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Posts Tagged ‘HIF-1’


Refined Warburg hypothesis -2.1.2

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

Refined Warburg Hypothesis -2.1.2

The Warburg discoveries from 1922 on, and the influence on metabolic studies for the next 50 years was immense, and then the revelations of the genetic code took precedence.  Throughout this period, however, the brilliant work of Briton Chance, a giant of biochemistry at the University of Pennsylvania, opened new avenues of exploration that led to a recent resurgence in this vital need for answers in cancer research. The next two series of presentations will open up this resurgence of fundamental metabolic research in cancer and even neurodegenerative diseases.

2.1.2.1 Cancer Cell Metabolism. Warburg and Beyond

Hsu PP, Sabatini DM
Cell, Sep 5, 2008; 134:703-707
http://dx.doi.org:/10.016/j.cell.2008.08.021

Described decades ago, the Warburg effect of aerobic glycolysis is a key metabolic hallmark of cancer, yet its significance remains unclear. In this Essay, we re-examine the Warburg effect and establish a framework for understanding its contribution to the altered metabolism of cancer cells.

It is hard to begin a discussion of cancer cell metabolism without first mentioning Otto Warburg. A pioneer in the study of respiration, Warburg made a striking discovery in the 1920s. He found that, even in the presence of ample oxygen, cancer cells prefer to metabolize glucose by glycolysis, a seeming paradox as glycolysis, when compared to oxidative phosphorylation, is a less efficient pathway for producing ATP (Warburg, 1956). The Warburg effect has since been demonstrated in different types of tumors and the concomitant increase in glucose uptake has been exploited clinically for the detection of tumors by fluorodeoxyglucose positron emission tomography (FDG-PET). Although aerobic glycolysis has now been generally accepted as a metabolic hallmark of cancer, its causal relationship with cancer progression is still unclear. In this Essay, we discuss the possible drivers, advantages, and potential liabilities of the altered metabolism of cancer cells (Figure 1). Although our emphasis on the Warburg effect reflects the focus of the field, we would also like to encourage a broader approach to the study of cancer metabolism that takes into account the contributions of all interconnected small molecule pathways of the cell.

Figure 1. The Altered Metabolism of Cancer Cells

Drivers (A and B). The metabolic derangements in cancer cells may arise either from the selection of cells that have adapted to the tumor microenvironment or from aberrant signaling due to oncogene activation. The tumor microenvironment is spatially and temporally heterogeneous, containing regions of low oxygen and low pH (purple). Moreover, many canonical cancer-associated signaling pathways induce metabolic reprogramming. Target genes activated by hypoxia inducible factor (HIF) decrease the dependence of the cell on oxygen, whereas Ras, Myc, and Akt can also upregulate glucose consumption and glycolysis. Loss of p53 may also recapitulate the features of the Warburg effect, that is, the uncoupling of glycolysis from oxygen levels. Advantages (C–E). The altered metabolism of cancer cells is likely to imbue them with several proliferative and survival advantages, such as enabling cancer cells to execute the biosynthesis of macromolecules (C), to avoid apoptosis (D), and to engage in local metabolite-based paracrine and autocrine signaling (E). Potential Liabilities (F and G). This altered metabolism, however, may also confer several vulnerabilities on cancer cells. For example, an upregulated metabolism may result in the build up of toxic metabolites, including lactate and noncanonical nucleotides, which must be disposed of (F). Moreover, cancer cells may also exhibit a high energetic demand, for which they must either increase flux through normal ATP-generating processes, or else rely on an increased diversity of fuel sources (G).

The Tumor Microenvironment Selects for Altered Metabolism

One compelling idea to explain the Warburg effect is that the altered metabolism of cancer cells confers a selective advantage for survival and proliferation in the unique tumor microenvironment. As the early tumor expands, it outgrows the diffusion limits of its local blood supply, leading to hypoxia and stabilization of the hypoxia-inducible transcription factor, HIF. HIF initiates a transcriptional program that provides multiple solutions to hypoxic stress (reviewed in Kaelin and Ratcliffe, 2008). Because a decreased dependence on aerobic respiration becomes advantageous, cell metabolism is shifted toward glycolysis by the increased expression of glycolytic enzymes, glucose transporters, and inhibitors of mitochondrial metabolism. In addition, HIF stimulates angiogenesis (the formation of new blood vessels) by upregulating several factors, including most prominently vascular endothelial growth factor (VEGF).

The oxygen levels within a tumor vary both spatially and temporally, and the resulting rounds of fluctuating oxygen levels potentially select for tumors that constitutively upregulate glycolysis. Interestingly, with the possible exception of tumors that have lost the von Hippel-Lindau protein (VHL), which normally mediates degradation of HIF, HIF is still coupled to oxygen levels, as evident from the heterogeneity of HIF expression within the tumor microenvironment (Wiesener et al., 2001; Zhong et al., 1999). Therefore, the Warburg effect—that is, an uncoupling of glycolysis from oxygen levels—cannot be explained solely by upregulation of HIF.

Recent work has demonstrated that the key components of the Warburg effect—increased glucose consumption, decreased oxidative phosphorylation, and accompanying lactate production—are also distinguishing features of oncogene activation. The signaling molecule Ras, a powerful oncogene when mutated, promotes glycolysis (reviewed in Dang and Semenza, 1999; Samanathan et al., 2005). Akt kinase, a well-characterized downstream effector of insulin signaling, reprises its role in glucose uptake and utilization in the cancer setting (reviewed in Manning and Cantley, 2007), whereas the Myc transcription factor upregulates the expression of various metabolic genes (reviewed in Gordan et al., 2007). The most parsimonious route to tumorigenesis may be activation of key oncogenic nodes that execute a proliferative program, of which metabolism may be one important arm. Moreover, regulation of metabolism is not exclusive to oncogenes. Loss of the tumor suppressor protein p53 prevents expression of the gene encoding SCO2 (the synthesis of cytochrome c oxidase protein), which interferes with the function of the mitochondrial respiratory chain (Matoba et al., 2006). A second p53 effector, TIGAR (TP53-induced glycolysis and apoptosis regulator), inhibits glycolysis by decreasing levels of fructose-2,6-bisphosphate, a potent stimulator of glycolysis and inhibitor of gluconeogenesis (Bensaad et al., 2006). Other work also suggests that p53-mediated regulation of glucose metabolism may be dependent on the transcription factor NF-κB (Kawauchi et al., 2008).
It has been shown that inhibition of lactate dehydrogenase A (LDH-A) prevents the Warburg effect and forces cancer cells to revert to oxidative phosphorylation in order to reoxidize NADH and produce ATP (Fantin et al., 2006; Shim et al., 1997). While the cells are respiratory competent, they exhibit attenuated tumor growth, suggesting that aerobic glycolysis might be essential for cancer progression. In a primary fibroblast cell culture model of stepwise malignant transformation through overexpression of telomerase, large and small T antigen, and the H-Ras oncogene, increasing tumorigenicity correlates with sensitivity to glycolytic inhibition. This finding suggests that the Warburg effect might be inherent to the molecular events of transformation (Ramanathan et al., 2005). However, the introduction of similar defined factors into human mesenchymal stem cells (MSCs) revealed that transformation can be associated with increased dependence on oxidative phosphorylation (Funes et al., 2007). Interestingly, when introduced in vivo these transformed MSCs do upregulate glycolytic genes, an effect that is reversed when the cells are explanted and cultured under normoxic conditions. These contrasting models suggest that the Warburg effect may be context dependent, in some cases driven by genetic changes and in others by the demands of the microenvironment. Regardless of whether the tumor microenvironment or oncogene activation plays a more important role in driving the development of a distinct cancer metabolism, it is likely that the resulting alterations confer adaptive, proliferative, and survival advantages on the cancer cell.

Altered Metabolism Provides Substrates for Biosynthetic Pathways

Although studies in cancer metabolism have largely been energy-centric, rapidly dividing cells have diverse requirements. Proliferating cells require not only ATP but also nucleotides, fatty acids, membrane lipids, and proteins, and a reprogrammed metabolism may serve to support synthesis of macromolecules. Recent studies have shown that several steps in lipid synthesis are required for and may even actively promote tumorigenesis. Inhibition of ATP citrate lyase, the distal enzyme that converts mitochondrial-derived citrate into cytosolic acetyl coenzyme A, the precursor for many lipid species, prevents cancer cell proliferation and tumor growth (Hatzivassiliou et al., 2005). Fatty acid synthase, expressed at low levels in normal tissues, is upregulated in cancer and may also be required for tumorigenesis (reviewed in Menendez and Lupu, 2007). Furthermore, cancer cells may also enhance their biosynthetic capabilities by expressing a tumor-specific form of pyruvate kinase (PK), M2-PK. Pyruvate kinase catalyzes the third irreversible reaction of glycolysis, the conversion of phosphoenolpyruvate (PEP) to pyruvate. Surprisingly, the M2-PK of cancer cells is thought to be less active in the conversion of PEP to pyruvate and thus less efficient at ATP production (reviewed in Mazurek et al., 2005). A major advantage to the cancer cell, however, is that the glycolytic intermediates upstream of PEP might be shunted into synthetic processes.

Biosynthesis, in addition to causing an inherent increase in ATP demand in order to execute synthetic reactions, should also cause a decrease in ATP supply as various glycolytic and Krebs cycle intermediates are diverted. Lipid synthesis, for example, requires the cooperation of glycolysis, the Krebs cycle, and the pentose phosphate shunt. As pyruvate must enter the mitochondria in this case, it avoids conversion to lactate and therefore cannot contribute to glycolysis-derived ATP. Moreover, whereas increased biosynthesis may explain the glucose hunger of cancer cells, it cannot explain the increase in lactic acid production originally described by Warburg, suggesting that lactate must also result from the metabolism of non-glucose substrates. Recently, it has been demonstrated that glutamine may be metabolized by the citric acid cycle in cancer cells and converted into lactate, producing NADPH for lipid biosynthesis and oxaloacetate for replenishment of Krebs cycle intermediates (DeBerardinis et al., 2007).

Metabolic Pathways Regulate Apoptosis

In addition to involvement in proliferation, altered metabolism may promote another cancer-essential function: the avoidance of apoptosis. Loss of the p53 target TIGAR sensitizes cancer cells to apoptosis, most likely by causing an increase in reactive oxygen species (Bensaad et al., 2006). On the other hand, overexpression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) prevents caspase-independent cell death, presumably by stimulating glycolysis, increasing cellular ATP levels, and promoting autophagy (Colell et al., 2007). Whether or not GAPDH plays a physiological role in the regulation of cell death remains to be determined. Intriguingly, Bonnet et al. (2007) have reported that treating cancer cells with dichloroacetate (DCA), a small molecule inhibitor of pyruvate dehydrogenase kinase, has striking effects on their survival and on xenograft tumor growth.

DCA, a currently approved treatment for congenital lactic acidosis, activates oxidative phosphorylation and promotes apoptosis by two mechanisms. First, increased flux through the electron transport chain causes depolarization of the mitochondrial membrane potential (which the authors found to be hyperpolarized specifically in cancer cells) and release of the apoptotic effector cytochrome c. Second, an increase in reactive oxygen species generated by oxidative phosphorylation upregulates the voltage-gated K+ channel, leading to potassium ion efflux and caspase activation. Their work suggests that cancer cells may shift their metabolism to glycolysis in order to prevent cell death and that forcing cancer cells to respire aerobically can counteract this adaptation.

Cancer Cells May Signal Locally in the Tumor Microenvironment

Cancer cells may rewire metabolic pathways to exploit the tumor microenvironment and to support cancer-specific signaling. Without access to the central circulation, it is possible that metabolites can be concentrated locally and reach suprasystemic levels, allowing cancer cells to engage in metabolite-mediated autocrine and paracrine signaling that does not occur in normal tissues. So called androgen-independent prostate cancers may only be independent from exogenous, adrenal-synthesized androgens. Androgen-independent prostate cancer cells still express the androgen receptor and may be capable of autonomously synthesizing their own androgens (Stanbrough et al., 2006).

Metabolism as an Upstream Modulator of Signaling Pathways

Not only is metabolism downstream of oncogenic pathways, but an altered upstream metabolism may affect the activity of signaling pathways that normally sense the state of the cell. Individuals with inherited mutations in succinate dehydrogenase and fumarate hydratase develop highly angiogenic tumors, not unlike those exhibiting loss of the VHL tumor suppressor protein that acts upstream of HIF (reviewed in Kaelin and Ratcliffe, 2008). The mechanism of tumorigenesis in these cancer syndromes is still contentious. However, it has been proposed that loss of succinate dehydrogenase and fumarate hydratase causes an accumulation of succinate or fumarate, respectively, leading to inhibition of the prolyl hydroxylases that mark HIF for VHL-mediated degradation (Isaacs et al., 2005; Pollard et al., 2005; Selak et al., 2005). In this rare case, succinate dehydrogenase and fumarate hydratase are acting as bona fide tumor suppressors.

There are many complex questions to be answered: Is it possible that cancer cells exhibit “metabolite addiction”? Are there unique cancer-specific metabolic pathways, or combinations of pathways, utilized by the cancer cell but not by normal cells? Are different stages of metabolic adaptations required for the cancer cell to progress from the primary tumor stage to invasion to metastasis? How malleable is cancer metabolism?

2.1.2.2 Cancer metabolism. The Warburg effect today

Ferreira LMR
Exp Molec Pathol 2010; 89:372-383.
http://dx.doi.org/10.1016/j.yexmp.2010.08.006

One of the first studies on the energy metabolism of a tumor was carried out, in 1922, in the laboratory of Otto Warburg. He established that cancer cells exhibited a specific metabolic pattern, characterized by a shift from respiration to fermentation, which has been later named the Warburg effect. Considerable work has been done since then, deepening our understanding of the process, with consequences for diagnosis and therapy. This review presents facts and perspectives on the Warburg effect for the 21st century.

Research highlights

► Warburg first established a tumor metabolic pattern in the 1920s. ► Tumors’ increased glucose uptake has been studied since then. ► Cancer bioenergetics’ study provides insights in all its hallmarks. ► New cancer diagnostic and therapeutic techniques focus on cancer metabolism.

Introduction
Contestation to Warburg’s ideas
Glucose’s uptake and intracellular fates
Lactate production and induced acidosis
Hypoxia
Impairment of mitochondrial function
Tumour microenvironment
Proliferating versus cancer cells
More on cancer bioenergetics – integration of metabolism
Perspectives

2.1.2.3 New aspects of the Warburg effect in cancer cell biology

Bensinger SJ, Cristofk HR
Sem Cell Dev Biol 2012; 23:352-361
http://dx.doi.org:/10.1016/j.semcdb.2012.02.003

Altered cellular metabolism is a defining feature of cancer [1]. The best studied metabolic phenotype of cancer is aerobic glycolysis–also known as the Warburg effect–characterized by increased metabolism of glucose to lactate in the presence of sufficient oxygen. Interest in the Warburg effect has escalated in recent years due to the proven utility of FDG-PET for imaging tumors in cancer patients and growing evidence that mutations in oncogenes and tumor suppressor genes directly impact metabolism. The goals of this review are to provide an organized snapshot of the current understanding of regulatory mechanisms important for Warburg effect and its role in tumor biology. Since several reviews have covered aspects of this topic in recent years, we focus on newest contributions to the field and reference other reviews where appropriate.

Highlights

► This review discusses regulatory mechanisms that contribute to the Warburg effect in cancer. ► We list cancers for which FDG-PET has established applications as well as those cancers for which FDG-PET has not been established. ► PKM2 is highlighted as an important integrator of diverse cellular stimuli to modulate metabolic flux and cancer cell proliferation. ► We discuss how cancer metabolism can directly influence gene expression programs. ► Contribution of aerobic glycolysis to the cancer microenvironment and chemotherapeutic resistance/susceptibility is also discussed.

Regulation of the Warburg effect

PKM2 integrates diverse signals to modulate metabolic flux and cell proliferation

PKM2 integrates diverse signals to modulate metabolic flux and cell proliferation

Fig. 1. PKM2 integrates diverse signals to modulate metabolic flux and cell proliferation

Metabolism can directly influence gene expression programs

Metabolism can directly influence gene expression programs

Fig. 2. Metabolism can directly influence gene expression programs. A schematic representation of how metabolism can intrinsically influence epigenetics resulting in durable and heritable gene expression programs in progeny.

2.1.2.4 Choosing between glycolysis and oxidative phosphorylation. A tumor’s dilemma

Jose C, Ballance N, Rossignal R
Biochim Biophys Acta 201; 1807(6): 552-561.
http://dx.doi.org/10.1016/j.bbabio.2010.10.012

A considerable amount of knowledge has been produced during the last five years on the bioenergetics of cancer cells, leading to a better understanding of the regulation of energy metabolism during oncogenesis, or in adverse conditions of energy substrate intermittent deprivation. The general enhancement of the glycolytic machinery in various cancer cell lines is well described and recent analyses give a better view of the changes in mitochondrial oxidative phosphorylation during oncogenesis. While some studies demonstrate a reduction of oxidative phosphorylation (OXPHOS) capacity in different types of cancer cells, other investigations revealed contradictory modifications with the upregulation of OXPHOS components and a larger dependency of cancer cells on oxidative energy substrates for anabolism and energy production. This apparent conflictual picture is explained by differences in tumor size, hypoxia, and the sequence of oncogenes activated. The role of p53, C-MYC, Oct and RAS on the control of mitochondrial respiration and glutamine utilization has been explained recently on artificial models of tumorigenesis. Likewise, the generation of induced pluripotent stem cells from oncogene activation also showed the role of C-MYC and Oct in the regulation of mitochondrial biogenesis and ROS generation. In this review article we put emphasis on the description of various bioenergetic types of tumors, from exclusively glycolytic to mainly OXPHOS, and the modulation of both the metabolic apparatus and the modalities of energy substrate utilization according to tumor stage, serial oncogene activation and associated or not fluctuating microenvironmental substrate conditions. We conclude on the importance of a dynamic view of tumor bioenergetics.

Research Highlights

►The bioenergetics of cancer cells differs from normals. ►Warburg hypothesis is not verified in tumors using mitochondria to synthesize ATP. ►Different oncogenes can either switch on or switch off OXPHOS. ►Bioenergetic profiling is a prerequisite to metabolic therapy. ►Aerobic glycolysis and OXPHOS cooperate during cancer progression.

  1. Cancer cell variable bioenergetics

Cancer cells exhibit profound genetic, bioenergetic and histological differences as compared to their non-transformed counterpart. All these modifications are associated with unlimited cell growth, inhibition of apoptosis and intense anabolism. Transformation from a normal cell to a malignant cancer cell is a multi-step pathogenic process which includes a permanent interaction between cancer gene activation (oncogenes and/or tumor-suppressor genes), metabolic reprogramming and tumor-induced changes in microenvironment. As for the individual genetic mapping of human tumors, their metabolic characterization (metabolic–bioenergetic profiling) has evidenced a cancer cell-type bioenergetic signature which depends on the history of the tumor, as composed by the sequence of oncogenes activated and the confrontation to intermittent changes in oxygen, glucose and amino-acid delivery.

In the last decade, bioenergetic studies have highlighted the variability among cancer types and even inside a cancer type as regards to the mechanisms and the substrates preferentially used for deriving the vital energy. The more popular metabolic remodeling described in tumor cells is an increase in glucose uptake, the enhancement of glycolytic capacity and a high lactate production, along with the absence of respiration despite the presence of high oxygen concentration (Warburg effect) [1]. To explain this abnormal bioenergetic phenotype pioneering hypotheses proposed the impairment of mitochondrial function in rapidly growing cancer cells [2].

