are induced in MCF-7 cells, a breast cancer cell line, following exposure to 12 Gy γ-irradiation. However, only CUGBP2 protein is induced, but COX-2 protein levels were not altered. Silencer RNA (siRNA)-mediated inhibition of CUGBP2 reversed the block in COX-2 protein expression. Furthermore, MCF-7 cells underwent apoptosis in response to radiation injury, which was also reversed by CUGBP2 siRNAs. These data suggest that CUGBP2 is a critical regulator of the apoptotic response to genotoxic injury in breast cancer cells.

Multiple and synergistic deregulations of apoptosis-controlling genes in pancreatic carcinoma cells
A Trauzold,1 S Schmiedel,1 C Röder,1 C Tams,1 M Christgen,1 S Oestern,1 A Arlt,2 S Westphal,1 M Kapischke,1 H Ungefroren,1 and H Kalthoff1,
Ibid 510-513.  Br J Cancer. 2003 Nov 3; 89(9): 1714–1721.
http://dx.doi.org/10.1038%2Fsj.bjc.6601330

CD95, TRAIL-R1 (tumor necrosis factor-related apoptosis inducing ligand-receptor 1) and TRAIL-R2 are members of the TNF-receptor family of transmembrane proteins that are capable of inducing apoptosis (Wiley et al, 1995Pitti et al, 1996Pan et al, 1997Peter et al, 1998). Following ligand binding, the receptors oligomerize and the pro-apoptotic molecules TRADD, FADD and FLICE/caspase-8 are recruited to their intracellular death domain forming the ‘death-inducing signaling complex’ (DISC) (Krammer, 1999). The subsequent events leading to apoptosis depend on the specific cell type being challenged. In type I cells the bulk induction of caspase-8 at the DISC leads to the direct activation of the effector caspase 3. In type II cells only little amounts of caspase-8 are activated at the DISC requiring the pro-apoptotic mitochondrial amplification loop for efficient caspase-3 activation (Scaffidi et al, 1998).
In Vivo Imaging of Chemotherapy-Induced Apoptosis in Human Cancers
T Belhocine, N Steinmetz, A Green, P Rigo
Ibid 525-529. http://dx.doi.org:/10.1196/annals.1299.097

Rationale. Induction of apoptosis in sensitive tumor cells is the main mechanism of action of chemotherapy agents in human cancers. Also, the assessment of drug-induced apoptosis soon after chemotherapy may be an early predictor of treatment efficacy. Patients and Methods. A phase I/II study was prospectively conducted in 15 patients presenting with proven lung cancers (n= 10), breast cancers (n= 2), and lymphomas (n= 3) to assess the value of the 99mTc-radiolabeled recombinant human (rh) Annexin V for imaging apoptosis immediately after completion of the first course of chemotherapy. Early Annexin V findings post-chemotherapy (day+1, day+2) were also compared to the tumor status at 6 to 12 weeks post-treatment.
Results. All lung and lymphoma patients with an increased tracer uptake post-treatment (n= 8) had either partial or complete tumor response. Five patients with no tracer uptake had progressive disease. However, two breast cancers had a response to treatment, although no significant tracer uptake was observed. Tumor response and survival time were significantly correlated with the 99mTc-labeled Annexin V uptake. No serious events related to tracer administration were noted. Conclusion. Preliminary results of this pilot study demonstrate the feasibility of the 99mTc-labeled Annexin V uptake for the in vivo imaging of apoptosis after one course of chemotherapy. If confirmed on larger series, these promising results may open new perspectives in the management of oncology patients.

In vivo photoacoustic imaging of chemotherapy-induced apoptosis in squamous cell carcinoma using a near-infrared caspase-9 probe.
Yang Q1Cui HCai SYang XForrest ML.
J Biomed Opt. 2011 Nov; 16(11):116026.
http://dx.doi.org:/10.1117/1.3650240

Anti-cancer drugs typically exert their pharmacological effect on tumors by inducing apoptosis, or programmed cell death, within the cancer cells. However, no tools exist in the clinic for detecting apoptosis in real time. Microscopic examination of surgical biopsies and secondary responses, such as morphological changes, are used to verify efficacy of a treatment. Here, we developed a novel near-infrared dye-based imaging probe to directly detect apoptosis with high specificity in cancer cells by utilizing a noninvasive photoacoustic imaging (PAI) technique. Nude mice bearing head and neck tumors received cisplatin chemotherapy (10 mg/kg) and were imaged by PAI after tail vein injection of the contrast agent. In vivo PAI indicated a strong apoptotic response to chemotherapy on the peripheral margins of tumors, whereas untreated controls showed no contrast enhancement by PAI. The apoptotic status of the mouse tumor tissue was verified by immunohistochemical techniques staining for cleaved caspase-3 p11 subunit. The results demonstrated the potential of this imaging probe to guide the evaluation of chemotherapy treatment.

Noninvasive imaging techniques are necessary for early cancer detection and evaluation of the chemotherapeutic effect on tumors. Current diagnostic imaging techniques generally include γ-scintigraphy, magnetic resonance imaging, computed tomography, and ultrasonography; however, these techniques only give morphological information on the tumor. These techniques do not report the biochemical response of the tumor to treatment and physical changes in the tumor in response to treatment may take days to weeks to fully manifest. Positron emission topography and SPECT can indirectly detect tumor response to treatment due to changes in metabolic activity and blood perfusion, respectively. However, no clinical imaging technique can directly detect the biochemical response, e.g., apoptosis, of tumors to treatment. Since apoptosis often occurs within in the first 18 to 36 h after treatment, direct imaging of apoptosis would rapidly indicate if there is a response in the tumor to chemotherapy.

Photoacoustic imaging (PAI) overcomes the spatial and resolution limitations of conventional imaging techniques at a relatively low cost,12 and it has shown its potential to monitor the growth of melanoma brain tumors3 and melanoma metastasis in sentinel lymph nodes.4 However, ascribed to the fact that PAI utilizes the optical absorption of tissues for contrast, it cannot differentiate normal from cancerous cells unless the cells are overexpressing chromomeric marker (e.g., melanomas) or labeled by reporter moieties as contrast agent to enhance the contrast between normal and pathological tissues. In this case, application of a contrast agent such as fluorochromes is expected to facilitate both the visualization of head and neck squamous cell carcinoma (HNSCC) cancer cells and their response to treatment in vivo by PAI.

We have synthesized a near-infrared fluorescent imaging probe – IR780-linker-Val-Ala-Glu(OMe)-FMK by conjugating a fluorochrome (IR780) to Z-Val-Ala-Glu (OMe), a cell permeable caspase inhibitor. The activation of caspase family of cysteine proteases has been recognized as a critical event of apoptosis, which is a physiological process of type I programmed cell death. Typically anti-cancer agents act on cancer cells to induce apoptosis, so apoptosis is a rapid and definite indicator of tumor response. For this reason, apoptosis is used in screening drug candidates in cell culture. The fluoromethyl ketone of the tripeptides valine, alanine, and O-methyle-glutamic acid [Val-Ala-Glu(OMe)-FMK] can specifically and irreversibly bind to the cysteine residue at the active site of caspase-9.5 Our preliminary in vitro cell-imaging test with prostate cancer DU 145 cells demonstrated the sensitivity of this imaging probe for cell apoptosis.6 In this study, we evaluated the application of IR780-linker-Val-Ala-Glu(OMe)-FMK for PAI to detect procaspase-9 activation caused by anticancer drug treatment in living nude mice bearing HNSCC tumors.

Increase in PA amplitude within the HNSCC tumor after intravenous injection of imaging agent

Increase in PA amplitude within the HNSCC tumor after intravenous injection of imaging agent

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3221716/bin/JBOPFO-000016-116026_1-g002.jpg

Fourteen micron (thickness) sections of the tumor tissue were stained with a goat primary polyclonal antibody for cleaved caspase-3 p11 subunit (Asp-175-Ser-176) and a donkey anti-goat secondary antibody with a fluorescein isothiocyanate (FITC) fluorophore (Santa Cruz Biotechnology Inc., Santa Cruz, California). Cell nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI).

Maximum amplitude projection images obtained from the PAI of the HNSCC tumor region shown in Fig. ​(Fig.2).2 were converted to grayscale images. The grayscale images at various time points were linearly aligned using the scale-invariant feature transform function of Fiji/ImageJA software (ver. 20110307, http://pacific.mpi-cbg.de/wiki/index.php/Fiji) (Fig. ​(Fig.3).3). Quantification of PA signal intensity within the tumor region was performed in triplet for each image by measuring the mean gray value (units: gray/pixel) of the circled tumor region. The extent of signal enhancement was calculated by normalizing the tumor signal against a background reading taken immediately before injection of the imaging agent (Fig. ​(Fig.2).2)

Apoptosis in the tumor tissues was independently verified by immunohistochemical staining for caspase 3, a downstream indicator of apoptosome-activated caspase-mediated apoptosis that would not cross-react with the caspase-9 PA probe. Figure ​(Figure4a) 4a represents a control section stained with the secondary antibody alone (autofluorescence of the tissue without apparent staining); while, Fig. ​(Fig.4b).4b shows the immunostaining of the caspase-3 p11 subunit (green) and the DAPI staining of cell nuclei. The intense green fluorescence in these sections suggests the wide spread apoptosis of cells in the tumor tissues after intravenous administration of high-dose cisplatin. In addition, cells on the peripheral of the tumor stained more strongly for caspase 3 (green fluorescence) compared to cells at the tumor interior. This was consistent with the PA imaging of apoptosis that showed strong apoptosis at the tumor peripheral, suggesting chemotherapeutics had penetrated the outer layers of the tumor and induced apoptosis.

Immunostaining for apoptosis in tumor

Immunostaining for apoptosis in tumor

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3221716/bin/JBOPFO-000016-116026_1-g004.jpg

Immunostaining for apoptosis in tumor. (a) Representative control section stained with secondary antibody alone and (b) tissue section of the HNSCC tumor stained for caspase-3 p11 subunit after cisplatin treatment.

I now consider mechanisms of metastasis as currently viewed.

Metastasis mechanisms.
Geiger TR1Peeper DS.
Biochim Biophys Acta. 2009 Dec; 1796(2):293-308.  http://dx.doi.org:/10.1016/j.bbcan.2009.07.006.

Metastasis, the spread of malignant cells from a primary tumor to distant sites, poses the biggest problem to cancer treatment and is the main cause of death of cancer patients. It occurs in a series of discrete steps, which have been modeled into a “metastatic cascade”. In this review, we comprehensively describe the molecular and cellular mechanisms underlying the different steps, including Epithelial-Mesenchymal Transition (EMT), invasion, anoikis, angiogenesis, transport through vessels and outgrowth of secondary tumors. Furthermore, we implement recent findings that have broadened and challenged the classical view on the metastatic cascade, for example the establishment of a “premetastatic niche”, the requirement of stem cell-like properties, the role of the tumor stroma and paracrine interactions of the tumor with cells in distant anatomical sites. A better understanding of the molecular processes underlying metastasis will conceivably present us with novel targets for therapeutic intervention.

Axis of evil: molecular mechanisms of cancer metastasis Thomas Bogenrieder1 and Meenhard Herlyn1
Oncogene (2003) 22, 6524–6536.
http://dx.doi.org:/doi:10.1038/sj.onc.1206757

Although the genetic basis of tumorigenesis may vary greatly between different cancer types, the cellular and molecular steps required for metastasis are similar for all cancer cells. Not surprisingly, the molecular mechanisms that propel invasive growth and metastasis are also found in embryonic development, and to a less perpetual extent, in adult tissue repair processes. It is increasingly apparent that the stromal microenvironment, in which neoplastic cells develop, profoundly influences many steps of cancer progression, including the ability of tumor cells to metastasize. In carcinomas, the influences of the microenvironment are mediated, in large part, by bidirectional interactions (adhesion, survival, proteolysis, migration, immune escape mechanisms lymph-/angiogenesis, and homing on target organs) between epithelial tumor cells and neighboring stromal cells, such as fibroblasts as well as endothelial and immune cells. In this review, we summarize recent advances in understanding the molecular mechanisms that govern this frequently lethal metastatic progression along an axis from primary tumor to regional lymph nodes to distant organ sites. Affected proteins include growth factor signaling molecules, chemokines, cell–cell adhesion molecules (cadherins, integrins) as well as extracellular proteases (matrix metalloproteinases). We then discuss promising new therapeutic approaches targeting the microenvironment. We note, however, that there is still too little knowledge of how the many events are coordinated and integrated by the cancer cell, with conspiratorial help by the stromal component of the host. Before drug development can proceed with a legitimate chance of success, significant gaps in basic knowledge need to be filled.

Metastases to regional lymph nodes are detected at diagnosis and surgery in approximately one-third of breast, colorectal, uterine cervix, and oral cavity and pharynx cancer patients, and one-quarter of esophageal, lung pancreas, gastric and bladder cancer patients (Greenlee et al., 2001). The high mortality rates associated with cancer are caused by the metastatic spread of tumor cells from the site of their origin. In fact, metastases are the cause of 90% of cancer deaths (Hanahan and Weinberg, 2000). The prognosis for a patient who is diagnosed with advanced invasive or metastatic disease remains little better than it was decades ago (Sporn, 1997). Tumor cells invade either the blood or lymphatic vessels to access the general circulation and then establish themselves in other (visceral) tissues. Ultimately, they become surgically unresectable, with pharmacological or radiological long-term control being uncommon (Stacker et al., 2002).