Although the increased consumption of glucose by tumor cells was confirmed in vivo by positron emission tomography (PET) using the glucose analog 2-(18F)-fluoro-2-deoxy-d-glucose (FDG), the actual utilization of glycolysis and oxidative phosphorylation (OXPHOS) cannot be evaluated with this technique. Nowadays, Warburg’s “aerobic-glycolysis” hypothesis has been challenged by a growing number of studies showing that mitochondria in tumor cells are not inactive per se but operate at low capacity [3] or, in striking contrast, supply most of the ATP to the cancer cells [4]. Intense glycolysis is effectively not observed in all tumor types. Indeed not all cancer cells grow fast and intense anabolism is not mandatory for all cancer cells. Rapidly growing tumor cells rely more on glycolysis than slowly growing tumor cells. This is why a treatment with bromopyruvate, for example is very efficient only on rapidly growing cells and barely useful to decrease the growth rate of tumor cells when their normal proliferation is slow. Already in 1979, Reitzer and colleagues published an article entitled “Evidence that glutamine, not sugar, is the major energy source for cultured Hela cells”, which demonstrated that oxidative phosphorylation was used preferentially to produce ATP in cervical carcinoma cells [5]. Griguer et al. also identified several glioma cell lines that were highly dependent on mitochondrial OXPHOS pathway to produce ATP [6]. Furthermore, a subclass of glioma cells which utilize glycolysis preferentially (i.e., glycolytic gliomas) can also switch from aerobic glycolysis to OXPHOS under limiting glucose conditions  [7] and [8], as observed in cervical cancer cells, breast carcinoma cells, hepatoma cells and pancreatic cancer cells [9][10] and [11]. This flexibility shows the interplay between glycolysis and OXPHOS to adapt the mechanisms of energy production to microenvironmental changes as well as differences in tumor energy needs or biosynthetic activity. Herst and Berridge also demonstrated that a variety of human and mouse leukemic and tumor cell lines (HL60, HeLa, 143B, and U937) utilize mitochondrial respiration to support their growth [12]. Recently, the measurement of OXPHOS contribution to the cellular ATP supply revealed that mitochondria generate 79% of the cellular ATP in HeLa cells, and that upon hypoxia this contribution is reduced to 30% [4]. Again, metabolic flexibility is used to survive under hypoxia. All these studies demonstrate that mitochondria are efficient to synthesize ATP in a large variety of cancer cells, as reviewed by Moreno-Sanchez [13]. Despite the observed reduction of the mitochondrial content in tumors [3][14][15][16][17][18] and [19], cancer cells maintain a significant level of OXPHOS capacity to rapidly switch from glycolysis to OXPHOS during carcinogenesis. This switch is also observed at the level of glutamine oxidation which can occur through two modes, “OXPHOS-linked” or “anoxic”, allowing to derive energy from glutamine or serine regardless of hypoxia or respiratory chain reduced activity [20].
While glutamine, glycine, alanine, glutamate, and proline are typically oxidized in normal and tumor mitochondria, alternative substrate oxidations may also contribute to ATP supply by OXPHOS. Those include for instance the oxidation of fatty-acids, ketone bodies, short-chain carboxylic acids, propionate, acetate and butyrate (as recently reviewed in [21]).

  1. Varying degree of mitochondrial utilization during tumorigenesis

In vivo metabolomic analyses suggest the existence of a continuum of bioenergetic remodeling in rat tumors according to tumor size and its rate of growth [22]. Peter Vaupel’s group showed that small tumors were characterized by a low conversion of glucose to lactate whereas the conversion of glutamine to lactate was high. In medium sized tumors the flow of glucose to lactate as well as oxygen utilization was increased whereas glutamine and serine consumption were reduced. At this stage tumor cells started with glutamate and alanine production. Large tumors were characterized by a low oxygen and glucose supply but a high glucose and oxygen utilization rate. The conversion of glucose to glycine, alanine, glutamate, glutamine, and proline reached high values and the amino acids were released [22]. Certainly, in the inner layers constituting solid tumors, substrate and oxygen limitation is frequently observed. Experimental studies tried to reproduce these conditions in vitro and revealed that nutrients and oxygen limitation does not affect OXPHOS and cellular ATP levels in human cervix tumor [23]. Furthermore, the growth of HeLa cells, HepG2 cells and HTB126 (breast cancer) in aglycemia and/or hypoxia even triggered a compensatory increase in OXPHOS capacity, as discussed above. Yet, the impact of hypoxia might be variable depending on cell type and both the extent and the duration of oxygen limitation.
In two models of sequential oncogenesis, the successive activation of specific oncogenes in non-cancer cells evidenced the need for active OXPHOS to pursue tumorigenesis. Funes et al. showed that the transformation of human mesenchymal stem cells increases their dependency on OXPHOS for energy production [24], while Ferbeyre et al. showed that cells expressing oncogenic RAS display an increase in mitochondrial mass, mitochondrial DNA, and mitochondrial production of reactive oxygen species (ROS) prior to the senescent cell cycle arrest [25]. Such observations suggest that waves of gene regulation could suppress and then restore OXPHOS in cancer cells during tumorigenesis [20]. Therefore, the definition of cancer by Hanahan and Weinberg [26] restricted to six hallmarks (1—self-sufficiency in growth signals, 2—insensitivity to growth-inhibitory (antigrowth) signals, 3—evasion of programmed cell death (apoptosis), 4—limitless replicative potential, 5—sustained angiogenesis, and 6—tissue invasion and metastases) should also include metabolic reprogramming, as the seventh hallmark of cancer. This amendment was already proposed by Tennant et al. in 2009 [27]. In 2006, the review Science published a debate on the controversial views of Warburg theory [28], in support of a more realistic description of cancer cell’s variable bioenergetic profile. The pros think that high glycolysis is an obligatory feature of human tumors, while the cons propose that high glycolysis is not exclusive and that tumors can use OXPHOS to derive energy. A unifying theory closer to reality might consider that OXPHOS and glycolysis cooperate to sustain energy needs along tumorigenesis [20]. The concept of oxidative tumors, against Warburg’s proposal, was introduced by Guppy and colleagues, based on the observation that breast cancer cells can generate 80% of their ATP by the mitochondrion [29]. The comparison of different cancer cell lines and excised tumors revealed a variety of cancer cell’s bioenergetic signatures which raised the question of the mechanisms underlying tumor cell metabolic reprogramming, and the relative contribution of oncogenesis and microenvironment in this process. It is now widely accepted that rapidly growing cancer cells within solid tumors suffer from a lack of oxygen and nutrients as tumor grows. In such situation of compromised energy substrate delivery, cancer cell’s metabolic reprogramming is further used to sustain anabolism (Fig. 1), through the deviation of glycolysis, Krebs cycle truncation and OXPHOS redirection toward lipid and protein synthesis, as needed to support uncontrolled tumor growth and survival [30] and [31]. Again, these features are not exclusive to all tumors, as Krebs cycle truncation was only observed in some cancer cells, while other studies indicated that tumor cells can maintain a complete Krebs cycle [13] in parallel with an active citrate efflux. Likewise, generalizations should be avoided to prevent over-interpretations.
Fig. 1. Energy metabolism at the crossroad between catabolism and anabolism.

Energy metabolism at the crossroad between catabolism and anabolism.

Energy metabolism at the crossroad between catabolism and anabolism.

The oncogene C-MYC participate to these changes via the stimulation of glutamine utilization through the coordinate expression of genes necessary for cells to engage in glutamine catabolism [30]. According to Newsholme EA and Board M [32] both glycolysis and glutaminolysis not only serve for ATP production, but also provide precious metabolic intermediates such as glucose-6-phosphate, ammonia and aspartate required for the synthesis of purine and pyrimidine nucleotides (Fig. 1). In this manner, the observed apparent excess in the rates of glycolysis and glutaminolysis as compared to the requirement for energy production could be explained by the need for biosynthetic processes. Yet, one should not reduce the shift from glycolysis to OXPHOS utilization to the sole activation of glutaminolysis, as several other energy substrates can be used by tumor mitochondria to generate ATP [21]. The contribution of these different fuels to ATP synthesis remains poorly investigated in human tumors.

  1. The metabolism of pre-cancer cells and its ongoing modulation by carcinogenesis

At the beginning of cancer, there might have been a cancer stem cell hit by an oncogenic event, such as alterations in mitogen signaling to extracellular growth factor receptors (EGFR), oncogenic activation of these receptors, or oncogenic alterations of downstream targets in the pathways that leads to cell proliferation (RAS–Raf–ERK and PI3K–AKT, both leading to m-TOR activation stimulating cell growth). Alterations of checkpoint genes controlling the cell cycle progression like Rb also participate in cell proliferation (Fig. 2) and this re-entry in the cell cycle implies three major needs to fill in: 1) supplying enough energy to grow and 2) synthesize building blocks de novo and 3) keep vital oxygen and nutrients available. However, the bioenergetic status of the pre-cancer cell could determine in part the evolution of carcinogenesis, as shown on mouse embryonic stem cells. In this study, Schieke et al. showed that mitochondrial energy metabolism modulates both the differentiation and tumor formation capacity of mouse embryonic stem cells [37]. The idea that cancer derives from a single cell, known as the cancer stem cell hypothesis, was introduced by observations performed on leukemia which appeared to be organized as origination from a primitive hematopoietic cell [38]. Nowadays cancer stem cells were discovered for all types of tumors [39][40][41] and [42], but little is known of their bioenergetic properties and their metabolic adaptation to the microenvironment. This question is crucial as regards the understanding of what determines the wide variety of cancer cell’s metabolic profile.

Impact of different oncogenes on tumor progression and energy metabolism remodeling.

Impact of different oncogenes on tumor progression and energy metabolism remodeling.

Fig. 2. Impact of different oncogenes on tumor progression and energy metabolism remodeling.

The analysis of the metabolic changes that occur during the transformation of adult mesenchymal stem cells revealed that these cells did not switch to aerobic glycolysis, but their dependency on OXPHOS was even increased [24]. Hence, mitochondrial energy metabolism could be critical for tumorigenesis, in contrast with Warburg’s hypothesis. As discussed above, the oncogene C-MYC also stimulates OXPHOS [30]. Furthermore, it was recently demonstrated that cells chronically treated with oligomycin repress OXPHOS and produce larger tumors with higher malignancy [19]. Likewise, alteration of OXPHOS by mutations in mtDNA increases tumorigenicity in different types of cancer cells [43][44] and [45].

Recently, it was proposed that mitochondrial energy metabolism is required to generate reactive oxygen species used for the carcinogenetic process induced by the K-RAS mutation [46]. This could explain the large number of mitochondrial DNA mutations found in several tumors. The analysis of mitochondria in human embryonic cells which derive energy exclusively from anaerobic glycolysis have demonstrated an immature mitochondrial network characterized by few organelles with poorly developed cristae and peri-nuclear distribution [47] and [48]. The generation of human induced pluripotent stem cell by the introduction of different oncogenes as C-MYC and Oct4 reproduced this reduction of mitochondrial OXPHOS capacity[49] and [50]. This indicates again the impact of oncogenes on the control of OXPHOS and might explain the existence of pre-cancer stem cells with different bioenergetic backgrounds, as modeled by variable sequences of oncogene activation. Accordingly, the inhibition of mitochondrial respiratory chain has been recently found associated with enhancement of hESC pluripotency [51].

Based on the experimental evidence discussed above, one can argue that 1) glycolysis is indeed a feature of several tumors and associates with faster growth in high glucose environment, but 2) active OXPHOS is also an important feature of (other) tumors taken at a particular stage of carcinogenesis which might be more advantageous than a “glycolysis-only” type of metabolism in conditions of intermittent shortage in glucose delivery. The metabolic apparatus of cancer cells is not fixed during carcinogenesis and might depend both on the nature of the oncogenes activated and the microenvironment. It was indeed shown that cancer cells with predominant glycolytic metabolism present a higher malignancy when submitted to carcinogenetic induction and analysed under fixed experimental conditions of high glucose [19]. Yet, if one grows these cells in a glucose-deprived medium they shift their metabolism toward predominant OXPHOS, as shown in HeLa cells and other cell types [9]. Therefore, one might conclude that glycolytic cells have a higher propensity to generate aggressive tumors when glucose availability is high. However, these cells can become OXPHOS during tumor progression [24] and [52]. All these observations indicate again the importance of maintaining an active OXPHOS metabolism to permit evolution of both embryogenesis and carcinogenesis, which emphasizes the importance of targeting mitochondria to alter this malignant process.

  1. Oncogenes and the modulation of energy metabolism

Several oncogenes and associated proteins such as HIF-1α, RAS, C-MYC, SRC, and p53 can influence energy substrate utilization by affecting cellular targets, leading to metabolic changes that favor cancer cell survival, independently of the control of cell proliferation. These oncogenes stimulate the enhancement of aerobic glycolysis, and an increasing number of studies demonstrate that at least some of them can also target directly the OXPHOS machinery, as discussed in this article (Fig. 2). For instance, C-MYC can concurrently drive aerobic glycolysis and/or OXPHOS according to the tumor cell microenvironment, via the expression of glycolytic genes or the activation of mitochondrial oxidation of glutamine [53]. The oncogene RAS has been shown to increase OXPHOS activity in early transformed cells [24][52] and [54] and p53 modulates OXPHOS capacity via the regulation of cytochrome c oxidase assembly [55]. Hence, carcinogenic p53 deficiency results in a decreased level of COX2 and triggers a shift toward anaerobic metabolism. In this case, lactate synthesis is increased, but cellular ATP levels remain stable [56]. The p53-inducible isoform of phosphofructokinase, termed TP53-induced glycolysis and apoptotic regulator, TIGAR, a predominant phosphatase activity isoform of PFK-2, has also been identified as an important regulator of energy metabolism in tumors [57].

  1. Tumor specific isoforms (or mutated forms) of energy genes

Tumors are generally characterized by a modification of the glycolytic system where the level of some glycolytic enzymes is increased, some fetal-like isozymes with different kinetic and regulatory properties are produced, and the reverse and back-reactions of the glycolysis are strongly reduced [60]. The GAPDH marker of the glycolytic pathway is also increased in breast, gastric, lung, kidney and colon tumors [18], and the expression of glucose transporter GLUT1 is elevated in most cancer cells. The group of Cuezva J.M. developed the concept of cancer bioenergetic signature and of bioenergetic index to describe the metabolic profile of cancer cells and tumors [18], [61], [64], [65]. This signature describes the changes in the expression level of proteins involved in glycolysis and OXPHOS, while the BEC index gives a ratio of OXPHOS protein content to glycolytic protein content, in good correlation with cancer prognostic[61]. Recently, this group showed that the beta-subunit of the mitochondrial F1F0-ATP synthase is downregulated in a large number of tumors, thus contributing to the Warburg effect [64] and [65]. It was also shown that IF1 expression levels were increased in hepatocellular carcinomas, possibly to prevent the hydrolysis of glytolytic ATP [66]. Numerous changes occur at the level of OXPHOS and mitochondrial biogenesis in human tumors, as we reviewed previously [67]. Yet the actual impact of these changes in OXPHOS protein expression level or catalytic activities remains to be evaluated on the overall fluxes of respiration and ATP synthesis. Indeed, the metabolic control analysis and its extension indicate that it is often required to inhibit activity beyond a threshold of 70–85% to affect the metabolic fluxes [68] and [69]. Another important feature of cancer cells is the higher level of hexokinase II bound to mitochondrial membrane (50% in tumor cells). A study performed on human gliomas (brain) estimated the mitochondrial bound HK fraction (mHK) at 69% of total, as compared to 9% for normal brain [70]. This is consistent with the 5-fold amplification of the type II HK gene observed by Rempel et al. in the rapidly growing rat AS-30D hepatoma cell line, relative to normal hepatocytes [71]. HKII subcellular fractionation in cancer cells was described in several studies [72][73] and [74]. The group led by Pete Pedersen explained that mHK contributes to (i) the high glycolytic capacity by utilizing mitochondrially regenerated ATP rather than cytosolic ATP (nucleotide channelling) and (ii) the lowering of OXPHOS capacity by limiting Pi and ADP delivery to the organelle [75] and [76].

All these observations are consistent with the increased rate of FDG uptake observed by PET in living tumors which could result from both an increase in glucose transport, and/or an increase in hexokinase activity. However, FDG is not a complete substrate for glycolysis (it is only transformed into FDG-6P by hexokinase before to be eliminated) and cannot be used to evidence a general increase in the glycolytic flux. Moreover, FDG-PET scan also gives false positive and false negative results, indicating that some tumors do not depend on, or do not have, an increased glycolytic capacity. The fast glycolytic system described above is further accommodated in cancer cells by an increase in the lactate dehydrogenase isoform A (LDH-A) expression level. This isoform presents a higher Vmax useful to prevent the inhibition of high glycolysis by its end product (pyruvate) accumulation. Recently, Fantin et al. showed that inhibition of LDH-A in tumors diminishes tumorigenicity and was associated with the stimulation of mitochondrial respiration [79]. The preferential expression of the glycolytic pyruvate kinase isoenzyme M2 (PKM2) in tumor cells, determines whether glucose is converted to lactate for regeneration of energy (active tetrameric form, Warburg effect) or used for the synthesis of cell building blocks (nearly inactive dimeric form) [80]. In the last five years, mutations in proteins of the respiratory system (SDH, FH) and of the TCA cycle (IDH1,2) leading to the accumulation of metabolite and the subsequent activation of HIF-1α were reported in a variety of human tumors [81], [82] and [83].

  1. Tumor microenvironment modulates cancer cell’s bioenergetics

It was extensively described how hypoxia activates HIF-1α which stimulates in turn the expression of several glycolytic enzymes such as HK2, PFK, PGM, enolase, PK, LDH-A, MCT4 and glucose transporters Glut 1 and Glut 3. It was also shown that HIF-1α can reduce OXPHOS capacity by inhibiting mitochondrial biogenesis [14] and [15], PDH activity [87] and respiratory chain activity [88]. The low efficiency and uneven distribution of the vascular system surrounding solid tumors can lead to abrupt changes in oxygen (intermittent hypoxia) but also energy substrate delivery. .. The removal of glucose, or the inhibition of glycolysis by iodoacetate led to a switch toward glutamine utilization without delay followed by a rapid decrease in acid release. This illustrates once again how tumors and human cancer cell lines can utilize alternative energy pathway such as glutaminolysis to deal with glucose limitation, provided the presence of oxygen. It was also observed that in situations of glucose limitation, tumor derived-cells can adapt to survive by using exclusively an oxidative energy substrate [9] and [10]. This is typically associated with an enhancement of the OXPHOS system. … In summary, cancer cells can survive by using exclusively OXPHOS for ATP production, by altering significantly mitochondrial composition and form to facilitate optimal use of the available substrate (Fig. 3). Yet, glucose is needed to feed the pentose phosphate pathway and generate ribose essential for nucleotide biosynthesis. This raises the question of how cancer cells can survive in the growth medium which do not contain glucose (so-called “galactose medium” with dialysed serum [9]). In the OXPHOS mode, pyruvate, glutamate and aspartate can be derived from glutamine, as glutaminolysis can replenish Krebs cycle metabolic pool and support the synthesis of alanine and NADPH [31]. Glutamine is a major source for oxaloacetate (OAA) essential for citrate synthesis. Moreover, the conversion of glutamine to pyruvate is associated with the reduction of NADP+ to NADPH by malic enzyme. Such NADPH is a required electron donor for reductive steps in lipid synthesis, nucleotide metabolism and GSH reduction. In glioblastoma cells the malic enzyme flux was estimated to be high enough to supply all of the reductive power needed for lipid synthesis [31].

Fig. 3. Interplay between energy metabolism, oncogenes and tumor microenvironment during tumorigenesis (the “metabolic wave model”).

Interplay between energy metabolism, oncogenes and tumor microenvironment

Interplay between energy metabolism, oncogenes and tumor microenvironment

While the mechanisms leading to the enhancement of glycolytic capacity in tumors are well documented, less is known about the parallel OXPHOS changes. Both phenomena could result from a selection of pre-malignant cells forced to survive under hypoxia and limited glucose delivery, followed by an adaptation to intermittent hypoxia, pseudo-hypoxia, substrate limitation and acidic environment. This hypothesis was first proposed by Gatenby and Gillies to explain the high glycolytic phenotype of tumors [91], [92] and [93], but several lines of evidence suggest that it could also be used to explain the mitochondrial modifications observed in cancer cells.

  1. Aerobic glycolysis and mitochondria cooperate during cancer progression

Metabolic flexibility considers the possibility for a given cell to alternate between glycolysis and OXPHOS in response to physiological needs. Louis Pasteur found that in most mammalian cells the rate of glycolysis decreases significantly in the presence of oxygen (Pasteur effect). Moreover, energy metabolism of normal cell can vary widely according to the tissue of origin, as we showed with the comparison of five rat tissues[94]. During stem cell differentiation, cell proliferation induces a switch from OXPHOS to aerobic glycolysis which might generate ATP more rapidly, as demonstrated in HepG2 cells [95] or in non-cancer cells[96] and [97]. Thus, normal cellular energy metabolism can adapt widely according to the activity of the cell and its surrounding microenvironment (energy substrate availability and diversity). Support for this view came from numerous studies showing that in vitro growth conditions can alter energy metabolism contributing to a dependency on glycolysis for ATP production [98].

Yet, Zu and Guppy analysed numerous studies and showed that aerobic glycolysis is not inherent to cancer but more a consequence of hypoxia[99].

Table 1. Impact of different oncogenes on energy metabolism

Impact of different oncogenes on energy metabolism.

Impact of different oncogenes on energy metabolism.

2.1.2.5 Mitohormesis

Yun J, Finkel T
Cell Metab May 2014; 19(5):757–766
http://dx.doi.org/10.1016/j.cmet.2014.01.011

For many years, mitochondria were viewed as semiautonomous organelles, required only for cellular energetics. This view has been largely supplanted by the concept that mitochondria are fully integrated into the cell and that mitochondrial stresses rapidly activate cytosolic signaling pathways that ultimately alter nuclear gene expression. Remarkably, this coordinated response to mild mitochondrial stress appears to leave the cell less susceptible to subsequent perturbations. This response, termed mitohormesis, is being rapidly dissected in many model organisms. A fuller understanding of mitohormesis promises to provide insight into our susceptibility for disease and potentially provide a unifying hypothesis for why we age.

Figure 1. The Basis of Mitohormesis. Any of a number of endogenous or exogenous stresses can perturb mitochondrial function. These perturbations are relayed to the cytosol through, at present, poorly understood mechanisms that may involve mitochondrial ROS as well as other mediators. These cytoplasmic signaling pathways and subsequent nuclear transcriptional changes induce various long-lasting cytoprotective pathways. This augmented stress resistance allows for protection from a wide array of subsequent stresses.

Figure 2. Potential Parallels between the Mitochondrial Unfolded Protein Response and Quorum Sensing in Gram-Positive Bacteria. In the C. elegans UPRmt response, mitochondrial proteins (indicated by blue swirls) are degraded by matrix proteases, and the oligopeptides that are generated are then exported through the ABC transporter family member HAF-1. Once in the cytosol, these peptides can influence the subcellular localization of the transcription factor ATFS-1. Nuclear ATFS-1 is capable of orchestrating a broad transcriptional response to mitochondrial stress. As such, this pathway establishes a method for mitochondrial and nuclear genomes to communicate. In some gram-positive bacteria, intracellularly generated peptides can be similarly exported through an ABC transporter protein. These peptides can be detected in the environment by a membrane-bound histidine kinases (HK) sensor. The activation of the HK sensor leads to phosphorylation of a response regulator (RR) protein that, in turn, can alter gene expression. This program allows communication between dispersed gram-positive bacteria and thus coordinated behavior of widely dispersed bacterial genomes.

Figure 3. The Complexity of Mitochondrial Stresses and Responses. A wide array of extrinsic and intrinsic mitochondrial perturbations can elicit cellular responses. As detailed in the text, genetic or pharmacological disruption of electron transport, incorrect folding of mitochondrial proteins, stalled mitochondrial ribosomes, alterations in signaling pathways, or exposure to toxins all appear to elicit specific cytoprotective programs within the cell. These adaptive responses include increased mitochondrial number (biogenesis), alterations in metabolism, increased antioxidant defenses, and augmented protein chaperone expression. The cumulative effect of these adaptive mechanisms might be an extension of lifespan and a decreased incidence of age-related pathologies.

2.1.2.6 Mitochondrial function and energy metabolism in cancer cells. Past overview and future perspectives

Mayevsky A
Mitochondrion. 2009 Jun; 9(3):165-79
http://dx.doi.org:/10.1016/j.mito.2009.01.009

The involvements of energy metabolism aspects of mitochondrial dysfunction in cancer development, proliferation and possible therapy, have been investigated since Otto Warburg published his hypothesis. The main published material on cancer cell energy metabolism is overviewed and a new unique in vivo experimental approach that may have significant impact in this important field is suggested. The monitoring system provides real time data, reflecting mitochondrial NADH redox state and microcirculation function. This approach of in vivo monitoring of tissue viability could be used to test the efficacy and side effects of new anticancer drugs in animal models. Also, the same technology may enable differentiation between normal and tumor tissues in experimental animals and maybe also in patients.

 Energy metabolism in mammalian cells

Fig. 1. Schematic representation of cellular energy metabolism and its relationship to microcirculatory blood flow and hemoglobin oxygenation.

Fig. 2. Schematic representation of the central role of the mitochondrion in the various processes involved in the pathology of cancer cells and tumors. Six issues marked as 1–6 are discussed in details in the text.

In vivo monitoring of tissue energy metabolism in mammalian cells

Fig. 3. Schematic presentation of the six parameters that could be monitored for the evaluation of tissue energy metabolism (see text for details).

Optical spectroscopy of tissue energy metabolism in vivo

Multiparametric monitoring system

Fig. 4. (A) Schematic representation of the Time Sharing Fluorometer Reflectometer (TSFR) combined with the laser Doppler flowmeter (D) for blood flow monitoring. The time sharing system includes a wheel that rotates at a speed of3000 rpm wit height filters: four for the measurements of mitochondrial NADH(366 nm and 450 nm)and four for oxy-hemoglobin measurements (585 nm and 577 nm) as seen in (C). The source of light is a mercury lamp. The probe includes optical fibers for NADH excitation (Ex) and emission (Em), laser Doppler excitation (LD in), laser Doppler emission (LD out) as seen in part E The absorption spectrum of Oxy- and Deoxy- Hemoglobin indicating the two wave length used (C).

Fig. 7. Comparison between mitochondrial metabolic states in vitro and the typical tissue metabolic states in vivo evaluated by NADH redox state, tissue blood flow and hemoglobin oxygenation as could be measured by the suggested monitoring system.

(very important)

2.1.2.7 Metabolic Reprogramming. Cancer Hallmark Even Warburg Did Not Anticipate

Ward PS, Thompson CB.
Cancer Cell 2012; 21(3):297-308
http://dx.doi.org/10.1016/j.ccr.2012.02.014

Cancer metabolism has long been equated with aerobic glycolysis, seen by early biochemists as primitive and inefficient. Despite these early beliefs, the metabolic signatures of cancer cells are not passive responses to damaged mitochondria but result from oncogene-directed metabolic reprogramming required to support anabolic growth. Recent evidence suggests that metabolites themselves can be oncogenic by altering cell signaling and blocking cellular differentiation. No longer can cancer-associated alterations in metabolism be viewed as an indirect response to cell proliferation and survival signals. We contend that altered metabolism has attained the status of a core hallmark of cancer.

The propensity for proliferating cells to secrete a significant fraction of glucose carbon through fermentation was first elucidated in yeast. Otto Warburg extended these observations to mammalian cells, finding that proliferating ascites tumor cells converted the majority of their glucose carbon to lactate, even in oxygen-rich conditions. Warburg hypothesized that this altered metabolism was specific to cancer cells, and that it arose from mitochondrial defects that inhibited their ability to effectively oxidize glucose carbon to CO2. An extension of this hypothesis was that dysfunctional mitochondria caused cancer (Koppenol et al., 2011). Warburg’s seminal finding has been observed in a wide variety of cancers. These observations have been exploited clinically using 18F-deoxyglucose positron emission tomography (FDG-PET). However, in contrast to Warburg’s original hypothesis, damaged mitochondria are not at the root of the aerobic glycolysis exhibited by most tumor cells. Most tumor mitochondria are not defective in their ability to carry out oxidative phosphorylation. Instead, in proliferating cells mitochondrial metabolism is reprogrammed to meet the challenges of macromolecular synthesis. This possibility was never considered by Warburg and his contemporaries.

Advances in cancer metabolism research over the last decade have enhanced our understanding of how aerobic glycolysis and other metabolic alterations observed in cancer cells support the anabolic requirements associated with cell growth and proliferation. It has become clear that anabolic metabolism is under complex regulatory control directed by growth factor signal transduction in non-transformed cells. Yet despite these advances, the repeated refrain from traditional biochemists is that altered metabolism is merely an indirect phenomenon in cancer, a secondary effect that pales in importance to the activation of primary proliferation and survival signals (Hanahan and Weinberg, 2011). Most proto-oncogenes and tumor suppressor genes encode components of signal transduction pathways. Their roles in carcinogenesis have traditionally been attributed to their ability to regulate the cell cycle and sustain proliferative signaling while also helping cells evade growth suppression and/or cell death (Hanahan and Weinberg, 2011). But evidence for an alternative concept, that the primary functions of activated oncogenes and inactivated tumor suppressors are to reprogram cellular metabolism, has continued to build over the past several years. Evidence is also developing for the proposal that proto-oncogenes and tumor suppressors primarily evolved to regulate metabolism.

We begin this review by discussing how proliferative cell metabolism differs from quiescent cell metabolism on the basis of active metabolic reprogramming by oncogenes and tumor suppressors. Much of this reprogramming depends on utilizing mitochondria as functional biosynthetic organelles. We then further develop the idea that altered metabolism is a primary feature selected for during tumorigenesis. Recent advances have demonstrated that altered metabolism in cancer extends beyond adaptations to meet the increased anabolic requirements of a growing and dividing cell. Changes in cancer cell metabolism can also influence cellular differentiation status, and in some cases these changes arise from oncogenic alterations in metabolic enzymes themselves.

Metabolism in quiescent vs. proliferating cells nihms-360138-f0001

Metabolism in quiescent vs. proliferating cells: both use mitochondria.
(A) In the absence of instructional growth factor signaling, cells in multicellular organisms lack the ability to take up sufficient nutrients to maintain themselves. Neglected cells will undergo autophagy and catabolize amino acids and lipids through the TCA cycle, assuming sufficient oxygen is available. This oxidative metabolism maximizes ATP production. (B) Cells that receive instructional growth factor signaling are directed to increase their uptake of nutrients, most notably glucose and glutamine. The increased nutrient uptake can then support the anabolic requirements of cell growth: mainly lipid, protein, and nucleotide synthesis (biomass). Excess carbon is secreted as lactate. Proliferating cells may also use strategies to decrease their ATP production while increasing their ATP consumption. These strategies maintain the ADP:ATP ratio necessary to maintain glycolytic flux. Green arrows represent metabolic pathways, while black arrows represent signaling.

Metabolism is a direct, not indirect, response to growth factor signaling nihms-360138-f0002

Metabolism is a direct, not indirect, response to growth factor signaling nihms-360138-f0002

Metabolism is a direct, not indirect, response to growth factor signaling.
(A) The traditional demand-based model of how metabolism is altered in proliferating cells. In response to growth factor signaling, increased transcription and translation consume free energy and decrease the ADP:ATP ratio. This leads to enhanced flux of glucose carbon through glycolysis and the TCA cycle for the purpose of producing more ATP. (B) Supply-based model of how metabolism changes in proliferating cells. Growth factor signaling directly reprograms nutrient uptake and metabolism. Increased nutrient flux through glycolysis and the mitochondria in response to growth factor signaling is used for biomass production. Metabolism also impacts transcription and translation through mechanisms independent of ATP availability.

Alterations in classic oncogenes directly reprogram cell metabolism to increase nutrient uptake and biosynthesis. PI3K/Akt signaling downstream of receptor tyrosine kinase (RTK) activation increases glucose uptake through the transporter GLUT1, and increases flux through glycolysis. Branches of glycolytic metabolism contribute to nucleotide and amino acid synthesis. Akt also activates ATP-citrate lyase (ACL), promoting the conversion of mitochondria-derived citrate to acetyl-CoA for lipid synthesis. Mitochondrial citrate can be synthesized when glucose-derived acetyl-CoA, generated by pyruvate dehydrogenase (PDH), condenses with glutamine-derived oxaloacetate (OAA) via the activity of citrate synthase (CS). mTORC1 promotes protein synthesis and mitochondrial metabolism. Myc increases glutamine uptake and the conversion of glutamine into a mitochondrial carbon source by promoting the expression of the enzyme glutaminase (GLS). Myc also promotes mitochondrial biogenesis. In addition, Myc promotes nucleotide and amino acid synthesis, both through direct transcriptional regulation and through increasing the synthesis of mitochondrial metabolite precursors.

Pyruvate kinase M2 (PKM2) expression in proliferating cells is regulated by signaling and mitochondrial metabolism to facilitate macromolecular synthesis. PKM2 is a less active isoform of the terminal glycolytic enzyme pyruvate kinase. It is also uniquely inhibited downstream of tyrosine kinase signaling. The decreased enzymatic activity of PKM2 in the cytoplasm promotes the accumulation of upstream glycolytic intermediates and their shunting into anabolic pathways. These pathways include the serine synthetic pathway that contributes to nucleotide and amino acid production. When mitochondrial metabolism is excessive, reactive oxygen species (ROS) from the mitochondria can feedback to inhibit PKM2 activity. Acetylation of PKM2, dependent on acetyl-CoA availability, may also promote PKM2 degradation and further contribute to increased flux through anabolic synthesis pathways branching off glycolysis.

IDH1 and IDH2 mutants convert glutamine carbon to the oncometabolite 2-hydroxyglutarate to dysregulate epigenetics and cell differentiation. (A) α-ketoglutarate, produced in part by wild-type isocitrate dehydrogenase (IDH), can enter the nucleus and be used as a substrate for dioxygenase enzymes that modify epigenetic marks. These enzymes include the TET2 DNA hydroxylase enzyme which converts 5-methylcytosine to 5-hydroxymethylcytosine, typically at CpG dinucleotides. 5-hydroxymethylcytosine may be an intermediate in either active or passive DNA demethylation. α-ketoglutarate is also a substrate for JmjC domain histone demethylase enzymes that demethylate lysine residues on histone tails. (B) The common feature of cancer-associated mutations in cytosolic IDH1 and mitochondrial IDH2 is the acquisition of a neomorphic enzymatic activity. This activity converts glutamine-derived α-ketoglutarate to the oncometabolite 2HG. 2HG can competitively inhibit α-ketoglutarate-dependent enzymes like TET2 and the JmjC histone demethylases, thereby impairing normal epigenetic regulation. This results in altered histone methylation marks, in some cases DNA hypermethylation at CpG islands, and dysregulated cellular differentiation.

Hypoxia and HIF-1 activation promote an alternative pathway for citrate synthesis through reductive metabolism of glutamine. (A) In proliferating cells under normoxic conditions, citrate is synthesized from both glucose and glutamine. Glucose carbon provides acetyl-CoA through the activity of PDH. Glutamine carbon provides oxaloacetate through oxidative mitochondrial metabolism dependent on NAD+. Glucose-derived acetyl-CoA and glutamine-derived oxaloacetate condense to form citrate via the activity of citrate synthase (CS). Citrate can be exported to the cytosol for lipid synthesis. (B) In cells proliferating in hypoxia and/or with HIF-1 activation, glucose is diverted away from mitochondrial acetyl-CoA and citrate production. Citrate can be maintained through an alternative pathway of reductive carboxylation, which we propose to rely on reverse flux of glutamine-derived α-ketoglutarate through IDH2. This reverse flux in the mitochondria would promote electron export from the mitochondria when the activity of the electron transport chain is inhibited because of the lack of oxygen as an electron acceptor. Mitochondrial reverse flux can be accomplished by NADH conversion to NADPH by mitochondrial transhydrogenase and the resulting NADPH use in α-ketoglutarate carboxylation. When citrate/isocitrate is exported to the cytosol, some may be metabolized in the oxidative direction by IDH1 and contribute to a shuttle that produces cytosolic NADPH.

A major paradox remaining with PKM2 is that cells expressing PKM2 produce more glucose-derived pyruvate than PKM1-expressing cells, despite having a form of the pyruvate kinase enzyme that is less active and more sensitive to inhibition. One way to get around the PKM2 bottleneck and maintain/enhance pyruvate production may be through an proposed alternative glycolytic pathway, involving an enzymatic activity not yet purified, that dephosphorylates PEP to pyruvate without the generation of ATP (Vander Heiden et al., 2010). Another answer to this paradox may emanate from the serine synthetic pathway. The decreased enzymatic activity of PKM2 can promote the accumulation of the 3-phosphoglycerate glycolytic intermediate that serves as the entry point for the serine synthetic pathway branch off glycolysis. The little studied enzyme serine dehydratase can then directly convert serine to pyruvate. A third explanation may lie in the oscillatory activity of PKM2 from the inactive dimer to active tetramer form. Regulatory inputs into PKM2 like tyrosine phosphorylation and ROS destabilize the tetrameric form of PKM2 (Anastasiou et al., 2011; Christofk et al., 2008b; Hitosugi et al., 2009), but other inputs present in glycolytic cancer cells like fructose-1,6-bisphosphate and serine can continually allosterically activate and/or promote reformation of the PKM2 tetramer (Ashizawa et al., 1991; Eigenbrodt et al., 1983). Thus, PKM2 may be continually switching from inactive to active forms in cells, resulting in an apparent upregulation of flux through anabolic glycolytic branching pathways while also maintaining reasonable net flux of glucose carbon through PEP to pyruvate. With such an oscillatory system, small changes in the levels of any of the above-mentioned PKM2 regulatory inputs can cause exquisite, rapid, adjustments to glycolytic flux. This would be predicted to be advantageous for proliferating cells in the setting of variable extracellular nutrient availability. The capability for oscillatory regulation of PKM2 could also provide an explanation for why tumor cells do not select for altered glycolytic metabolism upstream of PKM2 through deletions and/or loss of function mutations of other glycolytic enzymes.

IDH1 mutations at R132 are not simply loss-of-function for isocitrate and α-ketoglutarate interconversion, but also acquire a novel reductive activity to convert α-ketoglutarate to 2-hydroxyglutarate (2HG), a rare metabolite found at only trace amounts in mammalian cells under normal conditions (Dang et al., 2009). However, it still remained unclear if 2HG was truly a pathogenic “oncometabolite” resulting from IDH1 mutation, or if it was just the byproduct of a loss of function mutation. Whether 2HG production or the loss of IDH1 normal function played a more important role in tumorigenesis remained uncertain.

A potential answer to whether 2HG production was relevant to tumorigenesis arrived with the study of mutations in IDH2, the mitochondrial homolog of IDH1. Up to this point a small fraction of gliomas lacking IDH1 mutations were known to harbor mutations at IDH2 R172, the analogous residue to IDH1 R132 (Yan et al., 2009). However, given the rarity of these IDH2 mutations, they had not been characterized for 2HG production. The discovery of IDH2 R172 mutations in AML as well as glioma samples prompted the study of whether these mutations also conferred the reductive enzymatic activity to produce 2HG. Enzymatic assays and measurement of 2HG levels in primary AML samples confirmed that these IDH2 R172 mutations result in 2HG elevation (Gross et al., 2010; Ward et al., 2010).

It was then investigated if the measurement of 2HG levels in primary tumor samples with unknown IDH mutation status could serve as a metabolite screening test for both cytosolic IDH1 and mitochondrial IDH2 mutations. AML samples with low to undetectable 2HG were subsequently sequenced and determined to be IDH1 and IDH2 wild-type, and several samples with elevated 2HG were found to have neomorphic mutations at either IDH1 R132 or IDH2 R172 (Gross et al., 2010). However, some 2HG-elevated AML samples lacked IDH1 R132 or IDH2 R172 mutations. When more comprehensive sequencing of IDH1 and IDH2 was performed, it was found that the common feature of this remaining subset of 2HG-elevated AMLs was another mutation in IDH2, occurring at R140 (Ward et al., 2010). This discovery provided additional evidence that 2HG production was the primary feature being selected for in tumors.

In addition to intensifying efforts to find the cellular targets of 2HG, the discovery of the 2HG-producing IDH1 and IDH2 mutations suggested that 2HG measurement might have clinical utility in diagnosis and disease monitoring. While much work is still needed in this area, serum 2HG levels have successfully correlated with IDH1 R132 mutations in AML, and recent data have suggested that 1H magnetic resonance spectroscopy can be applied for 2HG detection in vivo for glioma (Andronesi et al., 2012; Choi et al., 2012; Gross et al., 2010; Pope et al., 2012). These methods may have advantages over relying on invasive solid tumor biopsies or isolating leukemic blast cells to obtain material for sequencing of IDH1 and IDH2. Screening tumors and body fluids by 2HG status also has potentially increased applicability given the recent report that additional IDH mutations can produce 2HG (Ward et al., 2011). These additional alleles may account for the recently described subset of 2HG-elevated chondrosarcoma samples that lacked the most common IDH1 or IDH2 mutations but were not examined for other IDH alterations (Amary et al., 2011). Metabolite screening approaches can also distinguish neomorphic IDH mutations from SNPs and sequencing artifacts with no effect on IDH enzyme activity, as well as from an apparently rare subset of loss-of-function, non 2HG-producing IDH mutations that may play a secondary tumorigenic role in altering cellular redox (Ward et al., 2011).

Will we find other novel oncometabolites like 2HG? We should consider basing the search for new oncometabolites on those metabolites already known to cause disease in pediatric inborn errors of metabolism (IEMs). 2HG exemplifies how advances in research on IEMs can inform research on cancer metabolism, and vice versa. Methods developed by those studying 2HG aciduria were used to demonstrate that R(-)-2HG (also known as D-2HG) is the exclusive 2HG stereoisomer produced by IDH1 and IDH2 mutants (Dang et al., 2009; Ward et al., 2010). Likewise, following the discovery of 2HG-producing IDH2 R140 mutations in leukemia, researchers looked for and successfully found germline IDH2 R140 mutations in D-2HG aciduria. IDH2 R140 mutations now account for nearly half of all cases of this devastating disease (Kranendijk et al., 2010). While interest has surrounded 2HG due to its apparent novelty as a metabolite not found in normal non-diseased cells, there are situations where 2HG appears in the absence of metabolic enzyme mutations. For example, in human cells proliferating in hypoxia, α-ketoglutarate can accumulate and be metabolized through an enhanced reductive activity of wild-type IDH2 in the mitochondria, leading to 2HG accumulation in the absence of IDH mutation (Wise et al., 2011). The ability of 2HG to alter epigenetics may reflect its evolutionary ancient status as a signal for elevated glutamine/glutamate metabolism and/or oxygen deficiency.