Although the genetic basis of tumorigenesis may vary greatly between different cancer types, the cellular and molecular steps required for metastasis are generally similar for all solid tumor cells (Woodhouse et al., 1997Liotta and Kohn, 2003). Not surprisingly, the molecular mechanisms that propel invasive growth and metastasis are also found in embryonic development, and, however to a less perpetual/chronic/aggressive/quantitatively different extent, in adult tissue maintenance (e.g. involving stem cell differentiation) and repair processes (‘tumors are wounds that do not heal’) (Dvorak, 1986). We now view cancer as a complex tissue resulting from disrupted organ homeostasis, rather than focusing on the cancer cell, and the genes within it, alone (Hanahan and Weinberg, 2000;Bissell and Radisky, 2001Bogenrieder and Herlyn, 2002Wiseman and Werb, 2002). Normal tissue homeostasis is maintained between epithelial cells and their microenvironment, such as fibroblasts, endothelial and immunocompetent cells, and the extracellular matrix (ECM). Similarly, during malignant transformation and progression, there are (however deregulated) reciprocal and conspirational interactions between the neoplastic cells and the adjacent stromal cells (Hsu et al., 2002). A series of recent investigations have shown that aberrations in the stroma can both precede and stimulate the development of cancers (reviewed in Bissell and Radisky, 2001Wiseman and Werb, 2002).

The process of metastasis involves an intricate interplay between altered cell adhesion, survival, proteolysis, migration, lymph-/angiogenesis (see articles in this issue by R Kerbel and P Campochiaro, pp. NNN–NNN), immune escape mechanisms, and homing on target organs (Table 1). However, there is still very little knowledge of how these events are coordinated by the cancer cell, with conspirational help by the stromal component (microenvironment) of the host (Clark, 1991Hsu et al., 2002). This process is usually said to be ‘uncontrolled’. As we shall see, however, it is by no means purely stochastic, but rather a finely tuned molecular machinery with active tumor cell–host collaboration. Thus, all explanations of ‘success’ of the metastatic axis contain a strong element of determinism. Whereas the early steps in the metastastic campaign are completed very efficiently, metastasis is an inefficient process in its later steps, especially the regulation of cancer cell growth at the secondary sites (Luzzi et al., 1998Cameron et al., 2000Chambers et al., 2002). Given that spread of the tumor to distant organs is usually lethal, more intense studies of these molecular mechanisms assume general importance to develop more effective anticancer strategies. In the following discussion of specific molecular mechanisms, we have often chosen to draw mainly from examples that pertain to melanoma progression, although similar processes are most likely also operating during oncogenesis of a wide range of cancers.

The classical metastatic cascade encompasses intravasation by tumor cells, their circulation in lymph and blood vascular systems, arrest in distant organs, extravasation, and growth into metastatic foci (Herlyn and Malkowicz, 1991;Woodhouse et al., 1997). Ann Chambers et al. (2001),(2002) have demonstrated in murine models that the limiting factor for metastasis formation is growth after extravasation (Figure 1a). Recently, this extravasation model has been challenged by Ruth Muschel and co-workers (Al-Mehdi et al., 2000Wong et al., 2002), who showed that tumor cells can readily proliferate after arrest in blood vessels, suggesting that extravasation is not a prerequisite for metastatic growth (Figure 1b). In a separate series of experiments, Mary Hendrix and co-workers described that tumor cells can even have endothelial cell-like functions and form channels that allow fluid flow (Maniotis et al., 1999Folberg et al., 2000) (Figure 1c). The group has identified some of the players, such as EphA2 and VE-cadherin, on aggressive melanoma cells that are shared with endothelial cells and that are likely involved in ‘vasculogenic mimicry’. Vasculogenic mimicry is the ability of aggressive cancer cells to form de novo vessel-like networks in vitro in the absence of endothelial cells or fibroblasts, concomitant with their expression of vascular-associated cellular marker (Sood et al., 2001,2002). Tumor cell plasticity is demonstrated by the ability of tumor cells to adopt a variety of phenotypes, including an endothelial phenotype (Sood et al., 2001, 2002). These exciting new findings underscore the plasticity of malignant cells from advanced tumor progression stages, and they require from tumor biologists a more dynamic view of the metastatic cascade. If the biological phenotype of metastasis must be portrayed flexibly, then we need a new ‘yardstick,’ a normal cell, to better characterize and understand the many faces of metastasis. We need to understand how the malignant cells exert cooperation from the normal cells. Our central hypothesis is that both normal and malignant cells utilize the same molecules for invasion, but that differences in downstream signaling events allow the tumor cells to dominate over normal cells in the microenvironment. This ‘dominant plasticity’ model of cancer metastasis takes into account the flexible response of malignant cells to microenvironmental pressures while maintaining dominance over the normal parenchymal and stromal cells.

Models of metastasis

Models of metastasis

http://www.nature.com/onc/journal/v22/n42/images/1206757f1.jpg

Models of metastasis. (a) According to Chambers and co-workers, only a very small population of injected cells (2%) form micrometastases, although over 87% are arrested in the liver. Furthermore, not all of the micrometastases persist, and the progressively growing metastases that kill the mice arise only from a small subset (0.02%) of the injected cells. (b) Muschel and co-workers recently proposed a new model for pulmonary metastasis in which endothelium-attached tumor cells that survived the initial apoptotic stimuli proliferate intravascularly. Thus, a principal tenet of this new model is that the extravasation of tumor cells is not a prerequisite for metastatic colony formation and that the initial proliferation takes place within the blood vessels. (c) The unique ability of aggressive tumor cells to generate patterned networks, similar to the patterned networks during embryonic vasculogenesis, and concomitantly to express vascular markers associated with endothelial cells, their precursors and other vascular cells has been termed ‘vasculogenic mimicry’ by Hendrix and co-workers

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Contributions to Cardiomyocyte Interactions and Signaling

Author and Curator: Larry H Bernstein, MD, FCAP

and

Curator: Aviva Lev-Ari, PhD, RN

Introduction

This is Part II of the ongoing research in the Lee Laboratory, concerned with Richard T Lee’s dissection of the underlying problems that will lead to a successful resolution of myocardiocyte regeneration unhampered by toxicity, and having a suffuciently sustained effect for an evaluation and introduction to the clinic.  This would be a milestone in the treatment of heart failure, and an alternative to transplantation surgery.  This second presentation focuses on the basic science work underpinning the therapeutic investigations.  It is work that, if it was unsupported and did not occur because of insufficient funding, the Part I story could not be told.

Cardiomyocyte hypertrophy and degradation of connexin43 through spatially restricted autocrine/paracrine heparin-binding EGF

J Yoshioka, RN Prince, H Huang, SB Perkins, FU Cruz, C MacGillivray, DA Lauffenburger, and RT Lee *
Cardiovascular Division, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA; and Biological Engineering Division, MIT, Cambridge, MA
PNAS 2005; 302(30):10622-10627.  http://pnas.org/cgi/doi/10.1073/pnas.0501198102

Growth factor signaling can affect tissue remodeling through autocrine/paracrine mechanisms. Recent evidence indicates that EGF receptor transactivation by heparin-binding EGF (HB-EGF) contributes to hypertrophic signaling in cardiomyocytes. Here, we show that HB-EGF operates in a spatially restricted circuit in the extracellular space within the myocardium, revealing the critical nature of the local microenvironment in intercellular signaling. This highly localized microenvironment of HB-EGF signaling was demonstrated with 3D morphology, consistent with predictions from a computational model of EGF signaling. HB-EGF secretion by a given cardiomyocyte in mouse left ventricles led to cellular hypertrophy and reduced expression of connexin43 in the overexpressing cell and in immediately adjacent cells but not in cells farther away. Thus, HB-EGF acts as an autocrine and local paracrine cardiac growth factor that leads to loss of gap junction proteins within a spatially confined microenvironment. These findings demonstrate how cells can coordinate remodeling with their immediate neighboring cells with highly localized extracellular EGF signaling. Within 3D tissues, cells must coordinate remodeling in response to stress or growth signals, and this communication may occur by direct contact or by secreted signaling molecules. Cardiac hypertrophy is a physiological response that enables the heart to adapt to an initial stress; however, hypertrophy can ultimately lead to the deterioration in cardiac function and an increase in cardiac arrhythmias. Although considerable progress has been made in elucidating the molecular pathogenesis of cardiac hypertrophy, the precise mechanisms guiding the hypertrophic process remain unknown. Recent evidence suggests that myocardial heparin-binding (HB)-epidermal growth factor participates in the hypertrophic response. In cardiomyocytes, hypertrophic stimuli markedly increase expression of the HB-EGF gene, suggesting that HB-EGF can act as an autocrine trophic factor that contributes to cellular growth. HB-EGF is first synthesized as a membrane-anchored form (proHB-EGF), and subsequent ectodo-main shedding at the cell surface releases the soluble form of HB-EGF. Soluble HB-EGF is a diffusible factor that can be captured by the receptors to activate the intracellular EGF receptor signaling cascade. Indeed, EGF receptor (EGFR) transactivation, triggered by shedding of HB-EGF from the cell surface, plays an important role in cardiac hypertrophy resulting from pressure overload in the aortic-banding model. EGFR activation can occur through autocrine and paracrine signaling. In autocrine signaling, a cell produces and responds to the same signaling molecules. Paracrine signaling molecules can target groups of distant cells or act as localized mediators affecting only cells in the immediate environment of the signaling cell. Thus, although locally produced HB-EGF may travel through the extra-cellular space, it may also be recaptured by the EGFR close to the point where it was released from the cell surface. The impact of spatially localized microenvironments of signaling could be extensive heterogeneous tissue remodeling, which can be particularly important in an electrically coupled tissue like myocardium. Interestingly, recent data suggest that EGF can regulate protea-some-dependent degradation of connexin43 (Cx43), a major trans-membrane gap junction protein, in liver epithelial cells, along with a rapid inhibition of cell–cell communication through gap junctions. One of the critical potential myocardial effects of HB-EGF could therefore be to increase degradation of Cx43 and reduce electrical stability of the heart. Reduced content of Cx43 is commonly observed in chronic heart diseases such as hypertrophy, myocardial infarction, and failure. Thus, we hypothesized that HB-EGF signals may operate in a spatially restricted local circuit in the extracellular space. We also hypothesized that HB-EGF secretion by a given cardiomyocyte could create a local remodeling microenvironment of decreased Cx43 within the myocardium. To explore whether HB-EGF signaling is highly spatially constrained, we took advantage of the nonuniform gene transfer to cardiac myocytes in vivo, normally considered a pitfall of gene therapy. We also performed computational modeling to predict HB-EGF dynamics and developed a 3D approach to measure cardiomyocyte hypertrophy.

Results

Autocrine HB-EGF and Cardiomyocyte Growth.

To assess the effects of gene transfer of HB-EGF on cardiomyocyte hypertrophy, cells were infected with adenoviral vectors expressing GFP alone (Ad-GFP) or HB-EGF and GFP (Ad-HB-EGF). At this level of infection, 99% of cardiomyocytes were transduced. The incidence of apoptotic cell death (sub-G1 fraction) was not different between Ad-GFP cells, suggesting that expression of GFP by the adenoviral vector was not cardiotoxic in these conditions. Western analysis by using an anti-HB-EGF antibody confirmed successful gene transfer of HB-EGF in cardiomyocytes (18 ± 5-fold, n = 4, P < 0.01); HB-EGF appeared electrophoretically as several bands from 15 to 30 kDa (Fig. 1A). The strongest band corresponds to the soluble 20-kDa form of HB-EGF. To confirm that Ad-HB-EGF results in cellular hypertrophy, cell size and protein synthesis were measured. Ad-HB-EGF enlarged cardiomyocytes compared with Ad-GFP-infected cells by phase-contrast microscopy (24 ± 10% increase in cell surface area, n = 27, P < 0.05) and with flow cytometry analysis (26 ± 10% increase of Ad-GFP infected cells, P < 0.01, Fig. 1B). Overexpression of HB-EGF increased total protein synthesis in cardiomyocytes as measured by [3H]leucine uptake (34 ± 6% of Ad-GFP, n = 6, P < 0.01, Fig. 1C). Uninfected cells within the same dish (and thus sharing the same culture media) did not develop hypertrophy. Additionally, medium from cultures previously infected with Ad-HB-EGF for 48 h was collected and applied to adenovirus-free cultures. Conditioned medium from Ad-HB-EGF-infected cardiomyocytes failed to stimulate hypertrophy in naive cardiomyocytes (Fig. 1C), and there were no significant differences in cell size between noninfected cells from Ad-GFP and Ad-HB-EGF dishes. These results suggest that HB-EGF acts primarily as an autocrine growth factor in cardiomyocytes in vitro.
Because the dilution factor in culture media is important for autocrine/paracrine signaling, we determined the concentration of soluble HB-EGF in the conditioned medium and the effective concentration to stimulate cardiomyocyte growth. HB-EGF levels in the conditioned medium from Ad-HB-EGF dishes were 258 ± 73 pg/ml (n = 4), whereas HB-EGF levels from Ad-GFP dishes (n = 8) were below the limit of detection (6.7 pg/ml). The addition of 300 pg/ml of exogenous recombinant HB-EGF into fresh media failed to stimulate hypertrophy in cardiomyocytes as measured by [3H]leucine uptake (-12 ± 5.0% compared with control, n = 5,P = not significant), but 2,000 pg/ml of recombinant HB-EGF did result in a significant effect (+24 ± 5.5% compared with control, n = 6, P < 0.05). This comparison implies that the local concentration of autocrine ligand is substantially greater than that indicated by a bulk measurement of conditioned media, consistent with previous experimental and theoretical studies.

Fig. 1. Effects of gene transfer of HB-EGF on rat neonatal cardiomyocyte growth.

(A) Cells were infected with adenoviral vectors expressing GFP (Ad-GFP), or HB-EGF and GFP (Ad-HB-EGF). Western analysis showed the successful gene transfer of HB-EGF. (8) FACS analysis of 5,000 cardiomyocytes demonstrated that overexpression of HB-EGF produced a 26 ± 10% increase in cell size that was significantly greater than the overex-pression of GFP. Bar graphs with errors represent mean ± SEM from three independent experiments. **, P < 0.01 vs. Ad-HB-EGF-nonin-fected cells and Ad-GFP nonin-fected cells. , P < 0.05 vs. Ad-GFP infected cells. (C) Overexpression of HB-EGF resulted in a 34 ± 6% increase in [3H]leucine uptake compared with Ad-GFP (n = 6), whereas conditioned medium from Ad-HB-EGF cells caused an only insignificant increase. **, P < 0.05 vs. Ad-GFP control and conditioned medium Ad-GFP.