With this broadened view of what constitutes an oncometabolite, one could argue that the discoveries of two other oncometabolites, succinate and fumarate, preceded that of 2HG. Loss of function mutations in the TCA cycle enzymes succinate dehydrogenase (SDH) and fumarate hydratase (FH) have been known for several years to occur in pheochromocytoma, paraganglioma, leiomoyoma, and renal carcinoma. It was initially hypothesized that these mutations contribute to cancer through mitochondrial damage producing elevated ROS (Eng et al., 2003). However, potential tumorigenic effects were soon linked to the elevated levels of succinate and fumarate arising from loss of SDH and FH function, respectively. Succinate was initially found to impair PHD2, the α-ketoglutarate-dependent enzyme regulating HIF stability, through product inhibition (Selak et al., 2005). Subsequent work confirmed that fumarate could inhibit PHD2 (Isaacs et al., 2005), and that succinate could also inhibit the related enzyme PHD3 (Lee et al., 2005). These observations linked the elevated HIF levels observed in SDH and FH deficient tumors to the activity of the succinate and fumarate metabolites. Recent work has suggested that fumarate may have other important roles that predominate in FH deficiency. For example, fumarate can modify cysteine residues to inhibit a negative regulator of the Nrf2 transcription factor. This post-translational modification leads to the upregulation of antioxidant response genes (Adam et al., 2011; Ooi et al., 2011).

There are still many unanswered questions regarding the biology of SDH and FH deficient tumors. In light of the emerging epigenetic effects of 2HG, it is intriguing that succinate has been shown to alter histone demethylase activity in yeast (Smith et al., 2007). Perhaps elevated succinate and fumarate resulting from SDH and FH mutations can promote tumorigenesis in part through epigenetic modulation.

Despite rapid technological advances in studying cell metabolism, we remain unable to reliably distinguish cytosolic metabolites from those in the mitochondria and other compartments. Current fractionation methods often lead to metabolite leakage. Even within one subcellular compartment, there may be distinct pools of metabolites resulting from channeling between metabolic enzymes. A related challenge lies in the quantitative measurement of metabolic flux; i.e., measuring the movement of carbon, nitrogen, and other atoms through metabolic pathways rather than simply measuring the steady-state levels of individual metabolites. While critical fluxes have been quantified in cultured cancer cells and methods for these analyses continue to improve (DeBerardinis et al., 2007; Mancuso et al., 2004; Yuan et al., 2008), many obstacles remain such as cellular compartmentalization and the reliance of most cell culture on complex, incompletely defined media.

Over the past decade, the study of metabolism has returned to its rightful place at the forefront of cancer research. Although Warburg was wrong about mitochondria, he was prescient in his focus on metabolism. Data now support the concepts that altered metabolism results from active reprogramming by altered oncogenes and tumor suppressors, and that metabolic adaptations can be clonally selected during tumorigenesis. Altered metabolism should now be considered a core hallmark of cancer. There is much work to be done.

2.1.2.8 A Role for the Mitochondrial Pyruvate Carrier as a Repressor of the Warburg Effect and Colon Cancer Cell Growth

Schell JC, Olson KA, …, Xie J, Egnatchik RA, Earl EG, DeBerardinis RJ, Rutter J.
Mol Cell. 2014 Nov 6; 56(3):400-13
http://dx.doi.org:/10.1016/j.molcel.2014.09.026

Cancer cells are typically subject to profound metabolic alterations, including the Warburg effect wherein cancer cells oxidize a decreased fraction of the pyruvate generated from glycolysis. We show herein that the mitochondrial pyruvate carrier (MPC), composed of the products of the MPC1 and MPC2 genes, modulates fractional pyruvate oxidation. MPC1 is deleted or underexpressed in multiple cancers and correlates with poor prognosis. Cancer cells re-expressing MPC1 and MPC2 display increased mitochondrial pyruvate oxidation, with no changes in cell growth in adherent culture. MPC re-expression exerted profound effects in anchorage-independent growth conditions, however, including impaired colony formation in soft agar, spheroid formation, and xenograft growth. We also observed a decrease in markers of stemness and traced the growth effects of MPC expression to the stem cell compartment. We propose that reduced MPC activity is an important aspect of cancer metabolism, perhaps through altering the maintenance and fate of stem cells.

Figure 2. Re-Expressed MPC1 and MPC2 Form a Mitochondrial Complex (A and B) (A) Western blot and (B) qRT-PCR analysis of the indicated colon cancer cell lines with retroviral expression of MPC1 (or MPC1-R97W) and/or MPC2. (C) Western blots of human heart tissue, hematologic cancer cells, and colon cancer cell lines with and without MPC1 and MPC2 re-expression. (D) Fluorescence microscopy of MPC1-GFP and MPC2-GFP overlaid with Mitotracker Red in HCT15 cells. Scale bar: 10 mm. (E) Blue-native PAGE analysis of mitochondria from control and MPC1/2-expressing cells. (F) Western blots of metabolic and mitochondrial proteins across four colon cancer cell lines with or without MPC1/2 expression

Figure 3. MPC Re-Expression Alters Mitochondrial Pyruvate Metabolism (A) OCR at baseline and maximal respiration in HCT15 (n = 7) and HT29 (n = 13) with pyruvate as the sole carbon source (mean ± SEM). (B and C) Schematic and citrate mass isotopomer quantification in cells cultured with D-[U-13C]glucose and unlabeled glutamine for 6 hr (mean ± SD, n = 2). (D) Glucose uptake and lactate secretion normalized to protein concentration (mean ± SD, n = 3). (E–G) (E) Western blots of PDH, phospho-PDH, and PDK1; (F) PDH activity assay and (G) CS activity assay with or without MPC1 and MPC2 expression (mean ± SD, n = 4). (H and I) Effects of MPC1/2 re-expression on mitochondrial membrane potential and ROS production (mean ± SD, n = 3). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 4. MPC Re-Expression Alters Growth under Low-Attachment Conditions (A) Cell number of control and MPC1/2 re-expressing cell lines in adherent culture (mean ± SD, n = 7). (B) Cell viability determined by trypan blue exclusion and Annexin V/PI staining (mean ± SD, n = 3). (C–F) (C) EdU incorporation of MPC re-expressing cell lines at 3 hr post EdU pulse. Growth in 3D culture evaluated by (D) soft agar colony formation (mean ± SD, n = 12, see also Table S1) and by ([E] and [F]) spheroid formation ± MPC inhibitor UK5099 (mean ± SEM, n = 12). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 7. MPC Re-Expression Alters the Cancer Initiating Cell Population (A) Western blot quantification of ALDHA and Lin28A from control or MPC re-expressing HT29 xenografts (mean ± SEM, n = 10). (B and C) Percentage of ALDHhi (n = 3) and CD44hi (n = 5) cells as determined by flow cytometry (mean ± SEM). (D) Western blot analysis of stem cell markers in control and MPC re-expressing cell lines. (E) Relative MPC1 and MPC2 mRNA levels in ALDH sorted HCT15 cells (n = 4,mean ± SEM). 2D growth of (F) whole-population HCT15 cells and (G) ALDH sorted cells. Area determined by ImageJ after crystal violet staining (mean ± SD, n = 6). (H and I) (H) Adherent and (I) spheroid growth of main population (MP) versus side population (SP) HCT15 cells. (mean ± SD, n = 6). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

Our demonstration that the MPC is lost or underexpressed in many cancers might provide clarifying context for earlier attempts to exploit metabolic regulation for cancer therapeutics. The PDH kinase inhibitor dichloroacetate, which impairs PDH phosphorylation and increases pyruvate oxidation, has been explored extensively as a cancer therapy (Bonnet et al., 2007; Olszewski et al., 2010). It has met with mixed results, however, and has typically failed to dramatically decrease tumor burden as a monotherapy (Garon et al., 2014;
Sanchez-Arago et al., 2010; Shahrzadetal.,2010). Is one possible reason for these failures that the MPC has been lost or inactivated, thereby limiting the metabolic effects of PDH activity? The inclusion of the MPC adds additional complexity to targeting cancer metabolism for therapy but has the potential to explain why treatments may be more effective in some studies than in others (Fulda et al., 2010; Hamanaka and Chandel, 2012; Tennant et al., 2010; Vander Heiden, 2011). The redundant measures to limit pyruvate oxidation make it easy to understand why expression of the MPC leads to relatively modest metabolic changes in cells grown in adherent culture conditions. While subtle, we observed a number of changes in metabolic parameters, all of which are consistent with enhanced mitochondrial pyruvate entry and oxidation. There are at least two possible explanations for the discrepancy that we observed between the impact on adherent and nonadherent cell proliferation. One hypothesis is that the stress of nutrient deprivation and detachment combines with these subtle metabolic effects to impair survival and proliferation.

2.1.2.9  ECM1 promotes the Warburg effect through EGF-mediated activation of PKM2

Lee KM, Nam K, Oh S, Lim J, Lee T, Shin I.
Cell Signal. 2015 Feb; 27(2):228-35
http://dx.doi.org:/10.1016/j.cellsig.2014.11.004

The Warburg effect is an oncogenic metabolic switch that allows cancer cells to take up more glucose than normal cells and favors anaerobic glycolysis. Extracellular matrix protein 1 (ECM1) is a secreted glycoprotein that is overexpressed in various types of carcinoma. Using two-dimensional digest-liquid chromatography-mass spectrometry (LC-MS)/MS, we showed that the expression of proteins associated with the Warburg effect was upregulated in trastuzumab-resistant BT-474 cells that overexpressed ECM1 compared to control cells. We further demonstrated that ECM1 induced the expression of genes that promote the Warburg effect, such as glucose transporter 1 (GLUT1), lactate dehydrogenase A (LDHA), and hypoxia-inducible factor 1 α (HIF-1α). The phosphorylation status of pyruvate kinase M2 (PKM-2) at Ser37, which is responsible for the expression of genes that promote the Warburg effect, was affected by the modulation of ECM1 expression. Moreover, EGF-dependent ERK activation that was regulated by ECM1 induced not only PKM2 phosphorylation but also gene expression of GLUT1 and LDHA. These findings provide evidence that ECM1 plays an important role in promoting the Warburg effect mediated by PKM2.

Fig. 1.ECM1 induces a metabolic shift toward promoting Warburg effect. (A) The levels of glucose uptake were examined with a cell-based assay. (B) Levels of lactate production were measured using a lactate assay kit. (C) Cellular ATP content was determined with a Cell Titer-Glo luminescent cell viability assay. Error bars represent mean ± SD of triplicate experiments (*p b 0.05, ***p b 0.0005).

Fig.2. ECM1 up-regulates expression of gene sassociated with the Warburg effect. (A) Cell lysates were analyzed by western blotting using antibodies specific for ECM1, LDHA, GLUT1,and actin (as a loading control). The intensities of the bands were quantified using 1D Scan software and plotted. (BandC) mRNA levels of each gene were determined by real-time PCR using specific primers. (D) HIF-1α-dependent transcriptional activities were examined using a hypoxia response element (HRE) reporter indual luciferase assays. Error bars represent mean ± SD of triplicate experiments (*p b 0.05, **p b 0.005, ***p b 0.0005).

Fig.3. ECM1-dependent upregulation of gene expression is not mediated byEgr-1.

Fig.4. ECM1 activates PKM2 via EGF-mediated ERK activation

Fig. 5. TheWarburg effect is attenuated by silencing of PKM2 in breast cancer cells

Recently, a non-glycolytic function of PKM2 was reported. Phosphorylated PKM2 at Ser37 is translocated into the nucleus after EGFR and ERK activation and regulates the expression of cyclin D1, c-Myc, LDHA, and GLUT1[19,37]. Here, we showed that ECM1 regulates the phosphorylation level and translocation of PKM2 via the EGFR/ ERK pathway. As we previously showed that ECM1 enhances the EGF response and increases EGFR expression through MUC1-dependent stabilization [17], it seemed likely that activation of the EGFR/ERK pathway by ECM1 is linked to PKM2 phosphorylation. Indeed, we show here that ECM1 regulates the phosphorylation of PKM2 at Ser37 and enhances the Warburg effect through the EGFR/ERK pathway. HIF-1α is known to be responsible for alterations in cancer cell metabolism [38] and our current studies showed that the expression level of HIF-1α is up-regulated by ECM1 (Fig. 2C and D). To determine the mechanism by which ECM1 upregulated HIF-1α expression, we focused on the induction of Egr-1 by EGFR/ERK signaling [39]. However, although Egr-1 expression was regulated by ECM1 we failed to find evidence that Egr-1 affected the expression of genes involved in the Warburg effect (Fig. 3C). Moreover, ERK-dependent PKM2 activation did not regulate HIF-1α expression in BT-474 cells (Fig. 4D and5B). These results suggested that the upregulation of HIF-1α by ECM1 is not mediated by the EGFR/ERK pathway.

Conclusions

In the current study we showed that ECM1 altered metabolic phenotypes of breast cancer cells toward promoting the Warburg effect.

Phosphorylation and nuclear translocation of PKM2 were induced by ECM1 through the EGFR/ERK pathway. Moreover, phosphorylated PKM2 increased the expression of metabolic genes such as LDHA and GLUT1, and promoted glucose uptake and lactate production. These findings provide a new perspective on the distinct functions of ECM1 in cancer cell metabolism. Supplementary data to this article can be found online at
http://dx.doi.org/10.1016/j.cellsig.2014.11.004

References

[1] R.A. Cairns, I.S. Harris, T.W. Mak, Cancer 11 (2011) 85–95.
[2] O. Warburg, Science 123 (1956) 309–314.
[3] G.L. Semenza, D.Artemov, A.Bedi, …, J. Simons, P. Taghavi, H. Zhong, Novartis Found. Symp. 240 (2001) 251–260 (discussion 260–254).
[4] N.C. Denko, Cancer 8 (2008) 705–713.
[5] C. Chen, N. Pore, A. Behrooz, F. Ismail-Beigi, A. Maity, J. Biol. Chem. 276 (2001) 9519–9525.
[6] J.Lum, T.Bui, M.Gruber, J.D.Gordan, R.J.DeBerardinis,.. ,C.B. Thompson, Genes Dev. 21 (2007) 1037–1049.
[7] J.T. Chi, Z. Wang, D.S. Nuyten, E.H. Rodriguez, .., P.O. Brown, PLoS Med.
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[8] G.L. Semenza, Cancer 3 (2003) 721–732.

2.1.2.10 Glutamine Oxidation Maintains the TCA Cycle and Cell Survival during impaired Mitochondrial Pyruvate Transport

Chendong Yang, B Ko, CT. Hensley,…, J Rutter, ME. Merritt, RJ. DeBerardinis
Molec Cell  6 Nov 2014; 56(3):414–424
http://dx.doi.org/10.1016/j.molcel.2014.09.025

Highlights

  • Mitochondria produce acetyl-CoA from glutamine during MPC inhibition
    •Alanine synthesis is suppressed during MPC inhibition
    •MPC inhibition activates GDH to supply pools of TCA cycle intermediates
    •GDH supports cell survival during periods of MPC inhibition

Summary

Alternative modes of metabolism enable cells to resist metabolic stress. Inhibiting these compensatory pathways may produce synthetic lethality. We previously demonstrated that glucose deprivation stimulated a pathway in which acetyl-CoA was formed from glutamine downstream of glutamate dehydrogenase (GDH). Here we show that import of pyruvate into the mitochondria suppresses GDH and glutamine-dependent acetyl-CoA formation. Inhibiting the mitochondrial pyruvate carrier (MPC) activates GDH and reroutes glutamine metabolism to generate both oxaloacetate and acetyl-CoA, enabling persistent tricarboxylic acid (TCA) cycle function. Pharmacological blockade of GDH elicited largely cytostatic effects in culture, but these effects became cytotoxic when combined with MPC inhibition. Concomitant administration of MPC and GDH inhibitors significantly impaired tumor growth compared to either inhibitor used as a single agent. Together, the data define a mechanism to induce glutaminolysis and uncover a survival pathway engaged during compromised supply of pyruvate to the mitochondria.

Yang et al, Graphical Abstract

Yang et al, Graphical Abstract

Graphical abstract

Figure 1. Pyruvate Depletion Redirects Glutamine Metabolism to Produce AcetylCoA and Citrate (A) Top: Anaplerosis supplied by [U-13C]glutamine. Glutamine supplies OAA via a-KG, while acetylCoA is predominantly supplied by other nutrients, particularly glucose. Bottom: Glutamine is converted to acetyl-CoA in the absence of glucosederived pyruvate. Red circles represent carbons arising from [U-13C]glutamine, and gray circles are unlabeled. Reductive carboxylation is indicated by the green dashed line. (B) Fraction of succinate, fumarate, malate, and aspartate containing four 13C carbons after culture of SFxL cells for 6 hr with [U-13C]glutamine in the presence or absence of 10 mM unlabeled glucose (Glc). (C) Mass isotopologues of citrate after culture of SFxL cells for 6 hr with [U-13C]glutamine and 10 mM unlabeled glucose, no glucose, or no glucose plus 6 mM unlabeled pyruvate (Pyr). (D) Citrate m+5 and m+6 after culture of HeLa or Huh-7 cells for 6 hr with [U-13C]glutamine and 10 mM unlabeled glucose, no glucose, or no glucose plus 6 mM unlabeled pyruvate. Data are the average and SD of three independent cultures. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 2. Isolated Mitochondria Convert Glutamine to Citrate (A) Western blot of whole-cell lysates (Cell) and preparations of isolated mitochondria (Mito) or cytosol from SFxL cells. (B) Oxygen consumption in a representative mitochondrial sample. Rates before and after addition of ADP/GDP are indicated. (C) Mass isotopologues of citrate produced by mitochondria cultured for 30 min with [U-13C] glutamine and with or without pyruvate.

Figure 3. Blockade of Mitochondrial Pyruvate Transport Activates Glutamine-Dependent Citrate Formation (A) Dose-dependent effects of UK5099 on citrate labeling from [U-13C]glucose and [U-13C]glutamine in SFxL cells. (B) Time course of citrate labeling from [U-13C] glutamine with or without 200 mM UK5099. (C) Abundance of total citrate and citrate m+6 in cells cultured in [U-13C]glutamine with or without 200 mM UK5099. (D) Mass isotopologues of citrate in cells cultured for 6 hr in [U-13C]glutamine with or without 10 mM CHC or 200 mM UK5099. (E) Effect of silencing ME2 on citrate m+6 after 6 hr of culture in [U-13C]glutamine. Relative abundances of citrate isotopologues were determined by normalizing total citrate abundance measured by mass spectrometry against cellular protein for each sample then multiplying by the fractional abundance of each isotopologue. (F) Effect of silencing MPC1 or MPC2 on formation of citrate m+6 after 6 hr of culture in [U-13C]glutamine. (G) Citrate isotopologues in primary human fibroblasts of varying MPC1 genotypes after culture in [U-13C]glutamine. Data are the average and SD of three independent cultures. *p < 0.05; **p < 0.01; ***p < 0.001. See also Figure S1.

Figure 4. Kinetic Analysis of the Metabolic Effects of Blocking Mitochondrial Pyruvate Transport (A) Summation of 13C spectra acquired over 2 min of exposure of SFxL cells to hyperpolarized [1-13C] pyruvate. Resonances are indicated for [1-13C] pyruvate (Pyr1), the hydrate of [1-13C]pyruvate (Pyr1-Hydr), [1-13C]lactate (Lac1), [1-13C]alanine (Ala1), and H[13C]O3 (Bicarbonate). (B) Time evolution of appearance of Lac1, Ala1, and bicarbonate in control and UK5099-treated cells. (C) Relative 13C NMR signals for Lac1, Ala1, and bicarbonate. Each signal is summed over the entire acquisition and expressed as a fraction of total 13C signal. (D) Quantity of intracellular and secreted alanine in control and UK5099-treated cells. Data are the average and SD of three independent cultures. *p < 0.05; ***p < 0.001. See also Figure S2.

Figure 5. Inhibiting Mitochondrial Pyruvate Transport Enhances the Contribution of Glutamine to Fatty Acid Synthesis (A) Mass isotopologues of palmitate extracted from cells cultured with [U-13C] glucose or [U-13C]glutamine, with or without 200 mM UK5099. For simplicity, only even-labeled isotopologues (m+2, m+4, etc.) are shown. (B) Fraction of lipogenic acetyl-CoA derived from glucose or glutamine with or without 200 mM UK5099. Data are the average and SD of three independent cultures. ***p < 0.001. See also Figure S3.

Figure 6. Blockade of Mitochondrial Pyruvate Transport Induces GDH (A) Two routes by which glutamate can be converted to AKG. Blue and green symbols are the amide (g) and amino (a) nitrogens of glutamine, respectively. (B) Utilization and secretion of glutamine (Gln), glutamate (Glu), and ammonia (NH4+) by SFxL cells with and without 200 mM UK5099. (C) Secretion of 15N-alanine and 15NH4+ derived from [a-15N]glutamine in SFxL cells expressing a control shRNA (shCtrl) or either of two shRNAs directed against GLUD1 (shGLUD1-A and shGLUD1-B). (D) Left: Phosphorylation of AMPK (T172) and acetyl-CoA carboxylase (ACC, S79) during treatment with 200 mM UK5099. Right: Steady-state levels of ATP 24 hr after addition of vehicle or 200 mM UK5099. (E) Fractional contribution of the m+6 isotopologue to total citrate in shCtrl, shGLUD1-A, and shGLUD1-B SFxL cells cultured in [U-13C]glutamine with or without 200 mM UK5099. Data are the average and SD of three independent cultures. *p < 0.05; **p < 0.01; ***p < 0.001. See also Figure S4.