 Effects of HB-EGF on Cx43 Content in Cultured Cardiomyocytes

Because EGF can induce degradation of the gap junction protein Cx43 in other cells, we then determined whether Cx43 is regulated by HB-EGF in cardiomyocytes. Fig. 2A shows a representative immunoblot from three separate experiments in which Cx43 migrated as three major bands at 46, 43, and 41 kDa, as reported in ref. 16. Overexpression of HB-EGF decreased total Cx43 content (27 ± 11% compared with Ad-GFP, n = 4, P < 0.05) without affecting the intercellular adhesion protein, N-cadherin. The phosphorylation of ERK1/2, an intracellular signaling kinase downstream of EGFR transactivation, was augmented by HB-EGF (3.2 ± 1.0-fold compared with Ad-GFP, n = 4, P < 0.05). Northern analysis showed that HB-EGF did not reduce Cx43 gene expression, suggesting that HB-EGF decreases Cx43 by posttranslational modification (Fig. 2B). AG 1478 (10 iLM), a specific inhibitor of EGFR tyrosine kinase, abolished the effect of HB-EGF on Cx43 (Fig. 2C), indicating that the decrease in Cx43 content depends on EGFR transactivation by HB-EGF. The conditioned medium from Ad-HB-EGF-infected cells did not change expression of Cx43 in naive cells, even though ERK1/2 was slightly activated by the conditioned medium (Fig. 1D). These data are consistent with the hypertrophy data presented above, demonstrating that HB-EGF can act as a predominantly autocrine factor both in hypertrophy and in the reduction of Cx43 content in cardiomyocytes.

Computational Analysis Predicts HB-EGF Autocrine/Paracrine Signaling in Vivo.

Although these in vitro experiments showed HB-EGF as a predominantly autocrine cardiac growth factor, HB-EGF signaling in vivo takes place in a very different environment. Therefore, we sought to determine the extent that soluble HB-EGF may travel in the interstitial space of the myocardium with a simple 2D model of HB-EGF diffusion (Fig. 3A). An approximate geometric representation of myocytes in cross-section is a square (15 x 15 iLm), with each of the corners occupied by a capillary (diameter 5 iLm). The cell shape was chosen so that the extracellular matrix width (0.5 iLm), in which soluble HB-EGF is free to diffuse, was constant around all tissue features. This model geometry is based on a square array of capillaries; although a hexagonal pattern of capillary distribution is commonly accepted, the results are not expected to be substantially different with this simpler construction, because both have four capillaries surrounding each myocyte. The model represents a single central cell that is releasing HB-EGF at a constant rate, Rgen, (approximated from the HB-EGF concentration measurement in conditioned medium) into the extracellular space. HB-EGF then can diffuse throughout this space, or enter a capillary and leave the system. This system is governed by
  • the diffusion equation at steady state (DV2C = 0),
  • the boundary condition for the ligand producing cell (—DVC = Rgen),
  • the boundary condition for all other cells (—DVC = 0), and
  • the capillary boundary condition (DVC = h(C — Cblood)).

C denotes HB-EGF concentration, D is the diffusivity constant, h is the mass transfer coefficient, and Cblood is the concentration of HB-EGF in the blood, approximated to be zero.  The numerical solution in Fig. 3B illustrates that HB-EGF remained localized around the cell which produced it and did not diffuse farther because of the sink-like effect of the capillaries. The maximum concentration of soluble HB-EGF achieved is 0.27 nM, which is near the threshold level of HB-EGF measured to stimulate cardiomyocyte growth (2,000 pg/ml). Therefore, the central HB-EGF-producing cell only signals to its four adjacent neighbors where the HB-EGF concentration reaches this threshold. However, if the model geometry is altered to reflect a 50% and 150% increase in cross-sectional area in all cells because of hypertrophy, estimated from 1 and 4 weeks of transverse aortic constriction, the maximum concentration achieved increases slightly to 0.29 nM and 0.37 nM, respectively. As the cell width increases, HB-EGF must diffuse farther to reach a capillary, exposing adjacent cells to a higher concentration during hypertrophy. However, no additional cells are exposed to HB-EGF. 

Fig. 3. Computational modeling of HB-EGF diffusion in the myocardium.

Red areas represent capillaries, green represents the HB-EGF ligand producing cell, pink represents adjacent cells, and white is an extracellular matrix where HB-EGF is free to diffuse. (A) The model geometry where HB-EGF is generated by the ligand-producing cell at a constant rate, Rgen, and diffuses throughout the extracellular space or enters a capillary and leaves the system with a mass transfer coefficient, h. (B) Numerical solution of the steady-state HB-EGF concentration profile with Rgen = 10 cell-1s-1, D = 0.7 µm2/s, and h = 0.02 µm/s, where concentration is shown by the color scale and height depicted. The maximum concentration achieved with the stated parameters was 0.27 nM from a capillary. Myocyte length was assumed to be 100 µm.

The driving force that determined the extent to which HB-EGF traveled was the rate of HB-EGF transfer into the capillaries and the diffusivity of HB-EGF. The exact mechanism of macromolecule transport into capillaries is unknown; however, it is most likely through diffusion, transcytosis, or a combination of the two. In the case of diffusion, the mass transfer coefficient governing the flux of HB-EGF through the capillary wall is coupled to the diffusivity of HB-EGF, whereas the terms are uncoupled for the case of transcytosis. Therefore, this model assessed transcytosis as a conservative scenario for HB-EGF localization. Parameter perturbation with uncoupled diffusion and capillary mass transfer showed that HB-EGF remained localized around the origin of production and diffused only to immediate neighbors for mass transfer coefficients >0.002 µm/s. For values <0.002 µm/s, HB-EGF diffused distances more than two cells away from the origin. Although the actual mass transfer coefficient of ligands in the size range of HB-EGF is unknown, values for O2 (0.02 µm/s, 0.032 kDa) (19) and LDL (1.7 x 10-5 µm/s, 2,000–3,000 kDa) (20) have been reported, and we assumed HB-EGF is in the upper end of that range due to its small size. HB-EGF also binds to EGFRs, the extracellular matrix, and cell surface heparan sulfate proteoglycans. EGFR binding and internalization could serve to further localize HB-EGF. The number of extracellular binding sites does not affect the steady-state HB-EGF concentration profile if this binding is reversible. However, these binding sites could serve to localize HB-EGF as the cell begins to produce the ligand by slowing the travel of HB-EGF to the capillaries in the approach to the steady state, or as a source of HB-EGF as the cell slows or stops HB-EGF production. At a diffusivity of 0.7 µm2/s (21), HB-EGF traveled only one cell away, but traveled approximately five cells away at 51.8 µm2/s (22), with a peak concentration below the estimated threshold for stimulating.

Overexpression of HB-EGF Causes Hypertrophy on the Infected Cell and Its Immediate Neighbor in Vivo.

To explore whether HB-EGF signals operate in a spatially restricted local circuit in the in vivo myocardial extracellular space as predicted by computational modeling, adenoviral vectors were injected directly into the left ventricular free wall in 26 male mice (Ad-GFP, n = 12; Ad-HB-EGF, n = 14). Of the 26 mice, 5 (4 Ad-GFP and 1 Ad-HB-EGF) mice died after the surgery. Gene expression was confirmed as positive cellular fluorescence in the presence of GFP, allowing determination of which cells were infected at 7 days (Fig. 4A). Immunohis-tochemical staining revealed that HB-EGF was localized on the Ad-HB-EGF-infected cell membrane or in the extracellular space around the overexpressing cell (Fig. 4A). For comparison, remote cells were defined as noninfected cells far (15–20 cell dimensions) from the adenovirus-infected area and in the same field as infected cells. Conventional 2D cross-sectional analysis blinded to treatment group (Fig. 4B) showed that Ad-GFP-infected cells (n = 102) resulted in no cellular hypertrophy compared with noninfected, adjacent (n = 92), or remote (n = 97) cells (2D myocyte cross-sectional area, 250 ± 7 versus 251 ± 7 or 255 ± 6 µm2, respectively). These data suggest that expression of GFP in these conditions does not cause cellular hypertrophy. However, overexpression of HB-EGF caused hypertrophy in both Ad-HB-EGF-infected cells (a 41 ± 5% increase of Ad-GFP-infected cells, n = 119, P < 0.01) and noninfected adjacent cells (a 33 ± 5% increase of Ad-GFP-adjacent cells, n = 97, P < 0.01) compared with remote cells (n = 109). Because 2D analysis of cardiomyocyte hypertrophy can be influenced by the plane of sectioning, we then developed a 3D histology approach that allowed reconstruction of cardiomyocytes in situ (Fig. 4C). We performed an independent 3D histology analysis of cardiomyocytes to determine cell volumes, blinded to treatment group (Fig. 4B). The volumes of both HB-EGF-infected cells (n = 19, 42,700 ± 4,000 µm3) and their adjacent cells (n = 11, 33,500 ± 3,300 µm3) were significantly greater than volumes of remote cells (n = 13, 18,600 ± 1,700 µm3, P < 0.01 vs. HB-EGF-infected cells and P < 0.05 vs. HB-EGF-adjacent cells, Fig. 4D). In contrast, cells treated with Ad-GFP (n = 12) showed no hypertrophy in the Ad-GFP-adjacent (n = 10) or remote cells (n = 9). These data demonstrate that HB-EGF acts as both an autocrine and local paracrine growth factor within myocardium, as predicted by computational modeling.

Degradation of Cx43 Through Local Autocrine/Paracrine HB-EGF

To determine whether the spatially confined effect of HB-EGF reduces local myocardial Cx43 in vivo, Cx43 was assessed with immunohistochemistry and confocal fluorescence imaging. Cells infected with Ad-HB-EGF had significant decreases in Cx43 immunoreactive signal compared with Ad-GFP cells, consistent with the results of in vitro immunoblotting (Fig. 5A). Quantitative digital image analyses of Cx43 in a total of 22 fields in 6 Ad-HB-EGF hearts and 19 fields in 4 Ad-GFP hearts were analyzed (Fig. 5B). Although Ad-GFP-infected cells showed immunoreactive Cx43 at the appositional membrane, overexpression of HB-EGF increased Cx43 in intracellular vesicle-like components (Fig. 5C), with reduced gap junction plaques (percent Cx43 area per cell area, 52 ± 8% of Ad-GFP control, P < 0.01). These data suggest that reduced expression of Cx43 can be attributed to an increased rate of internalization and degradation in gap junction plaques in cardiomyocytes. Interestingly, HB-EGF secretion by a given cardiomyocyte caused a 37 ± 13% reduction of Cx43 content in its adjacent cells compared with GFP controls (P < 0.05). As degradation of Cx43 may accompany structural changes with marked rearrangement of intercellular connections.  In contrast to Cx43, there was no significant difference in total area occupied by N-cadherin immunoreactive signal in between Ad-GFP (n = 19) and Ad-HB-EGF hearts (1.8 ± 0.5-fold compared with Ad-GFP, n = 17, P = not significant), indicating that HB-EGF has a selective effect on Cx43. Taken together, these data show that HB-EGF leads to cardiomyocyte hypertrophy and degradation of Cx43 in the infected cell and its immediately adjacent neighbors because of autocrine/ paracrine signaling. It should be noted, however, that quantifying the Cx43 from immunostaining could be limited by a nonlinear relation between the amount of Cx43 present and the area of staining.

Fig. 4. Effects of gene transfer of HB-EGF on cardiomyocyte hypertrophy in vivo.

(A) Adenoviral vectors (Ad-GFP or Ad-HB-EGF) were injected into the left ventricular free wall in mice. Myocytes were grouped as infected or noninfected on the basis of GFP fluorescence. Overex-pression of HB-EGF was confirmed by im-munohistochemistry. The presented image was pseudocolored with blue from that stained with Alexa Fluor 555 for the presence of HB-EGF. (Scale bars: 20 sm.) (B) 2D cross-sectional area of cardiomyo-cytes was measured in infected and non-infected cells in the same region of the same animal. Overexpression of HB-EGF caused cellular hypertrophy in both infected and adjacent cells. **, P < 0.01 vs. Ad-GFP infected; , P < 0.01 vs. Ad-HB-EGF remote; and §, P < 0.01 vs. Ad-GFP adjacent cells. GFP (infected 102 cells, adjacent 92 cells, and remote 97 cells from 5 mice), and HB-EGF (infected 119 cells, adjacent 97 cells, and remote 109 cells from 7 mice). The 3D histology also revealed cellular hypertrophy in both Ad-HB-EGF-infected cell and its adjacent cell. **, P < 0.01 vs. Ad-GFP infected; , P < 0.01; and *, P < 0.05 vs. Ad-HB-EGF remote cells. GFP (infected 12 cells, adjacent 10 cells, and remote 9 cells), and HB-EGF (infected 19 cells, adjacent 11 cells, and remote 13 cells). Statistical analysis was performed with one-way ANOVA. (C) Sample image of extracted myocytes in three dimensions.

Discussion

We have demonstrated in this study that HB-EGF secreted by cardiomyocytes leads to cellular growth and reduced expression of the principal ventricular gap junction protein Cx43 in a local autocrine/paracrine manner. Although proHB-EGF is biologically active as a juxtacrine growth factor that can signal to immediately neighboring cells in a nondiffusible mannerseveral studies have revealed the crucial role of metalloproteases in the enzymatic conversion of proHB-EGF to soluble HB-EGF, which binds to and activates the EGFR. Hypertrophic stimuli such as mechanical strain and G protein-coupled receptors agonists mediate cardiac hypertrophy through the shedding of membrane-bound proHB-EGF. Thus, an autocrine/paracrine loop, which requires the diffusible, soluble form of HB-EGF, is necessary for subsequent transactivation of the EGFR to produce the hypertrophic response.

To our knowledge, there have been no previous reports concerning the spatial extent of autocrine/paracrine ligand distribution and signaling in myocardial tissue. A theoretical analysis by Shvartsman et al. predicted, from computational modeling in an idealized cell culture environment, that autocrine ligands may remain highly localized, even within subcellular distances; this prediction has support from experimental data in the EGFR system. In contrast, a theoretical estimate by Francis and Palsson has suggested that cytokines might effectively communicate larger distances, approximated to be 200–300 m from the point of release. However, these studies have all focused on idealized cell culture systems, so our combined experimental and computational investigation here aimed at understanding both in vitro and in vivo situations offers insight.
Our computational model of diffusion in the extracellular space predicts that HB-EGF acts as both an autocrine and spatially restricted paracrine growth factor for neighboring cells. We studied the responses of the signaling cell and its immediate neighbors compared with more distant cells. For a paracrine signal to be delivered to its proper target, the secreted signaling molecules cannot diffuse too far; in vitro experiments, in fact, indicated that HB-EGF acts as a predominantly autocrine signal in cell culture, where diffusion into the medium is relatively unconstrained.
In contrast, in the extracellular space of the myocardium, HB-EGF is localized around the source of production because of tissue geometry, thereby acting in a local paracrine or autocrine manner only. Indeed, our results from in vivo gene transfer demonstrated that both the cell releasing soluble HB-EGF and its surrounding cells undergo hypertrophy. This localized conversation between neighboring cells may allow remodeling to be fine-tuned on a highly spatially restricted level within the myocardium and in other tissues.

Common genetic variation at the IL1RL1locus regulates IL-33/ST2 signaling

JE Ho, Wei-Yu Chen, Ming-Huei Chen, MG Larson, ElL McCabe, S Cheng, A Ghorbani, E Coglianese, V Emilsson, AD Johnson,….. CARDIoGRAM Consortium, CHARGE Inflammation Working Group, A Dehghan, C Lu, D Levy, C Newton-Cheh, CHARGE Heart Failure Working Group, …. JL Januzzi, RT Lee, and TJ Wang J Clin Invest Oct 2013; 123(10):4208-4218.  http://dx.doi.org/10.1172/JCI67119

Abstract and Introduction

The suppression of tumorigenicity 2/IL-33 (ST2/IL-33) pathway has been implicated in several immune and inflammatory diseases. ST2 is produced as 2 isoforms. The membrane-bound isoform (ST2L) induces an immune response when bound to its ligand, IL-33. The other isoform is a soluble protein (sST2) that is thought to be a decoy receptor for IL-33 signaling. Elevated sST2 levels in serum are associated with an increased risk for cardiovascular disease. We investigated the determinants of sST2 plasma concentrations in 2,991 Framing­ham Offspring Cohort participants. While clinical and environmental factors explained some variation in sST2 levels, much of the variation in sST2 production was driven by genetic factors. In a genome-wide associ­ation study (GWAS), multiple SNPs within IL1RL1 (the gene encoding ST2) demonstrated associations with sST2 concentrations. Five missense variants of IL1RL1 correlated with higher sST2 levels in the GWAS and mapped to the intracellular domain of ST2, which is absent in sST2. In a cell culture model, IL1RL1 missense variants increased sST2 expression by inducing IL-33 expression and enhancing IL-33 responsiveness (via ST2L). Our data suggest that genetic variation in IL1RL1 can result in increased levels of sST2 and alter immune and inflammatory signaling through the ST2/IL-33 pathway. Suppression of tumorigenicity 2 (ST2) is a member of the IL-1 receptor (IL-1R) family that plays a major role in immune and inflammatory responses. Alternative promoter activation and splicing produces both a membrane-bound protein (ST2L) and a soluble form (sST2). The transmembrane form of ST2 is selectively expressed on Th2- but not Th1-type T cells, and bind­ing of its ligand, IL-33, induces Th2 immune responses.  In contrast, the soluble form of ST2 acts as a decoy receptor by sequestering IL-33. The IL-33/ST2 pathway has important immunomodulatory effects. Clinically, the ST2/IL-33 signaling pathway participates in the pathophysiology of a number of inflammatory and immune diseases related to Th2 activation, including asthma, ulcera­tive colitis, and inflammatory arthritis. ST2 expression is also upregulated in cardiomyocytes in response to stress and appears to have cardioprotective effects in experimental studies. As a biomarker, circulating sST2 concentrations have been linked to worse prognosis in patients with heart failure, acute dyspnea, and acute coronary syndrome, and also predict mortality and incident cardiovascular events in individuals without existing cardiovascular disease. Both sST2 and its transmembrane form are encoded by IL-1R– like 1 (IL1RL1). Genetic variation in this pathway has been linked to a number of immune and inflammatory diseases. The contribution of IL1RL1 locus variants to interindividual variation in sST2 has not been investigated. The emergence of sST2 as an important predictor of cardiovascular risk and the important role outside of the ST2/IL-33 pathway in inflammatory diseases highlight the value of understanding genetic determinants of sST2. The fam­ily-based FHS cohort provides a unique opportunity to examine the heritability of sST2 and to identify specific variants involved using a genome-wide association study (GWAS). Thus, we per­formed a population-based study to examine genetic determinants of sST2 concentrations, coupled with experimental studies to elu­cidate the underlying molecular mechanisms.

Results

Clinical characteristics of the 2,991 FHS participants are presented in Supplemental Table 1 (supplemental material available online with this article; doi:10.1172/JCI67119DS1). The mean age of participants was 59 years, and 56% of participants were women. Soluble ST2 concentrations were higher in men compared with those in women (P < 0.001). Soluble ST2 concentrations were positively associated with age, systolic blood pressure, body-mass index, antihypertensive medication use, and diabetes mellitus (P < 0.05 for all). Together, these variables accounted for 14% of the variation in sST2 concentrations. The duration of hypertension or diabetes did not materially influence variation in sST2 concentra­tions. After additionally accounting for inflammatory conditions, clinical variables accounted for 14.8% of sST2 variation.

Heritability of sS72.

The age- and sex-adjusted heritability (h2) of sST2 was 0.45 (P = 5.3 x 10–16), suggesting that up to 45% of the vari­ation in sST2 not explained by clinical variables was attributable to genetic factors. Multivariable adjustment for clinical variables pre­viously shown to be associated with sST2 concentrations (21) did not attenuate the heritability estimate (adjusted h2 = 0.45, P = 8.2 x 10–16). To investigate the influence of shared environmental fac­tors, we examined the correlation of sST2 concentrations among 603 spousal pairs and found no significant correlation (r = 0.05, P = 0.25).

Genetic correlates of sS72.

We conducted a GWAS of circulating sST2 concentrations. Quantile-quantile, Manhattan, and regional linkage disequilibrium plots are shown in Supplemental Figures 1–3.  All genome-wide significant SNPs were located in a 400-kb linkage disequilibrium block that included IL1RL1 (the gene encoding ST2), IL1R1, IL1RL2, IL18R1, IL18RAP, and SLC9A4 (Figure 1). Results for 11 genome-wide significant “indepen­dent” SNPs, defined as pairwise r2 < 0.2, are shown in Table 1. In aggregate, these 11 “independent” genome-wide significant SNPs across the IL1RL1 locus accounted for 36% of heritability of sST2. In conditional analyses, 4 out of the 11 SNPs remained genome-wide significant, independent of each other (rs950880, rs13029918, rs1420103, and rs17639215), all within the IL1RL1 locus. The most significant SNP (rs950880, P = 7.1 x 10–94) accounted for 12% of the residual interindividual variability in circulating sST2 concentrations. Estimated mean sST2 concen­trations were 43% higher in major homozygotes (CC) compared with minor homozygotes (AA). Tree loci outside of the IL1RL1 locus had suggestive associations with sST2 (P < 1 x 10–6) and are displayed in Supplemental Table 3.

In silico association with expression SNPs.

The top 10 sST2 SNPs (among 11 listed in Table 1) were explored in collected gene expression databases. There were 5 genome-wide significant sST2 SNPs associated with gene expression across a variety of tissue types (Table 2). Specifically, rs13001325 was associated with IL1RL1 gene expression (the gene encoding both soluble and transmembrane ST2) in several subtypes of brain tissue (prefrontal cortex, P = 1.95 x 10–12; cerebellum, P = 1.54 x 10–5; visual cortex, P = 1.85 x 10–7). The CC genotype of rs13001325 was associated with a higher IL1RL1 gene expression level as well as a higher circulating sST2 concentration when compared with the TT genotype (Supplemental Figure 4). Other ST2 variants were significantly associated with IL18RAP (P = 8.50 x 10–41, blood) and IL18R1 gene expression (P = 2.99 x 10–12, prefrontal cortex).

In silico association with clinical phenotypes in published data

The G allele of rs1558648 was associated with lower sST2 concentra­tions in the FHS (0.88-fold change per G allele, P = 3.94 x 10–16) and higher all-cause mortality (hazard ratio [HR] 1.10 per G allele, 95% CI 1.03–1.16, P = 0.003) in the CHARGE consortium, which observed 8,444 deaths in 25,007 participants during an average fol­low-up of 10.6 years (22). The T allele of rs13019803 was associated with lower sST2 concentrations in the FHS (0.87-fold change per G allele, P = 5.95 x 10–20), higher mortality in the CHARGE consor­tium (HR 1.06 per C allele, 95% CI 1.01–1.12, P = 0.03), and higher risk of coronary artery disease (odds ratio 1.06, 95% CI 1.00–1.11, P = 0.035) in the CARDIoGRAM consortium, which included 22,233 individuals with coronary artery disease and 64,762 controls (23). In relating sST2 SNPs to other clinical phenotypes (including blood pressure, body-mass index, lipids, fasting glucose, natriuretic peptides, C-reactive protein, and echocardiographic traits) in pre­viously published studies, we found nominal associations with C-reactive protein for 2 SNPs (Supplemental Table 4).

Putative functional variants.

Using GeneCruiser, we examined nonsynonymous SNPs (nSNPs) (missense variants) that had at least suggestive association with sST2 (P < 1 x 10–4), includ­ing SNPs that served as proxies (r2 = 1.0) for nSNPs within the 1000 Genomes Pilot 1 data set (ref. 24 and Table 3). There were 6 missense variants located within the IL1RL1 gene, 5 of which had genome-wide significant associations with sST2 concen­trations, including rs6749114 (proxy for rs10192036, Q501K), rs4988956 (A433T), rs10204137 (Q501R), rs10192157 (T549I), rs10206753 (L551S), and rs1041973 (A78AE). Base substitutions and corresponding amino acid changes for these coding muta­tions are listed in Table 3. In combination, these 6 missense muta­tions accounted for 5% of estimated heritability, with an effect estimate of 0.23 (standard error [s.e.] 0.02, P = 2.4 x 10–20). When comparing major homozygotes with minor homozygotes, the esti­mated sST2 concentrations for these missense variants differed by 11% to 15% according to genotype (Supplemental Table 5). In conditional analyses, intracellular and extracellular variants appeared to be independently associated with sST2. For instance, in a model containing rs4988956 (A433T) and rs1041973 (A78E), both SNPs remained significantly associated with sST2 (P = 2.61 x 10–24 and P = 7.67 x 10–15, respectively). In total, missence variants added little to the proportion of sST2 variance explained by the 11 genome-wide significant nonmissense variants listed in Table 1. In relating these 6 missense variants to other clinical phenotypes in large consortia, we found an association with asthma for 4 out of the 6 variants (lowest P = 4.8 x 10–12 for rs10204137) (25).

Homology map of IL1RL1 missense variants and ST2 structure.

Of the 6 missense variants mapping to IL1RL1, 5 were within the cytoplas­mic Toll/IL-1R (TIR) domain of the transmembrane ST2 receptor (Figure 2A), and these intracellular variants are thus not part of the circulating sST2 protein. Of these cytoplasmic domain variants, A433T was located within the “box 2” region of sequence conserva­tion, described in the IL-1R1 TIR domain as important for IL-1 sig­naling . Q501R/K was within a conserved motif called “box 3,” but mutants of IL-1R1 in box 3 did not significantly affect IL-1 signaling in previous experiments (26). Both T549I and L551S were near the C terminus of the transmembrane ST2 receptor and were not predicted to alter signaling function based on previous exper­iments with the IL-1R . The A78E SNP was located within the extracellular domain of ST2 and is thus present in both the sST2 isoform and the transmembrane ST2 receptor. In models of the ST2/IL-33/IL-1RAcP complex derived from a crystal structure of the IL-1RII/IL-1β/IL-1RAcP complex (protein data bank ID 1T3G and 3O4O), A78E was predicted to be located on a surface loop within the first immunoglobulin-like domain (Figure 2B), distant from the putative IL-33 binding site or the site of interaction with IL-1RAcP. There were 2 rare extracellular variants that were not cap­tured in our GWAS due to low minor allele frequencies (A80E, MAF 0.008; A176T, MAF 0.002). Both were distant from the IL-33 bind­ing site on homology mapping and unlikely to affect IL-33 binding.

Functional effects of IL1RL1 missense variants on sST2 expression and promoter activity.