Figure 7. GDH Sustains Growth and Viability during Suppression of Mitochondrial Pyruvate Transport (A) Relative growth inhibition of shCtrl, shGLUD1A, and shGLUD1-B SFxL cells treated with 50 mM UK5099 for 3 days. (B) Relative growth inhibition of SFxL cells treated with combinations of 50 mM of the GDH inhibitor EGCG, 10 mM of the GLS inhibitor BPTES, and 200 mM UK5099 for 3 days. (C) Relative cell death assessed by trypan blue staining in SFxL cells treated as in (B). (D) Relative cell death assessed by trypan blue staining in SF188 cells treated as in (B) for 2 days. (E) (Left) Growth of A549-derived subcutaneous xenografts treated with vehicle (saline), EGCG, CHC, or EGCG plus CHC (n = 4 for each group). Data are the average and SEM. Right: Lactate abundance in extracts of each tumor harvested at the end of the experiment. Data in (A)–(D) are the average and SD of three independent cultures. NS, not significant; *p < 0.05; **p < 0.01; ***p < 0.001. See also Figure S5.

Mitochondrial metabolism complements glycolysis as a source of energy and biosynthetic precursors. Precursors for lipids, proteins, and nucleic acids are derived from the TCA cycle. Maintaining pools of these intermediates is essential, even under circumstances of nutrient limitation or impaired supply of glucose-derived pyruvate to the mitochondria. Glutamine’s ability to produce both acetyl-CoA and OAA allows it to support TCA cycle activity as a sole carbon source and imposes a greater cellular dependence on glutamine metabolism when MPC function or pyruvate supply is impaired. Other anaplerotic amino acids could also supply both OAA and acetyl-CoA, providing flexible support for the TCA cycle when glucose is limiting. Although fatty acids are an important fuel in some cancer cells (Caro et al., 2012), and fatty acid oxidation is induced upon MPC inhibition, this pathway produces acetyl-CoA but not OAA. Thus, fatty acids would need to be oxidized along with an anaplerotic nutrient in order to enable the cycle to function as a biosynthetic hub. Notably, enforced MPC overexpression also impairs growth of some tumors (Schell et al., 2014), suggesting that maximal growth may require MPC activity to be maintained within a narrow window. After decades of research on mitochondrial pyruvate transport, molecular components of the MPC were recently reported (Halestrap, 2012; Schell and Rutter, 2013). MPC1 and MPC2 form a heterocomplex in the inner mitochondrial membrane, and loss of either component impairs pyruvate import, leading to citrate depletion (Bricker et al., 2012; Herzig et al., 2012). Mammalian cells lacking functional MPC1 display normal glutamine-supported respiration (Bricker et al., 2012), consistent with our observation that glutamine supplies the TCA cycle in absence of pyruvate import. We also observed that isolated mitochondria produce fully labeled citrate from glutamine, indicating that this pathway operates as a self-contained mechanism to maintain TCA cycle function. Recently, two well-known classes of drugs have unexpectedly been shown to inhibit MPC. First, thiazolidinediones, commonly used as insulin sensitizers, impair MPC function in myoblasts (Divakaruni et al.,2013). Second, the phosphodiesterase inhibitor Zaprinast inhibits MPC in the retina and brain (Du et al., 2013b). Zaprinast also induced accumulation of aspartate, suggesting that depletion of acetyl-CoA impaired the ability of a new turn of the TCA cycle to be initiated from OAA; as a consequence, OAA was transaminated to aspartate. We noted a similar phenomenon in cancer cells, suggesting that UK5099 elicits a state in which acetyl-CoA supply is insufficient to avoid OAA accumulation. Unlike UK5099, Zaprinast did not induce glutamine-dependent acetyl-CoA formation. This may be related to the reliance of isolated retinas on glucose rather than glutamine to supply TCA cycle intermediates or the exquisite system used by retinas to protect glutamate from oxidation (Du et al., 2013a). Zaprinast was also recently shown to inhibit glutaminase (Elhammali et al., 2014), which would further reduce the contribution of glutamine to the acetyl-CoA pool.

Comment by reader –

The results from these studies served as a good
reason to attempt the vaccination of patients using p53-
derived peptides, and a several clinical trials are currently
in progress. The most advanced work used a long
synthetic peptide mixture derived from p53 (p53-SLP; ISA
Pharmaceuticals, Bilthoven, the Netherlands) (Speetjens
et al., 2009; Shangary et al., 2008; Van der Burg et al.,
2001). The vaccine is delivered in the adjuvant setting
and induces T helper type cells.

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Larry H Bernstein, MD, FCAP, Author and Curator

Chief, Scientific Communication

Leaders in Pharmaceutical Intelligence

with contributions from JEDS Rosalis, Brazil
and Radislov Rosov, Univ of Virginia, VA, USA

A Brief Curation of Proteomics, Metabolomics, and Metabolism

This article is a continuation of a series of elaborations of the recent and
accelerated scientific discoveries that are enlarging the scope of and
integration of biological and medical knowledge leading to new drug
discoveries.  The work that has led us to this point actually has roots
that go back 150 years.  The roots go back to studies in the mid-nineteenth century, with the emergence of microbiology, physiology,
pathology, botany, chemistry and physics, and the laying down of a
mechanistic approach divergent from descriptive observation in the
twentieth century. Medicine took on the obligation to renew the method
of training physicians after the Flexner Report (The Flexner Report of
1910 transformed the nature and process of medical education in America
with a resulting elimination of proprietary schools), funded by the Carnegie
Foundation.  Johns Hopkins University Medical School became the first to
adopt the model, as did Harvard, Yale, University of Chicago, and others.

The advances in biochemistry, genetics and genomics, were large, as was
structural organic chemistry in the remainder of the centrury.  The advances
in applied mathematics and in instrumental analysis opened a new gateway
into the 21st century with the Human Genome Project, the Proteome Library,
Signaling Pathways, and the Metabolomes – human, microbial, and plants.

shall elaborate on how the key processes of life are being elucidated as
these interrelated disciplines converge.  I shall not be covering in great
detail the contribution of the genetic code and transcripton because they
have been covered at great length in this series.

Part I.  The foundation for the emergence of a revitalized molecular
biology 
and biochemistry.

In a series of discussions with Jose des Salles Roselino (Brazil) over a
period of months we have come to an important line of reasoning. DNA
to protein link goes from triplet sequence to amino acid sequence. The
realm of genetics. Further, protein conformation, activity and function
requires that environmental and microenvironmental factors should be
considered (Biochemistry).  This has been opened in several articles
preceding this.

In the cAMP coupled hormonal response the transfer of conformation
from protein to protein is paramount. For instance, if your scheme goes
beyond cAMP, it will show an effect over a self-assembly (inhibitor
protein and protein kinase). Therefore, sequence alone does not
explain conformation, activity and function of regulatory proteins.
Recall that sequence is primar structure, determined by the translation
of the code, but secondary structure is determined by disulfide bonds.
There is another level of structure, tertiary structure, that is molded by
steric influences of near neighbors and by noncovalent attractions
and repulsions.

A few comments ( contributed by Assoc. Prof. JEDS Roselino) are in
order to stress the importance of self-assembly (Prigogine, R. A
Marcus, conformation energy) in a subject that is the best for this
connection. We have to stress again that in the cAMP
coupled hormonal response the transfer of conformation from
protein to protein is paramount. For instance, in case the
reaction sequence follows beyond the production of the
second messenger, as in the case of cAMP, this second
messenger will remove a self-assembly of inhibitor protein
with the enzyme protein kinase. Therefore, sequence alone
does not explain conformation, activity and function of
regulatory proteins. In this case, if this important mechanism
was not ignored, the work of Stanley Prusiner would most
certainly have been recognized earlier, and “rogue” proteins
would not have been seen as so rogue as some assumed.
For the general idea of importance of self-assembly versus
change in covalent modification of proteins (see R. A Kahn
and A. G Gilman (1984) J. Biol. Chem.  259(10), pp 6235-
6240. In this case, trimeric or dimeric G does not matter.
“Signaling transduction tutorial”.
G proteins in the G protein coupled-receptor proteins are
presented following a unidirectional series of arrows.
This is adequate to convey the idea of information being
transferred from outside the cell towards cell´s interior
(therefore, against the dogma that says all information
moves from DNA to RNA to protein.  It is important to
consider the following: The entire process is driven by
a very delicate equilibrium between possible conform-
ational states of the proteins. Empty receptors have very
low affinity for G proteins. On the other hand, hormone
bound receptors have a change in conformation that
allows increasing the affinity for the G-trimer. When
hormone receptors bind to G-trimers two things happen:

  1. Receptors transfer conformation information to
    the G-triplex and
  2. the G-triplex transfers information back to the
    complex hormone-receptor.

In the first case , the dissociated G protein exchanges
GDP for GTP and has its affinity for the cyclase increased,
while by the same interaction receptor releases the
hormone which then places the first required step for the
signal. After this first interaction step, on the second and
final transduction system step is represented by an
opposite arrow. When, the G-protein + GTP complex
interacts with the cyclase two things happen:

  1. It changes the cyclase to an active conformation
    starting the production of cAMP as the single
    arrow of the scheme. However, the interaction
    also causes a backward effect.
  2. It activates the GTPase activity of this subunit
    and the breakdown of GTP to GDP moves this 
    subunit back to the initial trimeric inactive
    state
     of G complex.

This was very well studied when the actions of cholera toxin
required better understanding. Cholera toxin changes the
GTPase subunit by ADP-ribosilation (a covalent and far more
stable change in proteins) producing a permanent conformation
of GTP bound G subunit. This keeps the cyclase in permanent
active conformation because ADP-ribosilation inhibits GTPase
activity required to put an end in the hormonal signal.

The study made while G-proteins were considered a dimer still
holds despite its limited vision of the real complexity of the
transduction system. It was also possible to get this very same
“freezing” in the active state using GTP stable analogues. This
transduction system is one of the best examples of the delicate
mechanisms of conformational interaction of proteins. Further-
more, this system also shows on the opposite side of our
reasoning scheme, how covalent changes are adequate for
more stable changes than those mediated by Van der Wall’s
forces between proteins. Yet, these delicate forces are the
same involved when Sc-Prion transfers its rogue
conformation to c-Prion proteins and other similar events.
The Jacob-Monod Model

A combination of genetic and biochemical experiments in
bacteria led to the initial recognition of

  1. protein-binding regulatory sequences associated with genes and
  2. proteins whose binding to a gene’s regulatory sequences
    either activate or repress its transcription.

These key components underlie the ability of both prokaryotic and
eukaryotic cells to turn genes on and off. The  experimental findings lead to a general model of bacterial transcription control.

Gene control serves to allow a single cell to adjust to changes in its
nutritional environment so that its growth and division can be optimized.
Thus, the prime focus of research has been on genes that encode
inducible proteins whose production varies depending on the nutritional
status of the cells. Its most characteristic and biologically far-reaching
purpose in eukaryotes, distinctive from single cell organisms is the
regulation of a genetic program that underlies embryological
development and tissue differentiation.

The principles of transcription have already been described in this
series under the translation of the genetic code into amino acids
that are the building blocks for proteins.

E.coli can use either glucose or other sugars such as the
disaccharide lactose as the sole source of carbon and energy.
When E. coli cells are grown in a glucose-containing medium,
the activity of the enzymes needed to metabolize lactose is
very low. When these cells are switched to a medium
containing lactose but no glucose, the activities of the lactose-metabolizing enzymes increase. Early studies showed that the
increase in the activity of these enzymes resulted from the
synthesis of new enzyme molecules, a phenomenon termed
induction. The enzymes induced in the presence of lactose
are encoded by the lac operon, which includes two genes, Z
and Y, that are required for metabolism of lactose and a third
gene. The lac Y gene encodes lactose permease, which spans the E. coli cell membrane and uses the energy available from
the electrochemical gradient across the membrane to pump
lactose into the cell. The lac Z gene encodes β-galactosidase,
which splits the disaccharide lactose into the monosaccharides
glucose and galactose, which are further metabolized through
the action of enzymes encoded in other operons. The third
gene encodes thiogalactoside transacetylase.

Synthesis of all three enzymes encoded in the lac operon is rapidly
induced when E. coli cells are placed in a medium containing lactose
as the only carbon source and repressed when the cells are switched
to a medium without lactose. Thus all three genes of the lac operon
are coordinately regulated. The lac operon in E. coli provides one
of the earliest and still best-understood examples of gene control.
Much of the pioneering research on the lac operon was conducted by
Francois Jacob, Jacques Monod, and their colleagues in the 1960s.

Some molecules similar in structure to lactose can induce expression
of the lacoperon genes even though they cannot be hydrolyzed by β-galactosidase. Such small molecules (i.e., smaller than proteins) are
called inducers. One of these, isopropyl-β-D-thiogalactoside,
abbreviated IPTG,is particularly useful in genetic studies of the lac
operon, because it can diffuse into cells and, it is not metabolized.
Insight into the mechanisms controlling synthesis of β-galactosidase
and lactose permease came from the study of mutants in which control
of β-galactosidase expression was abnormal and used a colorimetric
assay for β-galactosidase.

When the cells are exposed to chemical mutagens before plating on
X-gal/glucose plates, rare blue colonies appear, but when cells
from these blue colonies are recovered and grown in media containing
glucose, they overexpress all the genes of the lac operon. These cells
are called constitutive mutants because they fail to repress the lac
operon in media lacking lactose and instead continuously express the
enzymes, and the genes were mapped to a region on the E. coli
chromosome. This led to the conclusion that these cells had a defect
in a protein that normally repressed expression of the lac operon in
the absence of lactose, and that it blocks transcription by binding to
a site on the E. coli genome where transcription of the lac operon is
initiated. In addition, it binds to the lac repressor in the lactose
medium and decreases its affinity for the repressor-binding site
on the DNA causing the repressor to unbind the DNA. Thereby,
transcription of the lac operon is initiated, leading to synthesis of
β-galactosidase, lactose permease, and thiogalactoside
transacetylase.

 regulation of the lac operon by lac repressor

Jacob and Monod model of transcriptional regulation of the lac operon

Next, Jacob and Monod isolated mutants that expressed the lac operon
constitutively even when two copies of the wild-type lacI gene
encoding the lac repressor were present in the same cell, and the
constitutive mutations mapped to one end of the lac operon, as the
model predicted.  Further, there are rare cells that carry a mutation
located at the region, promoter, that block initiation of transcription by
RNA polymerase.

lac I+ gene is trans-acting, & encodes a protein, which binds to a lac operator

 lac I+ gene is trans-acting, & encodes a protein, which
binds to a lac operator

They further demonstrated that the two types of mutations lac I and
lac I+, were cis- and trans-acting, the latter encoding a protein that
binds to the lac operator. The cis-acting Oc mutations prevent
binding of the lac repressor to the operator, and  mutations in the
lac promoter are cis-acting, since they alter the binding site for RNA
polymerase. In general, trans-acting genes that regulate expression
of genes on other DNA molecules encode diffusible products. In
most cases these are proteins, but in some cases RNA molecules
can act in trans to regulate gene expression.

According to the Jacob and Monod model of transcriptional control,
transcription of the lac operon, which encodes three inducible
proteins, is repressed by binding of lac repressor protein to the
operator sequence.

 (Section 10.1Bacterial Gene Control: The Jacob-Monod Model.)
This book is accessible by the search feature.

Comment: This seminal work was done a half century ago. It was a
decade after the Watson-Crick model for DNA. The model is
elaborated for the Eukaryote in the examples that follow.

(The next two articles were called to my attention by R. Bosov at
University of Virginia).

An acetate switch regulates stress erythropoiesis

M Xu,  JS Nagati, Ji Xie, J Li, H Walters, Young-Ah Moon, et al.
Nature Medicine 10 Aug 2014(20): 1018–1026.
http://dx.doi.org:/10.1038/nm.3587

message: 1- ( -CH3 ) = Ln ( (1/sqrt(1-Acetate^2) –
sqrt oxalate))/ Ln(oxygen) – K(o)
rsb5n@virginia.edu

The hormone erythropoietin (EPO), synthesized in the kidney or liver
of adult mammals, controls erythrocyte production and is regulated by
the stress-responsive transcription factor hypoxia-inducible factor-2
(HIF-2).
 HIFα acetylation and efficient HIF-2–dependent EPO
induction during hypoxia requires  the lysine acetyltransferase CREB-binding protein (CBP) . These processes require acetate-dependent
acetyl CoA synthetase 2 (ACSS2) as follows.Acetate levels rise and
ACSS2 is required for HIF-2α acetylation, CBP–HIF-2α complex
formation, CBP–HIF-2α recruitment to the EPO enhancer and induction
of EPO gene expression
 in human Hep3B hepatoma cells and in EPO-generating organs of hypoxic or acutely anemic mice. In acutely anemic
mice, acetate supplementation augments stress erythropoiesis in an
ACSS2-dependent manner. Moreover, in acquired and inherited
chronic anemia mouse models, acetate supplementation increases
EPO expression
 and the resting hematocrit. Thus, a mammalian
stress-responsive acetate switch controls HIF-2 signaling and EPO
induction during pathophysiological states marked by tissue hypoxia.

Figure 1: Acss2 controls HIF-2 signaling in hypoxic cells.
Time course of endogenous HIF-2α acetylation during hypoxia following
immunoprecipitation (IP) of HIF-2α from whole-cell extracts and detection
of acetylated lysines by immunoblotting (IB).
http://www.nature.com/nm/journal/v20/n9/carousel/nm.3587-F1.jpg

Figure 2: Acss2 regulates hypoxia-induced renal Epo expression in mice.
http://www.nature.com/nm/journal/v20/n9/carousel/nm.3587-F2.jpg

Figure 3: Acute anemia induces Acss2-dependent HIF-2 signaling in mice.
http://www.nature.com/nm/journal/v20/n9/carousel/nm.3587-F3.jpg

Figure 4: An acetate switch regulates Cbp–HIF-2 interactions in cells.
(a) HIF-2α acetylation following immunoprecipitation of endogenous
HIF-2α and detection by immunoblotting with antibodies to acetylated
lysine or HIF-2α.
http://www.nature.com/nm/journal/v20/n9/carousel/nm.3587-F4.jpg

Figure 5: Acss2 signaling in cells requires intact HIF-2 acetylation.
http://www.nature.com/nm/journal/v20/n9/carousel/nm.3587-F5.jpg

Figure 6: Acetate facilitates recovery from anemia.

Acetate facilitates recovery from anemia

Acetate facilitates recovery from anemia

(a) Serial hematocrits of CD1 wild-type female mice after PHZ treatment, followed
by once daily per os (p.o.) supplementation with water vehicle (Veh; n = 7 mice),
GTA (n = 6 mice), GTB (n = 8 mice) or GTP (n = 7 mice) (single measurem…

http://www.nature.com/nm/journal/v20/n9/carousel/nm.3587-F6.jpg

see also-.
1. Bunn, H.F. & Poyton, R.O. Oxygen sensing and molecular adaptation to
hypoxia. Physiol. Rev. 76, 839–885 (1996).

  1. .Richalet, J.P. Oxygen sensors in the organism: examples of regulation
    under altitude hypoxia in mammals. Comp. Biochem. Physiol. A Physiol.
    118, 9–14 (1997).
  2. .Koury, M.J. Erythropoietin: the story of hypoxia and a finely regulated
    hematopoietic hormone. Exp. Hematol. 33, 1263–1270 (2005).
  3. Wang, G.L., Jiang, B.H., Rue, E.A. & Semenza, G.L. Hypoxia-inducible
    factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated
    by cellular O2 tension. Proc. Natl. Acad. Sci. USA92, 5510–5514 (1995).
  4. Chen, R. et al. The acetylase/deacetylase couple CREB-binding
    protein/sirtuin 1 controls hypoxia-inducible factor 2 signaling. J. Biol.
    Chem. 287, 30800–30811 (2012).
  5. .Papandreou, I., Cairns, R.A., Fontana, L., Lim, A.L. & Denko, N.C.
    HIF-1 mediates adaptation to hypoxia by actively down-regulating
    mitochondrial oxygen consumption. Cell Metab. 3,187–197 (2006).

14. Kim, J.W., Tchernyshyov, I., Semenza, G.L. & Dang, C.V. HIF-1-
mediated expression of pyruvate dehydrogenase kinase: a metabolic
switch required for cellular adaptation to hypoxia. Cell Metab. 3,
177–185 (2006).

16. Fujino, T., Kondo, J., Ishikawa, M., Morikawa, K. & Yamamoto, T.T.
Acetyl-CoA synthetase 2, a mitochondrial matrix enzyme involved in the
oxidation of acetate. J. Biol. Chem. 276,11420–11426 (2001).

17..Luong, A., Hannah, V.C., Brown, M.S. & Goldstein, J.L. Molecular
characterization of human acetyl-CoA synthetase, an enzyme regulated
by sterol regulatory element-binding proteins. J. Biol. Chem. 275,
26458–26466 (2000).