Since 5 of the IL1RL1 missense variants asso­ciated with sST2 levels mapped to the intracellular domain of ST2L and hence are not present on sST2 itself, we hypothesized that these missense variants exert effects via intracellular mecha­nisms downstream of ST2 transmembrane receptor signaling to regulate sST2 levels. To investigate the effect of IL1RL1 missense variants (identified by GWAS) on sST2 expression, stable cell lines expressing WT ST2L, IL1RL1 variants (A78E, A433T, T549I, Q501K, Q501R, and L551S), and a construct containing the 5 IL1RL1 intracellular domain variants (5-mut) were generated. Expression of ST2L mRNA and protein (detected in membrane fractions) was confirmed (Supplemental Figures 5 and 6). Eight different stable clones in each group were analyzed to reduce bias from clonal selection. Intracellular domain variants (A433T, T549I, Q501K, Q501R, L551S, and 5-mut), but not the extracellular domain variant (A78E), were associated with increased basal sST2 expression when compared with WT expression (P < 0.05 for all, Figure 3A). sST2 expression was highest in the 5-mut construct, suggesting that intracellular ST2L variants cooperatively regulate sST2 levels. This same pattern was consistent across different cell types (U937, Jurkat T, and A549 cells; Supplemental Figure 7). These findings suggest that intracellular domain variants of the transmembrane ST2 receptor may functionally regulate downstream signaling.   IL1RL1 transcription may occur via two alternative promoters (proximal vs. distal), which leads to differential expression of the soluble versus membrane-bound ST2 proteins. Similar to the sST2 protein expression results above, the intracellular domain variants, but not the extracellular domain variant, were associated with higher basal proximal promoter activity. Dis­tal promoter activity was also increased for most intracellular domain variants (Supplemental Figure 8).

IL1RL1 intracellular missense variants resulted in higher IL-33 pro­tein levels.

In addition to upregulation of sST2 protein levels, IL1RL1 intracellular missense variants caused increased basal IL-33 protein expression (Figure 3B), suggesting a possible autoregulatory loop whereby IL-33 signaling positively induces sST2 expression. IL-33 induced sST2 protein expression in cells expressing both WT and IL1RL1 missense variants. Interest­ingly, this effect was particularly pronounced in the A433T and Q501R variants (Supplemental Figure 9A).

Enhanced IL-33 responsiveness is mediated by IL-113 in A433T and Q501R variants.

Interaction among IL-33, sST2, and IL-113.

Inhibition of IL-113 by anti–IL-113 mAb reduced basal expression of sST2 (Supplemental Figure 11A). Blocking of IL-33 by sST2 did not reduce the induction of IL-113 levels by the IL1RL1 variants (Supplemental Figure 11B). Furthermore, inhibition of IL-113 by anti–IL-113 reduced the basal IL-33 levels. IL-33 itself upregulated sST2 levels, which in turn reduced IL-33 levels (Supplemental Figure 11C). Our results revealed that both IL-33 and IL-113 drive sST2 expression and that IL-113 acts as an upstream inducer of IL-33 and maintains IL-33 expression by intracellular IL1RL1 vari­ants (Supplemental Figure 11D). This suggests that IL1RL1 vari­ants upregulated sST2 mainly through IL-33 autoregulation and that the enhanced IL-33 responsiveness by A433T and Q501R was mediated by IL-113 upregulation.

IL1RL1 missense variants modulate ST2 signaling pathways

The effect of IL1RL1 missense variants on known ST2 downstream regulatory pathways, including NF-KB, AP-1/c-Jun, AKT, and STAT3 , was examined in the presence and absence of IL-33 (Figure 4 and Supplemental Figure 12). The IL1RL1 intracellular missense vari­ants (A433T, T549I, Q501K, Q501R, and L551S) were associated with higher basal phospho–NF-KB p65 and phospho–c-Jun levels (Figure 4, A and B). Consistent with enhanced IL-33 responsive­ness in A433T and Q501R cells, levels of IL-33–induced NF-KB and c-Jun phosphorylation were enhanced in these 2 variants (Figure 4, B and D). In contrast, A433T and Q501R variants showed lower basal phospho-AKT levels (Figure 4E). ……….. The majority of sST2 gene variants in our study were located within or near IL1RL1, the gene coding for both transmembrane ST2 and sST2. IL1RL1 resides within a linkage disequilibrium block of 400 kb on chromosome 2q12, a region that includes a number of other cytokines, including IL-18 receptor 1 (IL18R1) and IL-18 receptor accessory protein (IL18RAP). Polymorphisms in this gene cluster have been associated previously with a num­ber of immune and inflammatory conditions, including asthma, celiac disease, and type 1 diabetes mellitus . Many of these variants were associated with sST2 concentrations in our analysis (Supplemental Table 6). The immune effects of ST2 are corroborated by experimental evidence: membrane-bound ST2 is selectively expressed on Th2- but not Th1-type T helper cells, and activation of the ST2/IL-33 axis elaborates Th2 responses. In general, the allergic phenotypes above are thought to be Th2-mediated processes, in contrast to atherosclerosis, which appears to be a Th1-driven process.

Fig 2  Models of ST2 illustrate IL1RL1 missense variant locations.

Figure 2 Models of ST2 illustrate IL1RL1 missense variant locations.

Models of the (A) intracellular TIR domain (ST2-TIR) and the (B) extracellular domain (ST2-ECD) of ST2 (protein data bank codes 3O4O and 1T3G, respectively). Domains of ST2 are shown in yellow, with identified mis-sense SNP positions represented as red spheres and labels. Note that positions 549 and 551 are near the C terminus of ST2, which is not defined in the crystal structure (protein data bank ID 1T3G, shown as dashed black line in A). Arrows point toward the transmembrane domain, which is also not observed in crystal structures.

Fig 3 IL1RL1 intracellular missense variants resulted in higher sST2 and IL-33.

Figure 3   IL1RL1 intracellular missense variants resulted in higher sST2 and IL-33.

Media from KU812 cells expressing WT and IL1RL1 missense variants were collected for ELISA analysis of (A) sST2, (B) IL-33, and (C) IL-113 levels. Horizontal bars indicate mean values, and symbols represent indi­vidual variants. *P < 0.05, **P < 0.01 vs. WT. (D) Effect of anti–IL-113 mAb on IL-33–induced sST2 expression. Dashed line indicates PBS-treated cells as referent group. Error bars represent mean ± SEM from 2 independent experiments. *P < 0.05 vs. IL-33.

Fig 4  IL1RL1 missense variants modulated ST2 signaling pathways

Figure 4  IL1RL1 missense variants modulated ST2 signaling pathways. 

KU812 cells expressing WT or IL1RL1 variants were treated with PBS or IL-33. Levels of the following phosphorylated proteins were detected in cell lysates using ELISA: (A and B) phospho-NF-KB p65; (C and D) phospho-c-Jun activity; and (E and F) phospho-AKT. (A, C, and E) White bars represent basal levels, and (B, D, and F) gray bars represent relative fold increase (compared with PBS-treated group) after IL-33 treatment. *P < 0.05 vs. WT; **P < 0.01 vs. PBS-treated group. Dashed line in B, D, and F represents PBS-treated cells as referent group. Error bars represent mean ± SEM from 2 independent experiments. Fig 5   IL-33–induced sST2 expression is enhanced with mTOR inhibition and occurs via ST2L-dependent signaling.

Figure 5  IL-33–induced sST2 expression is enhanced with mTOR inhibition and occurs via ST2L-dependent signaling.

(A) sST2 mRNA expression in KU812 cells after treatment with DMSO, IL-33, or IL-33 plus signal inhibitors (wortmannin, LY294002, rapamycin, PD98059, SP60125, BAY11-7082, or SR11302). (B) ST2L mRNA and (C) sST2 mRNA expression in KU812 cells treated with PBS (white columns), rapamycin (rapa), anti-ST2 mAb, IL-33, IL-33 plus anti-ST2, IL-33 plus rapamycin, IL-33 plus rapamycin plus anti-ST2 mAb, or rapamycin plus anti-ST2. (D) IL33 mRNA expression in KU812 cells after treatment with DMSO, signal inhibitors, IL-33 plus signal inhibitors, and IL-1n plus signal inhibitors. *P < 0.05 vs. PBS-treated group; #P < 0.05 vs. IL-33–treated group; &P < 0.05 vs. IL-1n–treated group. Error bars represent mean ± SEM from 2 independent experiments. (E) A schematic model illustrating the regulation of sST2 expression by IL1RL1 missense variants through enhanced induction of IL-33 via enhanced NF-KB and AP-1 signaling and enhanced IL-33 responsiveness via increasing ST2L expression.

Quantitating subcellular metabolism with multi-isotope imaging mass spectrometry

ML Steinhauser, A Bailey, SE Senyo, C Guillermier, TS Perlstein, AP Gould, RT Lee, and CP Lechene
Department of Medicine, Divisions of Cardiovascular Medicine & Genetics, Brigham and Women’s Hospital, Harvard Medical School & Harvard Stem Cell Institute Division of Physiology and Metabolism, Medical Research Council National Institute for Medical Research, Mill Hill, London, UK National Resource for Imaging Mass Spectroscopy
Nature 2012;481(7382): 516–519.   http://dx. do.org/10.1038/nature10734

Mass spectrometry with stable isotope labels has been seminal in discovering the dynamic state of living matter, but is limited to bulk tissues or cells. We developed multi-isotope imaging mass spectrometry (MIMS) that allowed us to view and measure stable isotope incorporation with sub-micron resolution. Here we apply MIMS to diverse organisms, including Drosophila, mice, and humans. We test the “immortal strand hypothesis,” which predicts that during asymmetric stem cell division chromosomes containing older template DNA are segregated to the daughter destined to remain a stem cell, thus insuring lifetime genetic stability. After labeling mice with 15N-thymidine from gestation through post-natal week 8, we find no 15N label retention by dividing small intestinal crypt cells after 4wk chase. In adult mice administered 15N-thymidine pulse-chase, we find that proliferating crypt cells dilute label consistent with random strand segregation. We demonstrate the broad utility of MIMS with proof-of-principle studies of lipid turnover in Drosophila and translation to the human hematopoietic system. These studies show that MIMS provides high-resolution quantitation of stable isotope labels that cannot be obtained using other techniques and that is broadly applicable to biological and medical research. MIMS combines ion microscopy with secondary ion mass spectrometry (SIMS), stable isotope reporters, and intensive computation (Supplemental Fig 1). MIMS allows imaging and measuring stable isotope labels in cell domains smaller than one micron cubed. We tested the potential of MIMS to quantitatively track DNA labeling with 15N-thymidine in vitro. In proliferating fibroblasts, we detected label incorporation within the nucleus by an increase in the 15N/14N ratio above natural ratio (Fig 1a). The labeling pattern resembled chromatin with either stable isotope-tagged thymidine or thymidine analogs (Fig 1b). We measured dose-dependent incorporation of 15N-thymidine over three orders of magnitude (Fig 1d, Supplemental Fig 2). We also tracked fibroblast division after a 24-hour label-free chase (Fig 1d,e, Supplemental Fig 3). Cells segregated into two populations, one indistinguishable from control cells suggesting no division, the other with halving of label, consistent with one division during chase. We found similar results by tracking cell division in vivo in the small intestine (Fig 1f,g, Supplemental Figs 4–6). We measured dose-dependent 15N-thymidine incorporation within nuclei of actively dividing crypt cells (Fig 1g, Supplemental Fig 4), down to a dose of 0.1µg/ g (Supplemental Fig 2). The cytoplasm was slightly above natural ratio, likely due to low level soluble 15N-thymidine or mitochondrial incorporation (Supplemental Fig 2). We measured halving of label with each division during label-free chase (Supplemental Fig 6). We then tested the “immortal strand hypothesis,” a concept that emerged from autoradiographic studies and that predicted long-term label retaining cells in the small intestinal crypt. It proposes that asymmetrically dividing stem cells also asymmetrically segregate DNA, such that older template strands are retained by daughter cells that will remain stem cells and newer strands are passed to daughters committed to differentiation (Supplemental Fig 7)5,6. Modern studies continue to argue both for or against the hypothesis, leading to the suggestion that definitive resolution of the debate will require a new experimental approach. Although prior evidence suggests a concentration of label-retaining cells in the +4 anatomic position, we searched for DNA label retention irrespective of anatomic position or molecular identity. We labeled mice with 15N-thymidine for the first 8 wks of life when intestinal stem cells are proposed to form. After a 4-wk chase, mice received bromodeoxyuridine (BrdU) for 24h prior to sacrifice to identify proliferating cells(Fig 2a, Supplemental Fig 8: Exp 1), specifically crypt base columnar (CBC) cells and transit amplifying cells (TA) (Supplemental Fig 9), which cycle at a rate of one and two times per 24h, respectively (Supplemental Fig 10). All crypt cell nuclei were highly labeled upon completion of 15N-thymidine; after a four-week chase, however, we found no label retention by non-Paneth crypt cells (Fig 2b–f; n=3 mice, 136 crypts analysed). 15N-labeling in BrdU/15N+ Paneth and mesenchymal cells was equivalent to that measured at pulse completion (Fig2b,c) suggesting quiescence during the chase (values above 15N/14N natural ratio: Paneth pulse=107.8 +/− 5.0% s.e.m. n=51 vs Paneth pulse-chase=96.3+/−2.8% s.e.m. n=218; mesenchymal pulse=92.0+/−5.0% s.e.m. n=89 vs mesenchymal pulse-chase=90.5+/ −2.2% s.e.m. n=543). The number of randomly selected crypt sections was sufficient to detect a frequency as low as one label-retaining stem cell per crypt irrespective of anatomic location within the crypt. Because each anatomic level contains approximately 16 circumferentially arrayed cells, a 2-dimensional analysis captures approximately 1/8th of the cells at each anatomic position (one on each side of the crypt; Supplemental Fig 9a). Therefore, assuming only 1 label-retaining stem cell per crypt we should have found 17 label-retaining cells in the 136 sampled crypts (1/8th of 136); we found 0 (binomial test p<0.0001). The significance of this result held after lowering the expected frequency of label-retaining cells by 25% to account for the development of new crypts, a process thought to continue into adulthood. In three additional experiments, using shorter labeling periods and including in utero development, we also found no label-retaining cells in the crypt other than Paneth cells (Supplemental Fig 8, Exps 2–4).

Fig 1 post-natal human DNA synthesis in the heart

In recent years, several protocols have been developed experimentally in an attempt to identify novel therapeutic interventions aiming at the reduction of infarct size and prevention of short and long term negative ventricular remodeling following ischemic myocardial injury. Three main strategies have been employed and a significant amount of work is being conducted to determine the most effective form of action for acute ischemic heart failure. The delivery of bone marrow progenitor cells (BMCs) has been highly controversial, but recent clinical data have shown improvement in ventricular performance and clinical outcome. These observations have not changed the nature of the debate concerning the efficacy of this cell category for the human disease and the mechanisms involved in the impact of BMCs on cardiac structure and function. Whether BMCs transdifferentiate and acquire the cardiomyocyte lineage has faced strong opposition and data in favor and against this possibility have been reported. However, this is the only cell class which has been introduced in the treatment of heart failure in patients and large clinical trials are in progress.
Human embryonic stem cells (ESCs) have repeatedly been utilized in animal models to restore the acutely infarcted myocardium, but limited cell engraftment, modest ability to generate vascular structures, teratoma formation and the apparent transient beneficial effects on cardiac hemodynamics have questioned the current feasibility of this approach clinically. Tremendous efforts are being performed to reduce the malignant tumorigenic potential of ESCs and promote their differentiation into cardiomyocytes with the expectation that these extremely powerful cells may be applied to human beings in the future. Additionally, the study of ESCs may provide unique understanding of the mechanisms of embryonic development that may lead to therapeutic interventions in utero and the correction of congenital malformations.
The recognition that a pool of primitive cells with the characteristics of stem cells resides in the myocardium and that these cells form myocytes, ECs and SMCs has provided a different perspective of the biology of the heart and mechanisms of cardiac homeostasis and tissue repair. Regeneration implies that dead cells are replaced by newly formed cells restoring the original structure of the organ. In adulthood, this process occurs during physiological cell turnover, in the absence of injury. However, myocardial damage interferes with recapitulation of cell turnover and restitutio ad integrum of the organ. Because of the inability of the adult heart to regenerate itself after infarction, previous studies have promoted tissue repair by injecting exogenously expanded CPCs in proximity of the necrotic myocardium or by activating resident CPCs through the delivery of growth factors known to induce cell migration and differentiation. These strategies have attenuated ventricular dilation and the impairment in cardiac function and in some cases have decreased animal mortality.

Although various subsets of CPCs have been used to reconstitute the infarcted myocardium and different degrees of muscle mass regeneration have been obtained, in all cases the newly formed cardiomyocytes possessed fetal-neonatal characteristics and failed to acquire the adult cell phenotype. In the current study, to enhance myocyte growth and differentiation, we have introduced cell therapy together with the delivery of self-assembly peptide nanofibers to provide a specific and prolonged local myocardial release of IGF-1. IGF-1 increases CPC growth and survival in vitro and in vivo and this effect resulted here in a major increase in the formation of cardiomyocytes and coronary vessels, decreasing infarct size and restoring partly cardiac performance. This therapeutic approach was superior to the administration of CPCs or NF-IGF-1 only. Combination therapy appeared to be additive; it promoted myocardial regeneration through the activation and differentiation of resident and exogenously delivered CPCs. Additionally, the strategy implemented here may be superior to the utilization of BMCs for cardiac repair. CPCs are destined to form myocytes, and vascular SMCs and ECs and, in contrast to BMCs, do not have to transdifferentiate to acquire cardiac cell lineages. Transdifferentiation involves chromatin reorganization with activation and silencing of transcription factors and epigenetic modifications.

Selected References

  1. Hsieh PC, Davis ME, Gannon J, MacGillivray C, Lee RT. Controlled delivery of PDGF-BB for myocardial protection using injectable self-assembling peptide nanofibers. J Clin Invest 2006;116:237–248. [PubMed: 16357943]
  2. Davis ME, Hsieh PC, Takahashi T, Song Q, Zhang S, Kamm RD, Grodzinsky AJ, Anversa P, Lee RT. Local myocardial insulin-like growth factor 1 (IGF-1) delivery with biotinylated peptide nanofibers improves cell therapy for myocardial infarction. Proc Natl Acad Sci USA 2006;103:8155–8160. [PubMed: 16698918]
  3. Beltrami AP, Barlucchi L, Torella D, Baker M, Limana F, Chimenti S, Kasahara H, Rota M, Musso E, Urbanek K, Leri A, Kajstura J, Nadal-Ginard B, Anversa P. Adult cardiac stem cells are multipotent and support myocardial regeneration. Cell 2003;114:763–776. [PubMed: 14505575]
  4. Rota M, Padin-Iruegas ME, Misao Y, De Angelis A, Maestroni S, Ferreira-Martins J, Fiumana E, Rastaldo R, Arcarese ML, Mitchell TS, Boni A, Bolli R, Urbanek K, Hosoda T, Anversa P, Leri A, Kajstura J. Local activation or implantation of cardiac progenitor cells rescues scarred infarcted myocardium improving cardiac function. Circ Res 2008;103:107–116. [PubMed: 18556576]

Cardiac anatomy.

Figure 2.  Cardiac anatomy.

(A and B) Cardiac weights and infarct size. R and L correspond, respectively, to the number of myocytes remaining and lost after infarction. (C–G) LV dimensions. Sham-operated: SO. *Indicates P<0.05 vs SO; **vs untreated infarcts (UN); †vs infarcts treated with CPCs; ‡vs infarcts treated with NF-IGF-1.

Ventricular function

Figure 3.  Ventricular function.

Combination therapy (CPC-NF-IGF-1) attenuated the most the negative impact of myocardial infarction on cardiac performance. See Figure 2 for symbols.

Endothelial Cells Promote Cardiac Myocyte Survival and Spatial Reorganization: Implications for Cardiac Regeneration

Daria A. Narmoneva, Rada Vukmirovic, Michael E. Davis, Roger D. Kamm,  and Richard T. Lee
Cardiovascular Division, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, and the Division of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA
Circulation. 2004 August 24; 110(8): 962–968.        http://dx.doi.org/10.1161/01.CIR.0000140667.37070.07

Background

Endothelial-cardiac myocyte (CM) interactions play a key role in regulating cardiac function, but the role of these interactions in CM survival is unknown. This study tested the hypothesis that endothelial cells (ECs) promote CM survival and enhance spatial organization in a 3-dimensional configuration.

Methods and Results

Microvascular ECs and neonatal CMs were seeded on peptide hydrogels in 1 of 3 experimental configurations:

  1. CMs alone,
  2. CMs mixed with ECs (coculture), or
  3. CMs seeded on preformed EC networks (prevascularized).

Capillary-like networks formed by ECs promoted marked CM reorganization along the EC structures, in contrast to limited organization of CMs cultured alone. The presence of ECs markedly inhibited CM apoptosis and necrosis at all time points. In addition, CMs on preformed EC networks resulted in significantly less CM apoptosis and necrosis compared with simultaneous EC-CM seeding (P<0.01, ANOVA). Furthermore, ECs promoted synchronized contraction of CMs as well as connexin 43 expression.

Conclusions

These results provide direct evidence for a novel role of endothelium in survival and organization of nearby CMs. Successful strategies for cardiac regeneration may therefore depend on establishing functional CM-endothelium interactions.

Keywords:  endothelium; cardiomyopathy; heart failure; tissue

Introduction

Recent studies suggest that the mammalian heart possesses some ability to regenerate itself through several potential mechanisms, including generation of new cardiomyocytes (CMs) from extracardiac progenitors, CM proliferation, or fusion with stem cells with subsequent hybrid cell division. These mechanisms are insufficient to regenerate adequate heart tissue in humans, although some vertebrates can regenerate large volumes of injured myocardium.
Several approaches in cell transplantation and cardiac tissue engineering have been investigated as potential treatments to enhance cardiac function after myocardial injury. Implantation of skeletal muscle cells, bone marrow cells, embryonic stem cell-derived CMs, and myoblasts can enhance cardiac function. Cell-seeded grafts have been used instead of isolated cells for in vitro cardiac tissue growth or in vivo transplantation. These grafts can develop a high degree of myocyte spatial organization, differentiation, and spontaneous and coordinated contractions. On implantation in vivo, cardiac grafts can integrate into the host tissue and neovascularization can develop. However, the presence of scar tissue and the death of cells in the graft can limit the amount of new myocardium formed, most likely due to ischemia. Therefore, creating a favorable environment to promote survival of transplanted cells and differentiation of progenitor cells remains one of the most important steps in regeneration of heart tissue.
One of the key factors for myocardial regeneration is revascularization of damaged tissue. In the normal heart, there is a capillary next to almost every CM, and endothelial cells (ECs) outnumber cardiomyocytes by ≈3:1. Developmental biology experiments reveal that myocardial cell maturation and function depend on the presence of endocardial endothelium at an early stage. Experiments with inactivation or overexpression of vascular endothelial growth factor (VEGF) demonstrated that at later stages, either an excess or a deficit in blood vessel formation results in lethality due to cardiac dysfunction. Both endocardium and myocardial capillaries have been shown to modulate cardiac performance, rhythmicity, and growth. In addition, a recent study showed the critical importance of CM-derived VEGF in paracrine regulation of cardiac morphogenesis. These findings and others highlight the significance of interactions between CMs and endothelium for normal cardiac function. However, little is known about the specific mechanisms for these interactions, as well as the role of a complex, 3-dimensional organization of myocytes, ECs, and fibroblasts in the maintenance of healthy cardiac muscle.
The critical relation of CMs and the microvasculature suggests that successful cardiac regeneration will require a strategy that promotes survival of both ECs and CMs. The present study explored the hypothesis that ECs (both as preexisting capillary-like structures and mixed with myocytes at the time of seeding) promote myocyte survival and enhance spatial reorganization in a 3-dimensional configuration. The results demonstrate that CM interactions with ECs markedly decrease myocyte death and show that endothelium may be important not only for the delivery of blood and oxygen but also for the formation and maintenance of myocardial structure.

Methods

  • Three-Dimensional Culture
  • Immunohistochemistry and Cell Death Assays
  • Evaluation of Contractile Areas

Results

  • EC-CM Interactions Affect Myocyte Reorganization
  • ECs Improve Survival of CMs
  • Preformed Endothelial Networks Promote Coordinated, Spontaneous Contractions
  • ECs Promote Cx43 Expression

EC-CM Interactions Affect Myocyte Reorganization

To explore interactions between CMs and ECs in 3-dimensional culture, we used peptide hydrogels, a tissue engineering scaffold. Cells seeded on the surface of the hydrogel attach and then migrate into the hydrogel. When CMs alone were used, cells attached on day 1 and then formed small clusters of cells at days 3 and 7 (Figure 1). In contrast, when CMs were seeded together with ECs, cells formed interconnected linear networks, as commonly seen with ECs in 3-dimensional culture environments, with increasing spatial organization from day 1 to day 7 (Figure 1).

Figure 1.  ECs promote CM reorganization. 

When CMs were cultured alone (left column), they aggregated into sparse clusters. When CMs were cultured with ECs (center), cells organized into capillary-like networks. There was no difference in morphological appearance between coculture or prevascularized cultures (not shown) and ECs alone (right column). Bar=100 μm. Abbreviations are as defined in text.

To establish whether preformed endothelial networks enhanced the organization of myocytes, we also seeded ECs 1 day before myocytes were added. These ECs formed similar interconnected networks in the absence of myocytes; preforming the vascular network did not lead to significant differences in morphology (data not shown). Furthermore, to exclude the possibility that the increasing cell density of added ECs caused the spatial organization, we also performed control experiments with varying numbers and combinations of cells; there was no effect of doubling or halving cell numbers, indicating that the spatial organization effect was specifically due to ECs. To establish that both myocytes and ECs were forming networks together, we performed immunofluorescence studies with specific antibodies, as well as analysis of cross sections of CM-EC cocultures, whereby cells were labeled with CellTracker dyes before seeding. Immunofluorescent staining demonstrated that >95% of CMs were present within these networks, suggesting that CMs preferentially migrate to or survive better near ECs (Figure 2).

Figure 2.  CMs appear on outside of endothelial networks.

CMs appear on outside of endothelial networks. High-magnification, double-immunofluorescence image of structures formed in EC-CM coculture at day 7 demonstrating CMs (sarcomeric actinin, red) spread on top of ECs (von Willebrand factor, green) with no myocytes present outside structure. Bar=100 μm. Abbreviations are as defined in text.

The analysis of cross sections demonstrated the presence of what appeared to be EC-derived, tubelike structures (Figure 3), with myocytes spread on the outer part of the capillary wall. Along with the capillary-like structures, clusters of intermingled cells (both myocytes and ECs) not containing the lumen were also observed (not shown). However, when the lumen was present, ECs were always on the inner side and myocytes on the outer side of the structure.

Figure 3.  ECs form tubelike structures with myocytes spreading on outer wall.

Cross section of paraffin-embedded sample of 3-day coculture of myocytes (red) and ECs (green) incubated in CellTracker dye before seeding on hydrogel. Bar=50 μm. Abbreviations are as defined in text.

In CM-fibroblast cocultures, cells rapidly (within 24 hours) formed large clusters consisting of cells of both types (not shown). At later time points, fibroblast proliferation resulted in their migration outside the clusters and spreading on the hydrogel without any pattern. However, in contrast to EC-CM cocultures, CMs remained in the clusters and demonstrated only limited spreading. Immunofluorescent staining revealed that there was no orientation of myocytes relative to the fibroblasts in the clusters. In cultures with EC-conditioned medium, myocyte morphology and spatial organization remained similar to those of myocyte controls.