20 .Wellen, K.E. et al. ATP-citrate lyase links cellular metabolism to
histone acetylation. Science324, 1076–1080 (2009).

24. McBrian, M.A. et al. Histone acetylation regulates intracellular pH.
Mol. Cell 49, 310–321(2013).

Asymmetric mRNA localization contributes to fidelity and sensitivity
of spatially localized systems

Robert J Weatheritt, Toby J Gibson & M Madan Babu
Nature Structural & Molecular Biology 21, 833–839 (2014)
http://www.nature.com/nsmb/journal/v21/n9/abs/nsmb.2876.html 

Although many proteins are localized after translation, asymmetric
protein distribution is also achieved by translation after mRNA localization.
Why are certain mRNA transported to a distal location and translated
on-site? Here we undertake a systematic, genome-scale study of
asymmetrically distributed protein and mRNA in mammalian cells.
Our findings suggest that asymmetric protein distribution by mRNA
localization enhances interaction fidelity and signaling sensitivity
.
Proteins synthesized at distal locations frequently contain intrinsically
disordered segments. These regions are generally rich in assembly-
promoting modules and are often regulated by post-translational
modifications. Such proteins are tightly regulated but display distinct
temporal dynamics upon stimulation with growth factors. Thus, proteins
synthesized on-site may rapidly alter proteome composition and
act as dynamically regulated scaffolds to promote the formation
of reversible cellular assemblies. 
Our observations are consistent
across multiple mammalian species, cell types and developmental stages,
suggesting that localized translation is a recurring feature of cell
signaling and regulation.

Figure 1: Classification and characterization of TAS and DSS proteins.

The two major mechanisms for localizing proteins to distal sites in the cell

The two major mechanisms for localizing proteins to distal sites in the cell

(a)The two major mechanisms for localizing proteins to distal sites in the cell.
(b) Data sets used to identify groups of DSS and TAS transcripts, as well as
DSS and TAS proteins in mouse neuroblastoma cells

http://www.nature.com/nsmb/journal/v21/n9/carousel/nsmb.2876-F1.jpg

Figure 2: Structural analysis of DSS proteins reveals an enrichment
in disordered regions.

Distributions of the various structural properties of the DSS and TAS proteins of the mouse neuroblastoma data sets

Distributions of the various structural properties of the DSS and TAS proteins of the mouse neuroblastoma data sets

(a,b) Distributions of the various structural properties of the DSS and TAS
proteins of the mouse neuroblastoma data sets (a), the mouse pseudopodia,
the rat embryonic sensory neuron data set and the adult sensory neuron data set (b).…

http://www.nature.com/nsmb/journal/v21/n9/carousel/nsmb.2876-F2.jpg

Figure 3: Analysis of DSS proteins reveals an enrichment for linear motifs, phase-
transition (i.e., higher-order assembly) promoting segments and PTM sites that act
as molecular switches.

(a,b) Distributions of the various regulatory and structural properties of the DSS
and TAS proteins of the mouse neuroblastoma data sets
http://www.nature.com/nsmb/journal/v21/n9/carousel/nsmb.2876-F3.jpg

Figure 4: Dynamic regulation of DSS transcripts and proteins.

Dynamic regulation of DSS transcripts and proteins

Dynamic regulation of DSS transcripts and proteins

Genome-wide quantitative measurements of gene expression of DSS (n = 289)
and TAS (n = 1,292) proteins in mouse fibroblast cells. DSS transcripts and
proteins have a lower abundance and shorter half-lives

http://www.nature.com/nsmb/journal/v21/n9/carousel/nsmb.2876-F4.jpg

Figure 5: An overview of the potential advantages conferred by distal-site protein
synthesis, inferred from our analysis.

An overview of the potential advantages conferred by distal-site protein synthesis, inferred from our analysis

An overview of the potential advantages conferred by distal-site protein synthesis, inferred from our analysis

Turquoise and red filled circle represents off-target and correct interaction partners,
respectively. Wavy lines – a disordered region within a distal site synthesis protein.

http://www.nature.com/nsmb/journal/v21/n9/carousel/nsmb.2876-F5.jpg

The identification of asymmetrically localized proteins and transcripts.

The identification of asymmetrically localized proteins and transcripts

The identification of asymmetrically localized proteins and transcripts

An illustrative explanation of the resolution of the study and the concept of asymmetric
localization of proteins and mRNA. In this example, on the left a neuron is divided into
its cell body and axon terminal, and transcriptome/proteo…

http://www.nature.com/nsmb/journal/v21/n9/carousel/nsmb.2876-SF1.jpg

Graphs and boxplots of functional and structural properties for distal site synthesis
(DSS) proteins (red) and transport after synthesis (TAS) proteins (gray).
See Online Methods for details and legend of Figure 2 for a description of boxplots
and statistical tests.
http://www.nature.com/nsmb/journal/v21/n9/carousel/nsmb.2876-SF2.jpg

See also –
1. Martin, K.C. & Ephrussi, A. mRNA localization: gene expression in the spatial
dimension. Cell136, 719–730 (2009).

  1. Scott, J.D. & Pawson, T. Cell signaling in space and time: where proteins come
    together and when they’re apart. Science 326, 1220–1224 (2009).

4..Holt, C.E. & Bullock, S.L. Subcellular mRNA localization in animal cells
and why it matters.Science 326, 1212–1216 (2009).

  1. Jung, H., Gkogkas, C.G., Sonenberg, N. & Holt, C.E. Remote control of
    gene function by local translation. Cell 157, 26–40 (2014). 

Regulation of metabolism by hypoxia-inducible factor 1.   
Semenza GL.    Author information
Cold Spring Harb Symp Quant Biol. 2011;76:347-53.
http://dx.doi.org:/10.1101/sqb.2011.76.010678.

The maintenance of oxygen homeostasis is critical for survival, and the
master regulator of this process in metazoan species is hypoxia-inducible
factor 1 (HIF-1), which

  • controls both O(2) delivery and utilization.

Under conditions of reduced O(2) availability,

  • HIF-1 activates the transcription of genes, whose protein products
  • mediate a switch from oxidative to glycolytic metabolism.

HIF-1 is activated in cancer cells as a result of intratumoral hypoxia
and/or genetic alterations.

In cancer cells, metabolism is reprogrammed to

  • favor glycolysis even under aerobic conditions.

Pyruvate kinase M2 (PKM2) has been implicated in cancer growth and
metabolism, although the mechanism by which it exerts these effects is
unclear. Recent studies indicate that

PKM2 interacts with HIF-1α physically and functionally to

  1. stimulate the binding of HIF-1 at target genes,
  2. the recruitment of coactivators,
  3. histone acetylation, and
  4. gene transcription.

Interaction with HIF-1α is facilitated by

  • hydroxylation of PKM2 at proline-403 and -408 by PHD3.

Knockdown of PHD3

  • decreases glucose transporter 1, lactate dehydrogenase A, and
    pyruvate dehydrogenase kinase 1 expression;
  • decreases glucose uptake and lactate production; and
  • increases O(2) consumption.

The effect of PKM2/PHD3 is not limited to genes encoding metabolic
enzymes because VEGF is similarly regulated.

These results provide a mechanism by which PKM2

  • promotes metabolic reprogramming and

suggest that it plays a broader role in cancer progression than has
previously been appreciated.   PMID: 21785006   

Cadherins

Cadherins are thought to be the primary mediators of adhesion
between the cells
 of vertebrate animals, and also function in cell
adhesion in many invertebrates. The expression of numerous cadherins
during development is highly regulated, and the precise pattern of
cadherin expression plays a pivotal role in the morphogenesis of tissues
and organs. The cadherins are also important in the continued maintenance
of tissue structure and integrity. The loss of cadherin expression appears
to be highly correlated with the invasiveness of some types of tumors. Cadherin adhesion is also dependent on the presence of calcium ions
in the extracellular milieu.

The cadherin protein superfamily, defined as proteins containing a
cadherin-like domain, can be divided into several sub-groups. These include

  • the classical (type I) cadherins, which mediate adhesion at adherens junctions;
  • the highly-related type II cadherins;
  • the desmosomal cadherins found in desmosome junctions;
  • protocadherins, expressed only in the nervous system; and
  • atypical cadherin-like domain containing proteins.

Members of all but the atypical group have been shown to play a role
in intercellular adhesion.

Part II.  PKM2 and regulation of glycolysis

PKM2 regulates the Warburg effect and promotes ​HMGB1
release in sepsis

L Yang, M Xie, M Yang, Y Yu, S Zhu, W Hou, R Kang, …, & D Tang
Nature Communic 14 July 2014; 5(4436)
http://dx.doi.org/doi:10.1038/ncomms5436

Increasing evidence suggests the important role of metabolic reprogramming

  • in the regulation of the innate inflammatory response,

We provide evidence to support a novel role for the

  • ​pyruvate kinase M2 (​PKM2)-mediated Warburg effect,

namely aerobic glycolysis,

  • in the regulation of ​high-mobility group box 1 (​HMGB1) release. ​
  1. PKM2 interacts with ​hypoxia-inducible factor 1α (​HIF1α) and
  2. activates the ​HIF-1α-dependent transcription of enzymes necessary
    for aerobic glycolysis in macrophages.

Knockdown of ​PKM2, ​HIF1α and glycolysis-related genes

  • uniformly decreases ​lactate production and ​HMGB1 release.

Similarly, a potential ​PKM2 inhibitor, ​shikonin,

  1. reduces serum ​lactate and ​HMGB1 levels, and
  2. protects mice from lethal endotoxemia and sepsis.

Collectively, these findings shed light on a novel mechanism for

  • metabolic control of inflammation by
  • regulating ​HMGB1 release and

highlight the importance of targeting aerobic glycolysis in the treatment
of sepsis and other inflammatory diseases.

  1. Glycolytic inhibitor ​2-D G attenuates ​HMGB1 release by activated macrophages.
    http://www.nature.com/ncomms/2014/140714/ncomms5436/carousel/ncomms5436-f1.jpg
  2. Figure 2: Upregulated ​PKM2 promotes aerobic glycolysis and ​HMGB1
    release in activated macrophages.
    http://www.nature.com/ncomms/2014/140714/ncomms5436/carousel/ncomms5436-f2.jpg
  3. Figure 3: ​PKM2-mediated ​HIF1α activation is required for ​HMGB1
    release in activated macrophages.
    http://www.nature.com/ncomms/2014/140714/ncomms5436/carousel/ncomms5436-f3.jpg

 

ERK1/2-dependent phosphorylation and nuclear translocation of
PKM2 promotes the Warburg effect  

W Yang, Y Zheng, Y Xia, Ha Ji, X Chen, F Guo, CA Lyssiotis, & Zhimin Lu
Nature Cell Biology  2012 (27 June 2014); 14: 1295–1304
Corrigendum (January, 2013)  http://dx.doi.org:/10.1038/ncb2629

Pyruvate kinase M2 (PKM2) is upregulated in multiple cancer types and
contributes to the Warburg. We demonstrate that

  • EGFR-activated ERK2 binds directly to PKM2 Ile 429/Leu 431
  • through the ERK2 docking groove
  • and phosphorylates PKM2 at Ser 37, but
  • does not phosphorylate PKM1.

Phosphorylated PKM2 Ser 37

  1. recruits PIN1 for cis–trans isomerization of PKM2, which
  2. promotes PKM2 binding to importin α5
  3. and PKM2 translocates to the nucleus.

Nuclear PKM2 acts as

  • a coactivator of β-catenin to
  • induce c-Myc expression,

This is followed by

  1. the upregulation of GLUT1, LDHA and,
  2. in a positive feedback loop,
  • PTB-dependent PKM2 expression.

Replacement of wild-type PKM2 with

  • a nuclear translocation-deficient mutant (S37A)
  • blocks the EGFR-promoted Warburg effect
    and brain tumour development in mice.

In addition, levels of PKM2 Ser 37 phosphorylation

  • correlate with EGFR and ERK1/2 activity
    in human glioblastoma specimens.

Our findings highlight the importance of

  • nuclear functions of PKM2 in the Warburg effect
    and tumorigenesis.
  1. ERK is required for PKM2 nucleus translocation.
    http://www.nature.com/ncb/journal/v14/n12/carousel/ncb2629-f1.jpg
  2. ERK2 phosphorylates PKM2 Ser 37.
    http://www.nature.com/ncb/journal/v14/n12/carousel/ncb2629-f2.jpg
  3. Figure 3: PKM2 Ser 37 phosphorylation recruits PIN1.
    http://www.nature.com/ncb/journal/v14/n12/carousel/ncb2629-f3.jpg

 Pyruvate kinase M2 activators promote tetramer formation
and suppress tumorigenesis

D Anastasiou, Y Yu, WJ Israelsen, Jian-Kang Jiang, MB Boxer, B Hong, et al.
Nature Chemical Biology  11 Oct 2012; 8: 839–847

Cancer cells engage in a metabolic program to

  • enhance biosynthesis and support cell proliferation.

The regulatory properties of pyruvate kinase M2 (PKM2)

  • influence altered glucose metabolism in cancer.

The interaction of PKM2 with phosphotyrosine-containing proteins

  • inhibits PTM2 enzyme activity and
  • increases the availability of glycolytic metabolites
  • supporting cell proliferation.

This suggests that high pyruvate kinase activity may suppress
tumor growth
.

  1. expression of PKM1,  the pyruvate kinase isoform with high
    constitutive activity, or
  2. exposure to published small-molecule PKM2 activators
  • inhibits the growth of xenograft tumors.

Structural studies reveal that

  • small-molecule activators bind PKM2
  • at the subunit interaction interface,
  • a site that is distinct from that of the
    • endogenous activator fructose-1,6-bisphosphate (FBP).

However, unlike FBP,

  • binding of activators to PKM2 promotes
  • a constitutively active enzyme state that is resistant to inhibition
  • by tyrosine-phosphorylated proteins.

These data support the notion that small-molecule activation of PKM2
can interfere with anabolic metabolism

  1. PKM1 expression in cancer cells impairs xenograft tumor growth.
    http://www.nature.com/nchembio/journal/v8/n10/carousel/nchembio.1060-F1.jpg
  2. TEPP-46 and DASA-58 isoform specificity in vitro and in cells.
    TEPP-46 and DASA-58 isoform specificity in vitro and in cells.

    TEPP-46 and DASA-58 isoform specificity in vitro and in cells.

    (a) Structures of the PKM2 activators TEPP-46 and DASA-58. (b) Pyruvate kinase (PK) activity in purified recombinant human
    PKM1 or PKM2 expressed in bacteria in the presence of increasing
    concentrations of TEPP-46 or DASA-58. M1, PKM1;…
    http://www.nature.com/nchembio/journal/v8/n10/carousel/nchembio.1060-F2.jpg

  3. Activators promote PKM2 tetramer formation and prevent
    inhibition by phosphotyrosine signaling.
Activators promote PKM2 tetramer formation and prevent inhibition by phosphotyrosine signaling.

Activators promote PKM2 tetramer formation and prevent inhibition by phosphotyrosine signaling.

Sucrose gradient ultracentrifugation profiles of purified recombinant
PKM2 (rPKM2) and the effects of FBP and TEPP-46 on PKM2 subunit stoichiometry.
http://www.nature.com/nchembio/journal/v8/n10/carousel/nchembio.1060-F3.jpg

Figure 5: Metabolic effects of cell treatment with PKM2 activators.
(a) Effects of TEPP-46, DASA-58 (both used at 30 μM) or PKM1
expression on the doubling time of H1299 cells under normoxia
(21% O2) or hypoxia (1% O2). (b) Effects of DASA-58 on lactate
production from glucose. The P value shown was ca…
http://www.nature.com/nchembio/journal/v8/n10/carousel/nchembio.1060-F5.jpg

EGFR has a tumour-promoting role in liver macrophages during
hepatocellular carcinoma formation

H Lanaya, A Natarajan, K Komposch, L Li, N Amberg, …, & Maria Sibilia
Nature Cell Biology 31 Aug 2014   http://dx.doi.org:/10.1038/ncb3031

Tumorigenesis has been linked with macrophage-mediated chronic
inflammation and diverse signaling pathways, including the ​epidermal
growth factor receptor (​EGFR) pathway. ​EGFR is expressed in liver
macrophages in both human HCC and in a mouse HCC model. Mice
lacking ​EGFR in macrophages show impaired hepatocarcinogenesis,
Mice lacking ​EGFR in hepatocytes develop HCC owing to increased
hepatocyte damage and compensatory proliferation. EGFR is required
in liver macrophages to transcriptionally induce ​interleukin-6 following
interleukin-1 stimulation, which triggers hepatocyte proliferation and HCC.
Importantly, the presence of ​EGFR-positive liver macrophages in HCC
patients is associated with poor survival. This study demonstrates a

  • tumour-promoting mechanism for ​EGFR in non-tumour cells,
  • which could lead to more effective precision medicine strategies.
  1. HCC formation in mice lacking ​EGFRin hepatocytes or all liver cells.
    http://www.nature.com/ncb/journal/vaop/ncurrent/carousel/ncb3031-f1.jpg

2. EGFR expression in Kupffer cells/liver macrophages promotes HCC development.

EGFR c2a expression in Kupffer cells.liver macrophages promotes HCC development.

EGFR c2a expression in Kupffer cells.liver macrophages promotes HCC development.

http://www.nature.com/ncb/journal/vaop/ncurrent/carousel/ncb3031-f2.jpg

Hypoxia-inducible factor 1 activation by aerobic glycolysis implicates
the Warburg effect in carcinogenesis
.

Lu H1, Forbes RA, Verma A.
J Biol Chem. 2002 Jun 28;277(26):23111-5. Epub 2002 Apr 9

Cancer cells display high rates of aerobic glycolysis, a phenomenon
known historically as the Warburg effect. Lactate and pyruvate, the end
products of glycolysis, are highly produced by cancer cells even in the
presence of oxygen
.

Hypoxia-induced gene expression in cancer cells

  • has been linked to malignant transformation.

Here we provide evidence that lactate and pyruvate

  • regulate hypoxia-inducible gene expression
  • independently of hypoxia
  • by stimulating the accumulation of hypoxia-inducible Factor 1alpha
    (HIF-1alpha).

In human gliomas and other cancer cell lines,

  • the accumulation of HIF-1alpha protein under aerobic conditions
  • requires the metabolism of glucose to pyruvate that
  1. prevents the aerobic degradation of HIF-1alpha protein,
  2. activates HIF-1 DNA binding activity, and
  3. enhances the expression of several HIF-1-activated genes
  4. erythropoietin,
  5. vascular endothelial growth factor,
  6. glucose transporter 3, and
  7. aldolase A.

Our findings support a novel role for pyruvate in metabolic signaling
and suggest a mechanism by which

  • high rates of aerobic glycolysis
  • can promote the malignant transformation and
  • survival of cancer cells.PMID: 11943784

Part IV. Transcription control and innate immunity

 c-Myc-induced transcription factor AP4 is required for
host protection mediated by CD8+ T cells

C Chou, AK Pinto, JD Curtis, SP Persaud, M Cella, Chih-Chung Lin, … & T Egawa Nature Immunology 17 Jun 2014;   http://dx.doi.org:/10.1038/ni.2943

The transcription factor c-Myc is essential for

  • the establishment of a metabolically active and proliferative state
  • in T cells after priming,

We identified AP4 as the transcription factor

  • that was induced by c-Myc and
  • sustained activation of antigen-specific CD8+ T cells.

Despite normal priming,

  • AP4-deficient CD8+ T cells
  • failed to continue transcription of a broad range of
    c-Myc-dependent targets.

Mice lacking AP4 specifically in CD8+ T cells showed

  • enhanced susceptibility to infection with West Nile virus.

Genome-wide analysis suggested that

  • many activation-induced genes encoding molecules
  • involved in metabolism were shared targets of
  • c-Myc and AP4.

Thus, AP4 maintains c-Myc-initiated cellular activation programs

  • in CD8+ T cells to control microbial infection.
  1. AP4 is regulated post-transcriptionally in CD8+ T cells.

Microarray analysis of transcription factor–encoding genes with a difference
in expression of >1.8-fold in activated CD8+ T cells treated for 12 h with
IL-2 (100 U/ml; + IL-2) relative to their expression in activated CD8+ T cells…
http://www.nature.com/ni/journal/vaop/ncurrent/carousel/ni.2943-F1.jpg

2. AP4 is required for the population expansion of antigen specific
CD8+ T cells following infection with LCMV-Arm.

Expression of CD4, CD8α and KLRG1 (a) and binding of an
H-2Db–gp(33–41) tetramer and expression of CD8α, KLRG1 and
CD62L (b) in splenocytes from wild-type (WT) and Tfap4−/− mice,
assessed by flow cytometry 8 d after infection
http://www.nature.com/ni/journal/vaop/ncurrent/carousel/ni.2943-F2.jpg

3. AP4 is required for the sustained clonal expansion of CD8+ T cells
but  not for their initial proliferation.
http://www.nature.com/ni/journal/vaop/ncurrent/carousel/ni.2943-F3.jpg

  1. AP4 is essential for host protection against infection with WNV, in
    a CD8+ T cell–intrinsic manner.
AP4 is essential for host protection against infection with WNV, in a CD8+ T cell–intrinsic manner.