ECs Improve Survival of CMs

To test the hypothesis that ECs promote CM survival, we assessed apoptosis and necrosis in the 3-dimensional cultures. Quantitative analyses of CMs positive for TUNEL and necrosis staining demonstrated significantly decreased myocyte apoptosis and necrosis when cultured with ECs, compared with CM-only cultures (Figure 4, P<0.01). This effect was observed at all 3 time points, although the decreased necrosis was most pronounced at day 1. In addition, CMs seeded on the preformed EC networks had a lower rate of apoptosis at day 1 relative to same-time seeding cultures (P<0.05, post hoc test), suggesting that early EC-CM interactions provided by the presence of well-attached and prearranged ECs may further promote CM survival. In contrast to the ECs, cardiac fibroblasts did not affect myocyte survival (P>0.05, Figure 4), with ratios for myocyte apoptosis and necrosis in the myocyte-fibroblast cocultures being similar to those for myocyte-only controls. However, addition of EC-conditioned medium resulted in a significant decrease in apoptosis and necrosis ratios of myocytes (P<0.01). Interestingly, the effect of conditioned medium on myocyte necrosis was similar in magnitude to the effect of ECs, whereas myocyte apoptosis ratios in the conditioned-medium group were only partially decreased compared with those in the presence of ECs. These results suggest that the prosurvival effect of ECs on CMs may not only be merely due to the local interactions between myocytes and ECs during myocyte attachment but may also involve direct signaling between myocytes and ECs.

Figure 4.  ECs prolong survival of CMs

Top, dual immunostaining of CMs and EC-myocyte prevascularized groups at day 3 in culture, with TUNEL-positive cells in red; green indicates sarcomeric actinin; blue, DAPI. Bottom, presence of ECs decreased CM apoptosis and necrosis, both in coculture conditions and when cultures were prevascularized by seeding with ECs 1 day before CMs (mean±SD, P<0.01). EC-conditioned medium decreased myocyte apoptosis and necrosis (P<0.01), whereas fibroblasts did not have any effect (P>0.05). *Different from myocytes alone; **different from EC-myocyte coculture and pre-vascularized. Bar=100 μm. Abbreviations are as defined in text.

Preformed Endothelial Networks Promote Coordinated, Spontaneous Contractions

In the prevascularized group with preformed vascular structures, synchronized, spontaneous contractions of large areas (Figure 5, top panels) were detected as early as days 2 to 3after seeding, in contrast to the coculture group, wherein such contractions were observed on days 6 to 7. In CM-only cultures, beating of separate cells and small cell clusters was also detected at days 2 to 3, similar to that in the prevascularized group. However, the average area of synchronized beating at day 3 in the myocyte-only group (3.5±0.5×102 μm2) was nearly 3 orders of magnitude smaller than the synchronously contracting area in the prevascularized group (4.3±2.5×105 μm2, mean±SD, n=5). These data suggest that ECs promote synchronized CM contraction, particularly when vascular networks are already formed.

Figure 5.  ECs promote large-scale, synchronized contraction of CMs.

Left, phase-contrast video of beating areas in CM-only and prevascularized groups (day 3). Right, motion analysis of video showing regions of synchronized contractions (connected areas in purple are contracting synchronously) and nonmoving areas in blue. Bars=100 μm. Abbreviations are as defined in text.

ECs Promote Cx43 Expression

Staining for Cx43 showed striking differences in the distribution pattern of this gap junction protein between EC-CM cocultures and CMs cultured alone. In myocyte-only cultures, Cx43 expression was barely detectable at day 1 (not shown); at days 3 and 7, Cx43 expression was sparse throughout the cell clusters (Figure 6). In the presence of ECs (in both coculture and prevascularized groups), Cx43 staining was evident at day 1, both between ECs and distributed among CMs. As early as day 3 in culture, patches of localized junction-like Cx43, in addition to diffuse staining, were observed for myocytes in the coculture group (Figure 6). In the prevascularized group at day 3, wherein spontaneous contractions were already observed, more junction-like patches of Cx43 were observed compared with the coculture group, indicating electrical connections between myocytes (Figure 6). In addition to junctions between myocytes, there was also evidence of Cx43 localized at the interface between ECs and myocytes (Figure 6) detected in both the coculture group (at day 7) and the preculture group (as early as day 3). When myocytes and myocyte-EC coculture groups were cultured for 3 days with or without addition of 100 ng/mL of neutralizing anti-mouse VEGF antibody (R&D Systems), we observed no differences in either apoptosis or Cx43 staining between VEGF antibody-containing cultures and controls.

Figure 6. ECs promote Cx43 expression

Cultures at 3 days immunostained for Cx43 (red) and anti-sarcomeric actinin (green); nuclei are stained with DAPI (blue). For CMs alone (left), Cx43 staining is diffuse and sparse, with no evidence of gap junctions; for coculture (center), both diffuse (yellow arrow) and patchlike (thin, white arrow) Cx43 staining is observed; for prevascularized (right), increased patchlike staining indicates presence of gap junctions. Thick arrow-heads indicate junctions between myocytes and ECs. Bar=50 μm. Abbreviations are as defined in text.

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Endothelial-Cardiomyocyte Interactions in Cardiac Development and Repair: Implications for Cardiac Regeneration

Patrick C.H. Hsieh, Michael E. Davis, Laura K. Lisowski, and Richard T. Lee

Cardiovascular Division, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA
Annu Rev Physiol.    PMC 2009 September 30

The ongoing molecular conversation between endothelial cells and cardiomyocytes is highly relevant to the recent excitement in promoting cardiac regeneration. The ultimate goal of myocardial regeneration is to rebuild a functional tissue that closely resembles mature myocardium, not just to improve systolic function transiently. Thus, regenerating myocardium will require rebuilding the vascular network along with the cardiomyocyte architecture. Here we review evidence demonstrating crucial molecular interactions between endothelial cells and cardiomyocytes. We first discuss endothelial-cardiomyocyte interactions during embryonic cardiogenesis, followed with morphological and functional characteristics of endothelial-cardiomyocyte interactions in mature myocardium. Finally, we consider strategies exploiting endothelial-cardiomyocyte interplay for cardiac regeneration.

Signaling from Cardiomyocytes to Endothelial Cells

The examples of neuregulin-1, NF1, and PDGF-B demonstrate that signals from endothelial cells regulate the formation of primary myocardium. Similarly, signaling from myocardial cells to endothelial cells is also required for cardiac development. Two examples of myocardial-to-endothelial signaling are vascular endothelial growth factor (VEGF)-A and angiopoietin-1.

VASCULAR ENDOTHELIAL GROWTH FACTOR-A

VEGF-A is a key regulator of angiogenesis during embryogenesis. In mice, a mutation in VEGF-A causes endocardial detachment from an underdeveloped myocardium. A mutation in VEGF receptor-2 (or Flk-1) also results in failure of the endocardium and myocardium to develop (18). Furthermore, cardiomyocyte-specific deletion of VEGF-A results in defects in vasculogenesis/angiogenesis and a thinned ventricular wall, further confirming reciprocal signaling from the myocardial cell to the endothelial cell during cardiac development. Interestingly, this cardiomyocyte-selective VEGF-A-deletion mouse has underdeveloped myocardial microvasculature but preserved coronary artery structure, implying a different signaling mechanism for vasculogenesis/angiogenesis in the myocardium and in the epicardial coronary arteries.
Cardiomyocyte-derived VEGF-A also inhibits cardiac endocardial-to-mesenchymal transformation. This process is essential in the formation of the cardiac cushions and requires delicate control of VEGF-A concentration. A minimal amount of VEGF initiates endocardial-to-mesenchymal transformation, whereas higher doses of VEGF-A terminate this transformation. Interestingly, this cardiomyocyte-derived VEGF-A signaling for endocardial-to-mesenchymal transformation may be controlled by an endothelial-derived feedback mechanism through the calcineurin/NFAT pathway (24), demonstrating the importance of endothelial-cardiomyocyte interactions for cardiac morphogenesis.

ANGIOPOIETIN-1

Another mechanism of cardiomyocyte control of endothelial cells during cardiac development is the angiopoietin-Tie-2 system. Both angiopoietin-1 and angiopoietin-2 may bind to Tie-2 receptors in a competitive manner, but with opposite effects: Angiopoietin-1 activates the Tie-2 receptor and prevents vascular edema, whereas angiopoietin-2 blocks Tie-2 phosphorylation and increases vascular permeability. During angiogenesis/vasculogenesis, angiopoietin-1 is produced primarily by pericytes, and Tie-2 receptors are expressed on endothelial cells. Angiopoietin-1 regulates the stabilization and maturation of neovasculature; genetic deletion of angiopoietin-1 or Tie-2 causes a defect in early vasculogenesis/angiogenesis and is lethal.
Cardiac endocardium is one of the earliest vascular components (along with the dorsal aorta and yolk sac vessels) and the adult heart can be regarded as a fully vascularized organ, angiopoietin-Tie-2 signaling may also be required for early cardiac development. Indeed, mice with mutations in Tie-2 have underdeveloped endocardium and myocardium. These Tie-2 knockout mice display defects in the endocardium but have normal vascular morphology at E10.5, suggesting that the endocardial defect is the fundamental cause of death. In addition, a recent study showed that overexpression, and not deletion, of angiopoietin-1 from cardiomyocytes caused embryonic death between E12.5-15.5 due to cardiac hemorrhage. The mice had defects in the endocardium and myocardium and lack of coronary arteries, suggesting that, as with VEGF-A, a delicate control of angiopoietin-1 concentration is critical for early heart development.

ENDOTHELIAL-CARDIOMYOCYTE INTERACTIONS IN NORMAL CARDIAC FUNCTION

Cardiac Endothelial Cells Regulate Cardiomyocyte Contraction

The vascular endothelium senses the shear stress of flowing blood and regulates vascular smooth muscle contraction. It is therefore not surprising that cardiac endothelial cells—the endocardial endothelial cells as well as the endothelial cells of intramyocardial capillaries— regulate the contractile state of cardiomyocytes. Autocrine and paracrine signaling molecules released or activated by cardiac endothelial cells are responsible for this contractile response (Figure 2).

NITRIC OXIDE

Three different nitric oxide synthase isoenzymes synthesize nitric oxide (NO) from L-arginine. The neuronal and endothelial NO synthases (nNOS and eNOS, respectively) are expressed in normal physiological conditions, whereas the inducible NO synthase is induced by stress or cytokines. Like NO in the vessel, which causes relaxation of vascular smooth muscle, NO in the heart affects the onset of ventricular relaxation, which allows for a precise optimization of pump function beat by beat. Although NO is principally a paracrine effector secreted by cardiac endothelial cells, cardiomyocytes also express both nNOS and eNOS. Endothelial expression of eNOS exceeds that in cardiomyocytes by greater than 4:1. Cardiomyocyte autocrine eNOS signaling can regulate β-adrenergic and muscarinic control of contractile state.
Barouch et al. demonstrated that cardiomyocyte nNOS and eNOS may have opposing effects on cardiac structure and function. Using mice with nNOS or eNOS deficiency, they found that nNOS and eNOS have not only different localization in cardiomyocytes but also opposite effects on cardiomyocyte contractility; eNOS localizes to caveolae and inhibits L-type Ca2+ channels, leading to negative inotropy, whereas nNOS is targeted to the sarcoplasmic reticulum and facilitates Ca2+ release and thus positive inotropy (31). These results demonstrate that spatial confinement of different NO synthase isoforms contribute independently to the maintenance of cardiomyocyte structure and phenotype.
As indicated above, mutation of neuregulin or either of two of its cognate receptors, erbB2 and erbB4, causes embryonic death during mid-embryogenesis due to aborted development of myocardial trabeculation . Neuregulin also appears to play a role in fully developed myocardium. In adult mice, cardiomyocyte-specific deletion of erbB2 leads to dilated cardiomyopathy. Neuregulin from endothelial cells may induce a negative inotropic effect in isolated rabbit papillary muscles. This suggests that, along with NO, the neuregulin signaling pathway acts as an endothelial-derived regulator of cardiac inotropism.  In fact, the negative inotropic effect of neuregulin may require NO synthase because L-NMMA, an inhibitor of NO synthase, significantly attenuates the negative inotropy of neuregulin.

Studies to date indicate that cardiac regeneration in mammals may be feasible, but the response is inadequate to preserve myocardial function after a substantial injury. Thus, understanding how normal myocardial structure can be regenerated in adult hearts is essential. It is clear that endothelial cells play a role in cardiac morphogenesis and most likely also in survival and function of mature cardiomyocytes. Initial attempts to promote angiogenesis in myocardium were based on the premise that persistent ischemia could be alleviated. However, it is also possible that endothelial-cardiomyocyte interactions are essential in normal cardiomyocyte function and for protection from injury. Understanding the molecular and cellular mechanisms controlling these cell-cell interactions will not only enhance our understanding of the establishment of vascular network in the heart but also allow the development of new targeted therapies for cardiac regeneration by improving cardiomyocyte survival and maturation.

Endothelial-cardiomyocyte assembly

Figure 1.  Endothelial-cardiomyocyte assembly in adult mouse myocardium.
Normal adult mouse myocardium is stained with intravital perfusion techniques to demonstrate cardiomyocyte (outlined in red) and capillary (green; stained with isolectin-fluorescein) assembly. Nuclei are blue (Hoechst). Original magnification: 600X

Endothelial dysfunction

Intramyocardial Fibroblast – Myocyte Communication

Rahul Kakkar, M.D. and Richard T. Lee, M.D.
From the Cardiology Division, Massachusetts General Hospital and the Cardiovascular Division, Brigham and Women’s Hospital, Department of Medicine, Harvard Medical School, Boston, MA
Circ Res. 2010 January 8; 106(1): 47–57.    http://dx.doi.org/10.1161/CIRCRESAHA.109.207456

Cardiac fibroblasts have received relatively little attention compared to their more famous neighbors, the cardiomyocytes. Cardiac fibroblasts are often regarded as the “spotters”, nonchalantly watching the cardiomyocytes do the real weight-lifting, and waiting for a catastrophe that requires their actions. However, emerging data now reveal the fibroblast as not only a critical player in the response to injury, but also as an active participant in normal cardiac function.
Interest in cardiac fibroblasts has grown with the recognition that cardiac fibrosis is a prominent contributor to diverse forms of myocardial disease. In the early 1990’s, identification of angiotensin receptors on the surface of cardiac fibroblasts linked the renin-angiotensin-aldosterone system directly with pathologic myocardial and matrix extracellular remodeling.  Fibroblasts were also revealed as a major source of not only extracellular matrix, but the proteases that regulate and organize matrix. New research has uncovered paracrine and well as direct cell-to-cell interactions between fibroblasts and their cardiomyocyte neighbors, and cardiac fibroblasts appear to be dynamic participants in ventricular physiology and pathophysiology.
This review will focus on several aspects of fibroblast-myocyte communication, including mechanisms of paracrine communication.  Ongoing efforts at regeneration of cardiac tissue focus primarily on increasing the number of cardiomyocytes in damaged myocardium. Although getting cardiomyocytes into myocardium is an important goal, understanding intercellular paracrine communication between different cell types, including endothelial cells but also fibroblasts, may prove crucial to regenerating stable myocardium that responds to physiological conditions appropriately.

An area of active research in cardiovascular therapeutics is the attempt to engineer, ex vivo, functional myocardial tissue that may be engrafted onto areas of injured ventricle. Recent data suggests that the inclusion of cardiac fibroblasts in three-dimensional cultures greatly enhances the stability and growth of the nascent myocardium. Cardiac fibroblasts when included in polymer scaffolds seeded with myocytes and endothelial cells have the ability to promote and stabilize vascular structures. Naito and colleagues constructed three dimensional cultures of neonatal rat cell isolates on collagen type I and Matrigel (a basement membrane protein mixture), and isolates of a mixed cell population versus a myocyte-enriched population were compared. The mixed population cultures, which contained a higher fraction of cardiac fibroblasts than the myocyte-enriched cultures, displayed improved contractile force generation and greater inotropic response despite an equivalent overall cell number. Greater vascularity was also seen in the mixed-pool cultures.(160) Building on this, Nichol and colleagues demonstrated that in a self-assembling nanopeptide scaffold, embedded rat neonatal cardiomyocytes exhibit greater cellular alignment and reduced apoptosis when cardiac fibroblasts were included in the initial culture. A similar result was noted when polymer scaffolds were pre-treated with cardiac fibroblasts before myocyte seeding, suggesting a persistent paracrine effect. These data reinforce the concept that engineering functional myocardium, either in situ or ex vivo will require attention to the nature of cell-cell interactions, including fibroblasts.

To date, a broad initial sketch of cardiac fibroblast-myocyte interactions has been drawn. Future studies in this field will better describe these interactions. How do multiple paracrine factors interact to produce a cohesive and coordinated communication scheme? What are the changes in coordinated bidirectional signaling that during development promotes myocyte progenitor proliferation but have different roles in the adult? Might fibroblasts actually be required for improved cardiac repair and regeneration?
Recent studies have begun to apply genetic and cellular fate-mapping techniques to document the origins of cardiac fibroblasts, the dynamic nature of their population, and how that population may be in flux during time of injury or pressure overload. It is crucial to define on a more specific molecular basis the origins and fates of cardiac fibroblasts. Do fibroblasts that have been resident within the ventricle since development fundamentally differ from those that arise from endothelial transition or that infiltrate from the bone marrow during adulthood? Do fibroblasts with these different origins behave differently or take on different roles in the face of ventricular strain or injury?

Our understanding of the nature of the cardiac fibroblast is evolving from the concept of the fibroblast as a bystander that causes unwanted fibrosis to the picture of a more complex role of fibroblasts in the healthy as well as diseased heart. The pathways used by cardiac fibroblasts to communicate with their neighboring myocytes are only partially described, but the data to date indicate that these pathways will be important for cardiac repair and regeneration.

. Paracrine bidirectional cardiac fibroblast-myocyte crosstalk

Figure 2. Paracrine bidirectional cardiac fibroblast-myocyte crosstalk

Under biomechanical overload, cardiac fibroblasts and myocytes respond to an altered environment via multiple mechanisms including integrin-extracellular matrix interactions and renin-angiotensin-aldosterone axis activation. Cardiac fibroblasts increase synthesis of matrix proteins and secrete a variety of paracrine factors that can stimulate myocyte hypertrophy. Cardiac myocytes similarly respond by secreting a conglomerate of factors. Hormones such as TGFβ1, FGF-2, and the IL-6 family members LIF and CT-1 have all been implicated in this bidirectional fibroblast-myocyte hormonal crosstalk.

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Author and Curator: Ritu Saxena, Ph.D

Although cancer stem cells constitute only a small percentage of the tumor burden, their self-renewal capacity and possible link with recurrence of cancer post treatment makes them a sought after therapeutic target in cancer. The post on cancer stem cells published on the 22nd of March, 2013, describes the identity of CSCs, their functional characteristics, possible cell of origin and biomarkers. This post focuses on the therapeutic potential of CSCs, their resistance to conventional anti-tumor therapies and current therapeutic targets including biomarkers, signaling pathways and niches.

CSCs Are Resistant to conventional anticancer therapies including chemotherapy, radiotherapy and surgery that are used either alone or in combination. However, these strategies have failed several times to eradicate CSCs resulting in metastasis and relapse, hence, a fatal disease outcome.

The properties of CSCs that contribute to or lead to chemoresistance include:

Quiescent Phenotype

Chemotherapeutic agents target fast-growing cells; however, some CSCs that remain in the dormant or quiescent stage are spared from lethal damage. Later, when the dormant CSCs enter cell cycle, tumor proliferation is stimulated.

Antiapoptosis

Antiapoptotic proteins such as BCL-2 and some self-renewal pathways such as transforming growth factor β, Wnt/ β -catenin or BMI-1 are activated in CSCs. Consequently, DNA damage repair capability of CSCs is enhanced after genotoxic stress or activation of autocrine loops through the production of growth factors like epidermal growth factor (Moserle L, Cancer Lett, 1 Feb 2010;288(1):1-9).

Expression of Drug Efflux Pumps

CSCs express some proteins that have typically been known to contribute to multidrug resistance. The proteins are drug efflux pumps ABCC1, ABCG2 or MDR1. Multidrug resistance-associated proteins (ABCC subfamily) are members of the ATP-binding cassette (ABC) superfamily of transport proteins and act as cellular efflux transporters for a wide variety of substrates, in particular glutathione, glucuronide and sulfate conjugates of diverse compounds.

Radiotherapy is mainly used in breast cancer and glioblastoma multiforme. In glioblastoma multiforme, the properties of CSCs that contribute to radiotherapy resistance is the presence of CD133 marker. CD133+ CSCs preferentially activate DNA damage repair pathway and significantly induced checkpoint kinases that leads to reduced apoptosis in CSCs compared to the CD133- tumor cells (Bao S, Nature, 7 Dec 2006;444(7120):756-60).

Radiotherapy resistance in breast cancer is due to reduced levels of reactive oxygen species in CSCs. In addition, radiation resistance of progenitor cells in an immortalized breast cancer cell line was mediated by the Wnt/β catenin pathway proteins (Diehn M, et al, Nature, 9 Apr 2009;458(7239):780-3; Chen MS, et al, J Cell Sci, 1 Feb 2007;120(Pt 3):468-77).

As mentioned in the previous post on CSCs, CSC targeting therapy could either eliminate CSCs by either killing them after differentiating them from other tumor population, and/or by disrupting their niche. Efficient eradication of CSCs may require the combined ablation of CSCs themselves and their niches. Thus, identification of appropriate and specific markers of CSCs is crucial for targeting them and preventing tumor relapse. Table 1 (adapted from a review article on CSCs by Zhao et al) describes the currently used biomarkers for CSC-targeted therapy (Zhao L, et al, Eur Surg Res, 2012;49(1):8-15).

Table 1

Specific Target Cancer type Marker properties and therapy
Targeting cell markers
CD24+CD44+ESA+ Pancreatic cancer Pancreatic CSCs, elevated during tumorigenesis
CD44+CD24–ESA+ Breast cancer Breast CSCs
EpCAM high CD44+CD166+ Colorectal cancer
CD34+CD38– AML broad use as a target for chemotherapy
CD133+ Prostate cancer and breast cancer 5-transmembrane domain cell surface glycoprotein,also a marker for neuron epithelial, hematopoietic and endothelialprogenitor cells
Stro1+CD105+CD44+ Bone sarcoma
Nodal/activin Knockdown or pharmacological inhibition of its receptorAlk4/7 abrogated self-renewal capacity and in vivo tumorigenicity of CSCs.
Targeting signaling pathways
Hedgehog signaling Upregulated in several cancer types inhibitors: GDC-0449,PF04449913, BMS-833923, IPI-926 and TAK-441
Wnt/β-catenin signaling CML, squamous cell carcinoma Be required for CSC self-renewal and tumor growthinhibitors: PRI-724, WIF-1 and telomerase
Notch signaling Several cancer types An important regulator in normal development, adult stem cell maintenance,and tumorigenesis in multiple organs,inhibitors: RO4929097, BMS-906024, IPI-926 and MK0752
PI3K/Akt/PTEN/mTOR, Several cancer types The pathway is deregulated in many tumors and used to preferentially target CSCsinhibitors: temsirolimus, everolimus FDA-approved therapy for renal cell carcinoma
Targeting CSC Niche
Angiogenesis Niche Colon cancer, breast cancer, NSCLC Inhibitor: bevacizumab results in a disruption of the CSC niche, depleted vasculature and a dramatic reduction in the number of CSCs.
Hypoxia (HIF pathway) Ovarian cancer, lung cancer, cervical cancer Inhibitors: topotecan and digoxin have been approved for ovarian, lung and cervical cancer
Targeting Micro RNA
miR-200 family Inhibits EMT and cancer cell migration by direct targeting of E-cadherin transcriptional repressors ZEB1 and ZEB2
Let-7 family Regulates BT-IC stem cell-like properties by silencing more than one target
miR-124 Related to neuronal differentiation, targets laminin γ1 and integrin β1.
miR-21 Suppresses the self-renewal of embryonic stem cells

The challenge is to develop an effective treatment regimen that prevents survival, self-renewal and differentiation of CSCs and also disturbs their niche without damaging normal stem cells. In order to evaluate the efficiency of CSC-targeting therapies, in vitro models and mouse xenotransplantation models have been used for preclinical studies. Some potential CSC targeting agents in preclinical stages include notch inhibitors for glioblastoma stem cells and telomerase peptide vaccination after chemoradiotherapy of non-small cell lung cancer stem cells Stem Cells (Hovinga KE, et al, Jun 2010;28(6):1019-29; Serrano D, Mol Cancer, 9 Aug 2011;10:96). In addition, several phase II and phase III trials are currently underway to test CSC-targeting drugs focusing on efficacy and safety of treatment.

Reference:

Bao S, Nature, 7 Dec 2006;444(7120):756-60).

Diehn M, et al, Nature, 9 Apr 2009;458(7239):780-3

Chen MS, et al, J Cell Sci, 1 Feb 2007;120(Pt 3):468-77

Zhao L, et al, Eur Surg Res, 2012;49(1):8-15

Hovinga KE, et al, Jun 2010;28(6):1019-29

Serrano D, Mol Cancer, 9 Aug 2011;10:96

Pharmaceutical Intelligence posts:

https://pharmaceuticalintelligence.com/2013/03/22/in-focus-identity-of-cancer-stem-cells/ Author and curator: Ritu Saxena, PhD

https://pharmaceuticalintelligence.com/2012/08/15/to-die-or-not-to-die-time-and-order-of-combination-drugs-for-triple-negative-breast-cancer-cells-a-systems-level-analysis/ Authors: Anamika Sarkar, PhD and Ritu Saxena, PhD

https://pharmaceuticalintelligence.com/2013/03/07/the-importance-of-cancer-prevention-programs-new-perceptions-for-fighting-cancer/ Author: Ziv Raviv, PhD

https://pharmaceuticalintelligence.com/2013/03/03/treatment-for-metastatic-her2-breast-cancer/ Reporter: Larry H Bernstein, MD

https://pharmaceuticalintelligence.com/2013/03/02/recurrence-risk-for-breast-cancer/ Larry H Bernstein, MD

https://pharmaceuticalintelligence.com/2013/02/14/prostate-cancer-androgen-driven-pathomechanism-in-early-onset-forms-of-the-disease/ Curator: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/01/15/exploring-the-role-of-vitamin-c-in-cancer-therapy/ Curator: Ritu Saxena, PhD

https://pharmaceuticalintelligence.com/2013/01/12/harnessing-personalized-medicine-for-cancer-management-prospects-of-prevention-and-cure-opinions-of-cancer-scientific-leaders-httppharmaceuticalintelligence-com/ Curator: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/01/10/the-molecular-pathology-of-breast-cancer-progression/ Author and reporter: Tilda Barliya PhD

https://pharmaceuticalintelligence.com/2012/11/30/histone-deacetylase-inhibitors-induce-epithelial-to-mesenchymal-transition-in-prostate-cancer-cells/ Reporter and Curator: Stephen J. Williams, PhD

https://pharmaceuticalintelligence.com/2012/10/22/blood-vessel-generating-stem-cells-discovered/ Reporter: Ritu Saxena, PhD

https://pharmaceuticalintelligence.com/2012/10/17/stomach-cancer-subtypes-methylation-based-identified-by-singapore-led-team/ Reporter: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2012/09/17/natural-agents-for-prostate-cancer-bone-metastasis-treatment/ Reporter: Ritu Saxena, PhD

https://pharmaceuticalintelligence.com/2012/08/28/cardiovascular-outcomes-function-of-circulating-endothelial-progenitor-cells-cepcs-exploring-pharmaco-therapy-targeted-at-endogenous-augmentation-of-cepcs/ Aviva Lev-Ari, PhD, RN

 

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