AP4 is essential for host protection against infection with WNV, in a CD8+ T cell–intrinsic manner.

  •  Survival of Tfap4F/FCre− control mice (Cre−; n = 16) and
  • Tfap4F/FCD8-Cre+ mice (CD8-Cre+; n = 22) following infection with WNV.
    (b,c) Viral titers in the brain (b) and spleen (c) of Tfap4F/F Cre− and Tfap4F/F
    CD8-Cre+ mice  on day 9…
    http://www.nature.com/ni/journal/vaop/ncurrent/carousel/ni.2943-F4.jpg

AP4 is essential for the sustained expression of genes that are targets of c-Myc.

Normalized signal intensity (NSI) of endogenous transcripts in
Tfap4+/+ and Tfap4−/− OT-I donor T cells adoptively transferred into
host mice and assessed on day 4 after infection of the host with LM-OVA
(top), and that of ERCC controls
http://www.nature.com/ni/journal/vaop/ncurrent/carousel/ni.2943-F6.jpg

Sustained c-Myc expression ‘rescues’ defects of Tfap4−/− CD8+ T cells.
http://www.nature.com/ni/journal/vaop/ncurrent/carousel/ni.2943-F7.jpg

AP4 and c-Myc have distinct biological functions.
http://www.nature.com/ni/journal/vaop/ncurrent/carousel/ni.2943-SF7.jpg

Mucosal memory CD8+ T cells are selected in the periphery
by an MHC class I molecule

Y Huang, Y Park, Y Wang-Zhu, …A Larange, R Arens, & H Cheroutre

Nature Immunology 2 Oct 2011; 12: 1086–1095
http://dx.doi.org:/10.1038/ni.2106

The presence of immune memory at pathogen-entry sites is a prerequisite
for protection. We show that the non-classical major histocompatibility
complex (MHC) class I molecule

  • thymus leukemia antigen (TL),
  • induced on dendritic cells interacting with CD8αα on activated CD8αβ+ T cells,
  • mediated affinity-based selection of memory precursor cells.

Furthermore, constitutive expression of TL on epithelial cells

  • led to continued selection of mature CD8αβ+ memory T cells.

The memory process driven by TL and CD8αα

  • was essential for the generation of CD8αβ+ memory T cells in the intestine and
  • the accumulation of highly antigen-sensitive CD8αβ+ memory T cells
  • that form the first line of defense at the largest entry port for pathogens.

The metabolic checkpoint kinase mTOR is essential for IL-15 signaling during the development and activation of NK cells.

Marçais A, Cherfils-Vicini J, Viant C, Degouve S, Viel S, Fenis A, Rabilloud J,
Mayol K, Tavares A, Bienvenu J, Gangloff YG, Gilson E, Vivier E,Walzer T.
Nat Immunol. 2014 Aug; 15(8):749-757. Epub 2014 Jun 29
http://dx.doi.org:/10.1038/ni.2936  .    PMID: 24973821

Interleukin 15 (IL-15) controls

  • both the homeostasis and the peripheral activation of natural killer (NK) cells.

We found that the metabolic checkpoint kinase

  • mTOR was activated and boosted bioenergetic metabolism
  • after exposure of NK cells to high concentrations of IL-15,

whereas low doses of IL-15 triggered

  • only phosphorylation of the transcription factor STAT5.

mTOR

  • stimulated the growth and nutrient uptake of NK cells and
  • positively fed back on the receptor for IL-15.

This process was essential for

  • sustaining NK cell proliferation during development and
  • the acquisition of cytolytic potential during inflammation
    or viral infection.

The mTORC1 inhibitor rapamycin 

  • inhibited NK cell cytotoxicity both in mice and humans;
    • this probably contributes to the immunosuppressive
      activity of this drug in different clinical settings.

The Critical Role of IL-15-PI3K-mTOR Pathway in Natural Killer Cell
Effector Functions.
Nandagopal NAli AKKomal AKLee SH.   Author information
Front Immunol. 2014 Apr 23; 5:187. eCollection 2014.
http://dx.doi.org:/10.3389/fimmu.2014.00187

Natural killer (NK) cells were so named for their uniqueness in killing
certain tumor and virus-infected cells without prior sensitization.
Their functions are modulated in vivo by several soluble immune mediators;

  • interleukin-15 (IL-15) being the most potent among them in
    enabling NK cell homeostasis, maturation, and activation.

During microbial infections,

  • NK cells stimulated with IL-15 display enhanced cytokine responses.

This priming effect has previously been shown with respect to increased
IFN-γ production in NK cells

  • upon IL-12 and IL-15/IL-2 co-stimulation.
  • we explored if this effect of IL-15 priming 
  • can be extended to various other cytokines and
  • observed enhanced NK cell responses to stimulation
    • with IL-4, IL-21, IFN-α, and IL-2 in addition to IL-12.
  • we also observed elevated IFN-γ production in primed NK cells

Currently, the fundamental processes required for priming and

  • whether these signaling pathways work collaboratively or
    independently 

    • for NK cell functions are poorly understood.

We examined IL-15 effects on NK cells in which

  • the pathways emanating from IL-15 receptor activation
    • were blocked with specific inhibitors
    • To identify the key signaling events for NK cell priming,

Our results demonstrate that

the PI3K-AKT-mTOR pathway is critical for cytokine responses
in IL-15 primed NK cells. 

This pathway is also implicated in a broad range of

  • IL-15-induced NK cell effector functions such as
    • proliferation and cytotoxicity.

Likewise, NK cells from mice

  • treated with rapamycin to block the mTOR pathway
  • displayed defects in proliferation, and IFN-γ and granzyme B productions
  • resulting in elevated viral burdens upon murine cytomegalovirus infection.

Taken together, our data demonstrate

  • the requirement of PI3K-mTOR pathway
    • for enhanced NK cell functions by IL-15, thereby
  • coupling the metabolic sensor mTOR to NK cell anti-viral responses.

KEYWORDS: IL-15; JAK–STAT pathway; mTOR pathway; natural killer cells; signal transduction

Part V. Predicting Therapeutic Targets 

New discovery approach accelerates identification of potential cancer treatments
 Laura Williams, Univ. of Michigan   09/30/2014
http://www.rdmag.com/news/2014/09/new-discovery-approach-accelerates-identification-potential-cancer-treatments

Researchers at the Univ. of Michigan have described a new approach to
discovering potential cancer treatments that

  • requires a fraction of the time needed for more traditional methods.

They used the platform to identify

  • a novel antibody that is undergoing further investigation as a potential
    treatment for breast, ovarian and other cancers.

In research published online in the Proceedings of the National Academy
of Sciences
, researchers in the laboratory of Stephen Weiss at the U-M Life
Sciences Institute detail an approach

  • that replicates the native environment of cancer cells and
  • increases the likelihood that drugs effective against the growth of
    tumor cells in test tube models
  • will also stop cancer from growing in humans.

The researchers have used their method

  • to identify an antibody that stops breast cancer tumor growth in animal models, and
  • they are investigating the antibody as a potential treatment in humans.

“Discovering new targets for cancer therapeutics is a long and tedious undertaking, and

  • identifying and developing a potential drug to specifically hit that
    target without harming healthy cells is a daunting task,” Weiss said.
  • “Our approach allows us to identify potential therapeutics
    • in a fraction of the time that traditional methods require.”

The researchers began by

  • creating a 3-D “matrix” of collagen, a connective tissue molecule very similar to that found
    • surrounding breast cancer cells in human patients.
  • They then embedded breast cancer cells into the collagen matrix,
    • where the cells grew as they would in human tissue.

The investigators then injected the cancer-collagen tissue composites into mice that then

  • recognize the human cancer cells as foreign tissue.
    • Much in the way that our immune system generates antibodies
      to fight infection,
  • the mice began to generate thousands of antibodies directed against
    the human cancer cells.
  • These antibodies were then tested for the ability to stop the growth
    of the human tumor cells.

“We create an environment in which cells cultured in the laboratory ‘think’
they are growing in the body and then

  • rapidly screen large numbers of antibodies to see if any exert
    anti-cancer effects,” Weiss said.
  • “This allows us to select promising antibodies very quickly and then

They discovered a particular antibody, 4C3, which was able to

  • almost completely stop the proliferation of the breast cancer cells.

They then identified the molecule on the cancer cells that the antibody targets.

The antibody can be further engineered to generate

  • humanized monoclonal antibodies for use in patients

“We still need to do a lot more work to determine how effective 4C3 might be as a
treatment for breast and other cancers, on its own or in conjunction with other
therapies,” Weiss said. “But we have enough data to warrant further pursuit,
and are expanding our efforts to use this discovery platform to find similarly promising antibodies.”

Source: Univ. of Michigan

  1. Jose Eduardo de Salles Roselino

    Larry,
    I think you have made a great effort in order to connect basic ideas of metabolic regulation with those of gene expression control “modern” mechanisms.
    Yet, I do not think that at this stage it will be clear for all readers. At least, for the great majority of the readers. The most important factor I my opinion, is derived from the fact that modern readers considers that metabolic regulation deals with so called “housekeeping activities” of the cell. Something that is of secondary, tertiary or even less level of relevance.
    My idea, that you have mentioned in the text when you write at the beginning, the word biochemistry, in order to resume it, derives from the reading of What is life together with Prof. Leloir . For me and also, for him, biochemistry comprises a set of techniques and also a framework of reasoning about scientific results. As a set of techniques, Schrodinger has considered that it will lead to better understanding of genetics and of physiology as a two legs structure supporting the future progress related to his time (mid-forties). For Leloir, the key was the understanding of chemical reactivity and I agree with him. However, as I was able to talk and discuss it with him in detail, we should also take into account levels of stabilities of macromolecules and above all, regulation of activities and function (this is where) Pasteur effect that I was studying in Leloir´s lab at that time, 1970-72, gets into the general picture.
    Regulation for complex living beings , that also have cancer cell as a great topic of research problem can be understood through the understanding of two quite different results when opposition with lack of regulation is taken into account or experimentally elicited. The most clearly line of experiments can follow the Pasteur Effect as the intracellular result best seen when aerobiosis is compared with anaerobiosis as conditions in which maintenance of ATP levels and required metabolic regulation (Energy charge D.E, Atkinson etc) is studied. Another line of experiments is one that takes into account the extracellular result or for instance the homeostatic regulation of blood glucose levels. The blood glucose level is the most conspicuous and related to Pasteur Effect regulatory event that can be studied in the liver taking into account both final results tested or compared regarding its regulation, ATP levels maintenance (intracellular) and blood glucose maintenance (extracellular).
    My key idea is to consider that the same factors that elicits fast regulatory responses also elicits the slow energetic expensive regulatory responses. The biologic logic behind this common root is the ATP economy. In case, the regulatory stimulus fades out quickly the fast regulatory responses are good enough to maintain life and the time requiring, energetic costly responses will soon be stopped cutting short the ATP expenditure. In case, the stimulus last for long periods of time the fast responses are replaced by adaptive responses that in general will follow the line of cell differentiation mechanisms with changes in gene expression etc.
    The change from fast response mechanisms to long lasting developmentally linked ones is not sharp. Therefore, somehow, cancer cells becomes trapped into a metastable regulatory mechanism that prevents cell differentiation and reinforces those mechanisms linked to its internal regulatory goals. This metastable mechanism takes advantage from the fact that other cells, tissues and organs will take good care of homeostatic mechanisms that provide for their nutritional needs. In the case of my Hepatology work you will see a Piruvate kinase that does not responds to homeostatic signals .

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Curator & Author: Larry H. Bernstein, MD, FCAP

Leaders in Pharmaceutical Intelligence

Subtitle: Nitric Oxide, Peroxinitrite, and NO donors in Renal Function Loss

Summary: The criticality of renal function is traced to the emergence of animal forms from the sea to land. It also becomes acutely and/or chronically dysfunctional in metabolic, systemic inflammatory and immunological diseases of man. We have already described the key role that nitric oxide and the NO synthases play in reduction of oxidative stress, and we have seen that a balance has to be struck between pro- and anti-oxidative as well as inflammatory elements for avoidance of diseases, specifically involving the circulation, but effectively not limited to any organ system. In this discussion we shall look at kidney function, NO and NO donors. This is an extension of a series of posts on NO and NO related disorders.

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Part I. The evolution of kidney structure and Function Evolution of kidney function

In fish the nerves that activate breathing take a short journey from an ancient part of the brain, the brain stem, to the throat and gills. For the ancient tadpole, the nerve controlling a reflex related to hiccup in man served a useful purpose, allowing the entrance to the lung to remain open when breathing air but closing it off when gulping water – which would then be directed only to the gills.

For humans and other mammals it provides a bit of evidence of our common ancestry. DNA evidence has pinned iguanas and chameleons as the closest relatives to snakes. In utero, we develop three separate kidneys in succession, absorbing the first two before we wind up with the embryonic kidney that will become our adult kidney. The first two of these reprise embryonic kidneys of ancestral forms, and in the proper evolutionary order.

The pronephric kidney does not function in human and other mammalian embryos. It disappears and gives rise to the Mesonephric kidney. This kidney filters wastes from the blood and excretes them to the outside of the body via a pair of tubes called the mesonephric ducts (also “Wolffian ducts”). The mesonephric kidney goes on to develop into the adult kidney of fish and amphibians.

This kidney does function for a few weeks in the human embryo, but then disappears as our final kidney forms, which is the Metanephric kidney. This begins developing about five weeks into gestation, and consists of an organ that filters wastes from the blood and excretes them to the outside through a pair ureters. In the embryo, the wastes are excreted directly into the amniotic fluid. The metanephric kidney is the final adult kidney of reptiles, birds, and mammals.

The first two kidneys resemble, in order, those of primitive aquatic vertebrates (lampreys and hagfish) and aquatic or semiaquatic vertebrates (fish and amphibians): an evolutionary order.

The explanation, then, is that we go through developmental stages that show organs resembling those of our ancestors. Take a step back and we see that fresh water fish have glomerular filtration. Cardiac contraction provides the pressure to force the water, small molecules, and ions into the glomerulus as nephric filtrate. The essential ingredients are then reclaimed by the tubules, returning to the blood in the capillaries surrounding the tubules. The amphibian kidney also functions chiefly as a device for excreting excess water.

But the problem is to conserve water, not eliminate it. The frog adjusts to the varying water content of its surroundings by adjusting the rate of filtration at the glomerulus. When blood flow through the glomerulus is restricted, a renal portal system is present to carry away materials reabsorbed through the tubules. Bird kidneys function like those of reptiles (from which they are descended). Uric acid is also their chief nitrogenous waste. All mammals share our use of urea as their chief nitrogenous waste. Urea requires much more water to be excreted than does uric acid. Mammals produce large amounts of nephric filtrate but are able to reabsorb most of this in the tubules. But even so, humans lose several hundred ml each day in flushing urea out of the body.

In his hypothesis of the evolution of renal function Homer Smith proposed that the formation of glomerular nephron and body armor had been adequate for the appearance of primitive vertebrates in fresh water and that the adaptation of homoiotherms to terrestrial life was accompanied by the appearance of the loop of Henle.

In the current paper, the increase in the arterial blood supply and glomerular filtration rate and the sharp elevation of the proximal reabsorption are viewed as important mechanisms in the evolution of the kidney. The presence of glomeruli in myxines and of nephron loops in lampreys suggests that fresh water animals used the preformed glomerular apparatus of early vertebrates, while mechanisms of urinary concentration was associated with the subdivision of the kidney into the renal cortex and medulla. The principles of evolution of renal functions can be observed at several levels of organizations in the kidney.

Natochin YV. Evolutionary aspects of renal function. Kidney International 1996; 49: 1539–1542; doi:10.1038/ki.1996.220. Smith HW: From Fish to Philosopher. Boston, Little, Brown, 1953.

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The Kidney: Anatomy and Physiology

The kidney lies in the lower abdomen capped by the adrenal glands. It has an outer cortex and an inner medulla. The basic unit is the nephron, which filters blood at the glomerulus, and not only filters urine eliminating mainly urea, also uric acid, and other nitrogenous waste, but also reabsorbs Na+ in exchange for H+/(reciprocal K+) through the carbonic anhydrase of the epithelium. In addition, it serves as a endocrine organ and receptor through the renin-angiotensin/aldosterone system, sensitivity to water loss controlled by antidiuretic hormone, and is sensitive to the natriuretic peptides of the heart. The kidney is an elegant structure with a high concentration of glomeruli in the cortex, and in the medulla one finds a U-shaped tube that is critical in a countercurrent multiplier system with a descending limb, Loop of Henley, and ascending limb.

As the filtrate flows through the glomerulus into the descending limb, there is reabsorption of glucose and of H+ by the carbonic anhydrase conversion to water and CO2, except with serious acidemia, in which K+ is reabsorbed with H+ loss to the filtrate, resulting in a hyperkalemia. In the descending limb Na+ is absorbed into the interstitium, and the hypertonic interstitium draws water back for circulation, regulated by the action of ADH on the epithelium of the ascending limb. The result in terms of basic urinary clearance, the volume of urine loss is moderated by the amount needed for circulation (10 units of whole blood) without dehydration, and an amount sufficient for metabolite loss (including drug metabolites). The urine flows into the kidney pelvis and flow down the ureters.

The renal blood flow needs mention. The blood reaches the glomerulus by way of the afferent arteriole and leaves by way of the efferent arteriole. In a book by the Harvard Pathologist Shields Warren on diabetes he made a distinction between hypertension and diabetes in that efferent arteriolar sclerosis is present in both, but diabetes is uniquely identified by afferent arteriolar sclerosis. In diabetes you also have a typical glomerulosclerosis, which might be related to the same hyalinization found in the pancreatic islets – a secondary amyloidosis.

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English: Nephron, Diagram of the urine formati...

English: Nephron, Diagram of the urine formation. The number inside tubular urin concentration in mOsm/l – when ADH acts Polski: Nefron, Schemat tworzenia moczu. Cyfry wewnątrz kanalików oznaczają lokalne stężenie w mOsm/l – gdy działa ADH (dochodzi do zagęszczania moczu). (Photo credit: Wikipedia)

Loop of Henle (Grey's Anatomy book)

Loop of Henle (Grey’s Anatomy book) (Photo credit: Wikipedia)

Frontal section through the kidney

Frontal section through the kidney (Photo credit: Wikipedia)

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_ Part IIa. Nitric Oxide role in renal tubular epithelial cell function Tubulointerstitial Nephritides

As part of the exponential growth in our understanding of nitric oxide (NO) in health and disease over the past 2 decades, the kidney has become appreciated as a major site where NO may play a number of important roles. Although earlier work on the kidney focused more on effects of NO at the level of larger blood vessels and glomeruli, there has been a rapidly growing body of work showing critical roles for NO in tubulointerstitial disease. In this review we discuss some of the recent contributions to this important field.

Mattana J, Adamidis A, Singhal PC. Nitric oxide and tubulointerstitial nephritides. Seminars in Nephrology 2004; 24(4):345-353.

Nitric oxide donors and renal tubular (subepithelial) matrix

Nitric oxide (NO) and its metabolite, peroxynitrite (ONOO-), are involved in renal tubular cell injury. If NO/ONOO- has an effect to reduce cell adhesion to the basement membrane, does this effect contribute to tubular obstruction and would it be partially responsible for the harmful effect of NO on the tubular epithelium during acute renal failure (ARF)?

Wangsiripaisan A, et al. examined the effect of the NO donors

  • [1] (z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1- ium-1, 2-diolate (DETA/NO),
  • [2] spermine NONOate (SpNO), and
  • [3] the ONOO- donor 3-morpholinosydnonimine (SIN-1) on

cell-matrix adhesion to collagen types I and IV, and also fibronectin using three renal tubular epithelial cell lines:

  • [1] LLC-PK1,
  • [2] BSC-1, and
  • [3] OK.

It was only the exposure to SIN-1 that caused a dose-dependent impairment in cell-matrix adhesion.

Similar results were obtained in the different cell types and matrix proteins. The effect of SIN-1 (500 microM) on LLC-PK1 cell adhesion was not associated with either cell death or alteration of matrix protein and was attenuated by either

  • [1] the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide,
  • [2] the superoxide scavenger superoxide dismutase, or
  • [3] the ONOO- scavenger uric acid in a dose-dependent manner.

These investigators concluded in this seminal paper that ONOO- generated in the tubular epithelium during ischemia/reperfusion has the potential to impair the adhesion properties of tubular cells, which then may contribute to the tubular obstruction in ARF.

Wangsiripaisan A, Gengaro PE, Nemenoff RA, Ling H, et al. Effect of nitric oxide donors on renal tubular epithelial cell-matrix adhesion. Kidney Int 1999; 55(6):2281-8.

Coexpressed Nitric Oxide Synthase and Apical β1 Integrins

In sepsis-induced acute renal failure, actin cytoskeletal alterations result in shedding of proximal tubule epithelial cells (PTEC) and tubular obstruction.

This study examined the hypothesis that inflammatory cytokines, released early in sepsis, cause PTEC cytoskeletal damage and alter integrin-dependent cell-matrix adhesion. The question of whether the intermediate nitric oxide (NO) modulates these cytokine effects was also examined. After exposure of human PTEC to tumor necrosis factor-α, interleukin-1α, and interferon-γ, the actin cytoskeleton was disrupted and cells became elongated, with extension of long filopodial processes.

Cytokines induced shedding of viable, apoptotic, and necrotic PTEC, which was dependent on NO synthesized by inducible NO synthase (iNOS) produced as a result of cytokine actions on PTEC. Basolateral exposure of polarized PTEC monolayers to cytokines induced maximal NO-dependent cell shedding, mediated in part through NO effects on cGMP. Cell shedding was accompanied by dispersal of basolateral β1 integrins and E-cadherin, with corresponding upregulation of integrin expression in clusters of cells elevated above the epithelial monolayer.

These cells demonstrated coexpression of iNOS and apically redistributed β1 integrins. These authors point out that the major ligand involved in cell anchorage was laminin, probably through interactions with the integrin α3β1.

This interaction was downregulated by cytokines but was not dependent on NO. They posulate a mechanism by which inflammatory cytokines induce PTEC damage in sepsis, in the absence of hypotension and ischemia.

Glynne PA, Picot J and Evans TJ. Coexpressed Nitric Oxide Synthase and Apical β1 Integrins Influence Tubule Cell Adhesion after Cytokine-Induced Injury. JASN 2001; 12(11): 2370-2383.

Potentiation by Nitric Oxide of Apoptosis in Renal Proximal Tubule Cells

Proximal tubular epithelial cells (PTEC) exhibit a high sensitivity to undergo apoptosis in response to proinflammatory stimuli and immunosuppressors and participate in the onset of several renal diseases. This study examined the expression of inducible nitric oxide (NO) synthase after challenge of PTEC with bacterial cell wall molecules and inflammatory cytokines and analyzed the pathways that lead to apoptosis in these cells by measuring changes in the mitochondrial transmembrane potential and caspase activation.

The data show that the apoptotic effects of proinflammatory stimuli mainly were due to the expression of inducible NO synthase. Cyclosporin A and FK506 inhibited partially NO synthesis.

However, both NO and immunosuppressors induced apoptosis, probably through a common mechanism that involved the irreversible opening of the mitochondrial permeability transition pore. Activation of caspases 3 and 7 was observed in cells treated with high doses of NO and with moderate concentrations of immunosuppressors.

The conclusion is that the cooperation between NO and immunosuppressors that induce apoptosis in PTEC might contribute to the renal toxicity observed in the course of immunosuppressive therapy.

Hortelano S, Castilla M, Torres AM, Tejedor A, and Bosca L.  Potentiation by Nitric Oxide of Cyclosporin A and FK506- Induced Apoptosis in Renal Proximal Tubule Cells. J Am Soc Nephrol 2000; 11: 2315–2323.

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Part IIb. Related studies with ROS and/or RNS on nonrenal epithelial cells

Reactive nitrogen species block cell cycle re-entry Endogenous sources of reactive nitrogen species (RNS) act as second messengers in a variety of cell signaling events, whereas environmental sources of RNS like nitrogen dioxide (NO2) inhibit cell survival and growth through covalent modification of cellular macromolecules. Murine type II alveolar cells arrested in G0 by serum deprivation were exposed to either NO2 or SIN-1, a generator of RNS, during cell cycle re-entry.

In serum-stimulated cells, RNS blocked cyclin D1 gene expression, resulting in cell cycle arrest at the boundary between G0 and G1. Dichlorofluorescin diacetate (DCF) fluorescence indicated that RNS induced sustained production of intracellular hydrogen peroxide (H2O2), which normally is produced only transiently in response to serum growth factors.

Loading cells with catalase prevented enhanced DCF fluorescence and rescued cyclin D1 expression and S phase entry.

These studies indicate environmental RNS interfere with cell cycle re-entry through an H2O2-dependent mechanism that influences expression of cyclin D1 and progression from G0 to the G1 phase of the cell cycle.

Yuan Z, Schellekens H, Warner L, Janssen-Heininger Y, Burch P, Heintz NH. Reactive nitrogen species block cell cycle re-entry through sustained production of hydrogen peroxide. Am J Respir Cell Mol Biol. 2003;28(6):705-12. Epub 2003 Jan 10.

Peroxynitrite modulates MnSOD gene expression

Peroxynitrite (ONOO-) is a strong oxidant derived from nitric oxide (‘NO) and superoxide (O2.-), reactive nitrogen (RNS) and oxygen species (ROS) present in inflamed tissue. Other oxidant stresses, e.g., TNF-alpha and hyperoxia,   induce mitochondrial, manganese-containing superoxide dismutase (MnSOD) gene expression.   3-morpholinosydnonimine HCI (SIN-1) (10 or 1000 microM) increased MnSOD mRNA, but did not change hypoxanthine guanine phosphoribosyl transferase (HPRT) mRNA.   Authentic peroxynitrite (ONOO ) (100-500 microM) also increased MnSOD mRNA but did not change constitutive HPRT mRNA expression.   ONOO stimulated luciferase gene expression driven by a 2.5 kb fragment of the rat MnSOD gene 5′ promoter region.

MnSOD gene induction due to ONOO- was

  • [1] inhibited effectively by L-cysteine (10 mM) and
  • [2] partially inhibited by N-acetyl cysteine (NAC)(50 mM) or
  • [3] pyrrole dithiocarbamate (10 mM).

.NO from 1-propanamine, 3-(2-hydroxy-2-nitroso-1-propylhydrazine) (PAPA NONOate) (100 or 1000 microM) did not change MnSOD or HPRT mRNA, nor did either H202 or NO2-, breakdown products of SIN-1 and ONOO, have any effect on MnSOD mRNA expression; ONOO- and SIN-1 also did not increase detectable MnSOD protein content or increase MnSOD enzymatic activity.

Nevertheless, increased steady state [O2.-] in the presence of .NO yields ONOO , and ONOO has direct, stimulatory effects on MnSOD transcript expression driven at the MnSOD gene 5′ promoter region inhibited completely by L-cysteine and partly by N-acetyl cysteine in lung epithelial cells. This raises a question of whether the same effect is seen in renal tubular epithelium.

Jackson RM, Parish G, Helton ES. Peroxynitrite modulates MnSOD gene expression in lung epithelial cells. Free Radic Biol Med. 1998; 25(4-5):463-72.

Comparative impacts of glutathione peroxidase-1 gene knockout on oxidative stress

Selenium-dependent glutathione peroxidase-1 (GPX1) protects against reactive-oxygen-species (ROS)-induced oxidative stress in vivo, but its role in coping with reactive nitrogen species (RNS) is unclear. Primary hepatocytes were isolated from GPX1-knockout (KO) and wild-type (WT) mice to test protection of GPX1 against cytotoxicity of

  • [1] superoxide generator diquat (DQ),
  • [2]NO donor S-nitroso-N-acetyl-penicillamine (SNAP) and
  • [3] peroxynitrite generator 3-morpholinosydnonimine (SIN-1).

Treating cells with SNAP in addition to DQ produced synergistic cytotoxicity that minimized differences in apoptotic cell death and oxidative injuries between the KO and WT cells. Less protein nitrotyrosine was induced by 0.05-0.5 mM DQ+0.25 mM SNAP in the KO than in the WT cells.

Total GPX activity in the WT cells was reduced by 65 and 25% by 0.5 mM DQ+0.1 mM SNAP and 0.5 mM DQ, respectively. Decreases in Cu,Zn-superoxide dismutase (SOD) activity and increases in Mn-SOD activity in response to DQ or DQ+SNAP were greater in the KO cells than in the WT cells.

The study indicates GPX1 was more effective in protecting hepatocytes against oxidative injuries mediated by ROS alone than by ROS and RNS together, and knockout of GPX1 did not enhance cell susceptibility to RNS-associated cytotoxicity. Instead, it attenuated protein nitration induced by DQ+SNAP.

To better understand the mechanism(s) underlying nitric oxide (. NO)-mediated toxicity, in the presence and absence of concomitant oxidant exposure, postmitotic terminally differentiated NT2N cells (which are incapable of producing . NO) were exposed to [1]PAPA-NONOate (PAPA/NO) and [2] 3-morpholinosydnonimine (SIN-1).

Exposure to SIN-1, which generated peroxynitrite (ONOO) in the range of 25-750 nM/min, produced a concentration- and time-dependent delayed cell death.   In contrast, a critical threshold concentration (>440 nM/min) was required for . NO to produce significant cell injury.   There is a largely necrotic lesion after ONOO exposure and an apoptotic-like morphology after . NO exposure.

Cellular levels of reduced thiols correlated with cell death, and pretreatment with N-acetylcysteine (NAC) fully protected from cell death in either PAPA/NO or SIN-1 exposure. NAC given within the first 3 h posttreatment further delayed cell death and increased the intracellular thiol level in SIN-1 but not . NO-exposed cells.

Cell injury from . NO was independent of cGMP, caspases, and superoxide or peroxynitrite formation.   Overall, exposure of non-. NO-producing cells to . NO or peroxynitrite results in delayed cell death, which, although occurring by different mechanisms,   appears to be mediated by the loss of intracellular redox balance.

Gow AJ, Chen Q, Gole M, Themistocleous M, Lee VM, Ischiropoulos H. Two distinct mechanisms of nitric oxide-mediated neuronal cell death show thiol dependency. Am J Physiol Cell Physiol. 2000; 278(6):C1099-107.

NO2 effect on phosphatidyl choline   Nitrogen dioxide (NO2) inhalation affects the extracellular surfactant as well as the structure and function of type II pneumocytes.

The studies had differences in oxidant concentration, duration of exposure, and mode of NO2 application. This study evaluated the influence of the NO2 application mode on the phospholipid metabolism of type II pneumocytes. Rats were exposed to identical NO2 body doses (720 ppm x h), which were applied continuously (10 ppm for 3 d), intermittently (10 ppm for 8 h per day, for 9 d), and repeatedly (10 ppm for 3 d, 28 d rest, and then 10 ppm for 3 d). Immediately after exposure, type II cells were isolated and evaluated for cell yield, vitality, phosphatidylcholine (PC) synthesis, and secretion.

Type II pneumocyte cell yield was only increased from animals that had been continuously exposed to NO2, but vitality of the isolated type II pneumocytes was not affected by the NO2 exposure modes. Continuous application of 720 ppm x h NO2 resulted in increased activity of the cytidine-5-diphosphate (CDP)-choline pathway.   After continuous NO2 application,

  • [1] specific activity of choline kinase,
  • [2] cytidine triphosphate (CTP):cholinephosphate cytidylyltransferase,
  • [3] uptake of choline, and
  • [4] pool sizes of CDP-choline and PC   were significantly increased over those of controls.

Intermittent application of this NO2 body dose provoked less increase in PC synthesis and the synthesis parameters were comparable to those for cells from control animals after repeated exposure. Whereas PC synthesis in type II cells was stimulated by NO2, their secretory activity was reduced.   Continuous exposure reduced the secretory activity most, whereas intermittent exposure nonsignificantly reduced this activity as compared with that of controls. The repeated application of NO2 produced no differences.

The authors conclude that…. type II pneumocytes adapt to NO2 atmospheres depending on the mode of its application, at least for the metabolism of PC and its secretion from isolated type II pneumocytes.

The reader asks whether this effect could also be found in renal epithelial cells, for which PC is not considered vital as for type II pneumocytes and possibly related to surfactant activity in the lung.

Müller B, Seifart C, von Wichert P, Barth PJ. Adaptation of rat type II pneumocytes to NO2: effects of NO2 application mode on phosphatidylcholine metabolism. Am J Respir Cell Mol Biol. 1998; 18(5): 712-20.

iNOS involved in immediate response to anaphylaxis

The generation of large quantities of nitric oxide (NO) is implicated in the pathogenesis of anaphylactic shock. The source of NO, however, has not been established and conflicting results have been obtained when investigators have tried to inhibit its production in anaphylaxis.

This study analyzed the expression of inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) in a mouse model of anaphylaxis.   BALB/c mice were sensitized and challenged with ovalbumin to induce anaphylaxis. Tissues were removed from the heart and lungs, and blood was drawn at different time points during the first 48 hours after induction of anaphylaxis. The Griess assay was used to measure nitric oxide generation.

Nitric oxide synthase expression was examined by reverse transcriptase polymerase chain reaction and immunohistochemistry. A significant increase in iNOS mRNA expression and nitric oxide production was evident as early as 10 to 30 minutes after allergen challenge in both heart and lungs.

In contrast, expression of eNOS mRNA was not altered during the course of the experiment. The results support involvement of iNOS in the immediate physiological response of anaphylaxis.

Sade K, Schwartz IF, Etkin S, Schwartzenberg S, et al. Expression of Inducible Nitric Oxide Synthase in a Mouse Model of Anaphylaxis. J Investig Allergol Clin Immunol 2007; 17(6):379-385.

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Part IIc. Additional Nonrenal Related NO References

1. Nitrogen dioxide induces death in lung epithelial cells in a density-dependent manner. Persinger RL, Blay WM, Heintz NH, Hemenway DR, Janssen-Heininger YM. Am J Respir Cell Mol Biol. 2001 May;24(5):583-90. PMID: 11350828 [PubMed – indexed for MEDLINE] Free Article

2. Molecular mechanisms of nitrogen dioxide induced epithelial injury in the lung. Persinger RL, Poynter ME, Ckless K, Janssen-Heininger YM. Mol Cell Biochem. 2002 May-Jun;234-235(1-2):71-80. Review. PMID: 12162462 [PubMed – indexed for MEDLINE]

3. Nitric oxide and peroxynitrite-mediated pulmonary cell death. Gow AJ, Thom SR, Ischiropoulos H. Am J Physiol. 1998 Jan;274(1 Pt 1):L112-8. PMID: 9458808 [PubMed – indexed for MEDLINE] Free Article

4. Mitogen-activated protein kinases mediate peroxynitrite-induced cell death in human bronchial epithelial cells. Nabeyrat E, Jones GE, Fenwick PS, Barnes PJ, Donnelly LE. Am J Physiol Lung Cell Mol Physiol. 2003 Jun;284(6):L1112-20. Epub 2003 Feb 21. PMID: 12598225 [PubMed – indexed for MEDLINE] Free Article

5. Peroxynitrite inhibits inducible (type 2) nitric oxide synthase in murine lung epithelial cells in vitro. Robinson VK, Sato E, Nelson DK, Camhi SL, Robbins RA, Hoyt JC. Free Radic Biol Med. 2001 May 1;30(9):986-91. PMID: 11316578 [PubMed – indexed for MEDLINE]

6. Nitric oxide-mediated chondrocyte cell death requires the generation of additional reactive oxygen species. Del Carlo M Jr, Loeser RF. Arthritis Rheum. 2002 Feb;46(2):394-403. PMID: 11840442 [PubMed – indexed for MEDLINE]

7. Colon epithelial cell death in 2,4,6-trinitrobenzenesulfonic acid-induced colitis is associated with increased inducible nitric-oxide synthase expression and peroxynitrite production.

Yue G, Lai PS, Yin K, Sun FF, Nagele RG, Liu X, Linask KK, Wang C, Lin KT, Wong PY. J Pharmacol Exp Ther. 2001 Jun;297(3):915-25. PMID: 11356911 [PubMed – indexed for MEDLINE] Free Article

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Part IIIa. Acute renal failure   Acute renal failure (ARF), characterized by sudden loss of the ability of the kidneys to [1] excrete wastes, [2] concentrate urine, [3] conserve electrolytes, and [4] maintain fluid balance, is a frequent clinical problem, particularly in the intensive care unit, where it is associated with a mortality of between 50% and 80%.

This clinical entity was described as an acute loss of kidney function that occurred in severely injured crush victims because of histological evidence for patchy necrosis of renal tubules at autopsy. In the clinical setting, the terms ATN and acute renal failure (ARF) are frequently used interchangeably. However, ARF does not include increases in blood urea due to [1] reversible renal vasoconstriction (prerenal azotemia) or [2] urinary tract obstruction (postrenal azotemia). Acute hemodialysis was first used clinically during the Korean War in 1950 to treat military casualties, and this led to a decrease in mortality of the ARF clinical syndrome from about 90% to about 50%.   In the half century that has since passed, much has been learned about the pathogenesis of ischemic and nephrotoxic ARF in experimental models, but there has been very little improvement in mortality. This may be explained by changing demographics: [1] the age of patients with ARF continues to rise, and [2] comorbid diseases are increasingly common in this population. Both factors may obscure any increased survival related to improved critical care. Examining the incidence of ARF in several military conflicts does, however, provide some optimism. The incidence of ARF in seriously injured casualties decreased between World War II and the Korean War, and again between that war and the Vietnam War, despite the lack of any obvious difference in the severity of the injuries. What was different was the rapidity of the fluid resuscitation of the patients? Fluid resuscitation on the battlefield with the rapid evacuation of the casualties to hospitals by helicopter began during the Korean War and was optimized further during the Vietnam War. For seriously injured casualties the incidence of ischemic ARF was one in 200 in the Korean War and one in 600 in the Vietnam War. This historical sequence of events suggests that early intervention could prevent the occurrence of ARF, at least in military casualties.   In experimental studies it has been shown that progression from an azotemic state associated with renal vasoconstriction and intact tubular function (prerenal azotemia) to established ARF with tubular dysfunction occurs if the renal ischemia is prolonged. Moreover, early intervention with fluid resuscitation was shown to prevent the progression from prerenal azotemia to established ARF. Diagnostic evaluation of ARF One important question, therefore, is how to assure that an early diagnosis of acute renal vasoconstriction can be made prior to the occurrence of tubular dysfunction, thus providing the potential to prevent progression to established ARF. In this regard, past diagnostics relied on observation of the patient response to a fluid challenge: [1] decreasing levels of blood urea nitrogen (BUN) indicated the presence of reversible vasoconstriction, [2] while uncontrolled accumulation of nitrogenous waste products, i.e., BUN and serum creatinine, indicated established ARF.

This approach, however, frequently led to massive fluid overload in the ARF patient with resultant

  • [1] pulmonary congestion,
  • [2] hypoxia, and
  • [3] premature need for mechanical ventilatory support and/or hemodialysis.

On this background the focus turned to an evaluation of urine sediment and urine chemistries to differentiate between renal vasoconstriction with intact tubular function and established ARF.

It was well established that if tubular function was intact, renal vasoconstriction was associated with enhanced tubular sodium reabsorption. Specifically, the fraction of filtered sodium that is rapidly reabsorbed by normal tubules of the vasoconstricted kidney is greater than 99%.

Thus, when nitrogenous wastes, such as creatinine and urea, accumulate in the blood due to a fall in glomerular filtration rate (GFR) secondary to renal vasoconstriction with intact tubular function, the fractional excretion of filtered sodium (FENa = [(urine sodium × plasma creatinine) / (plasma sodium × urine creatinine)]) is less than 1%. An exception to this physiological response of the normal kidney to vasoconstriction is when the patient is receiving a diuretic, including mannitol, or has glucosuria, which decreases tubular sodium reabsorption and increases FENa.

It has recently been shown in the presence of diuretics that a rate of fractional excretion of urea (FEurea) of less than 35 indicates intact tubular function, thus favoring renal vasoconstriction rather than established ARF as a cause of the azotemia.

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English: Physiology of Nephron

English: Physiology of Nephron (Photo credit: Wikipedia)

Structures of the kidney: 1.Renal pyramid 2.In...

Structures of the kidney: 1.Renal pyramid 2.Interlobar artery 3.Renal artery 4.Renal vein 5.Renal hilum 6.Renal pelvis 7.Ureter 8.Minor calyx 9.Renal capsule 10.Inferior renal capsule 11.Superior renal capsule 12.Interlobar vein 13.Nephron 14.Minor calyx 15.Major calyx 16.Renal papilla 17.Renal column (no distinction for red/blue (oxygenated or not) blood, arteriole is between capilaries and larger vessels (Photo credit: Wikipedia)

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Mechanisms of ARF

Based on the foregoing comments, this discussion of mechanisms of ARF will not include nitrogenous-waste accumulation due to renal vasoconstriction with intact tubular function (prerenal azotemia) or urinary tract obstruction (postrenal azotemia). The mechanisms of ARF involve both vascular and tubular factors. An ischemic insult to the kidney will in general be the cause of the ARF. While a decrease in renal blood flow with diminished oxygen and substrate delivery to the tubule cells is an important ischemic factor, it must be remembered that a relative increase in oxygen demand by the tubule is also a factor in renal ischemia.

Approximately 30–70% of these shed epithelial tubule cells in the urine are viable and can be grown in culture. Recent studies using cellular and molecular techniques have provided information relating to the structural abnormalities of injured renal tubules that occur both in vitro and in vivo. In vitro studies using chemical anoxia have revealed abnormalities in the proximal tubule cytoskeleton that are associated with translocation of Na+/K+-ATPase from the basolateral to the apical membrane.

A comparison of cadaveric transplanted kidneys with delayed versus prompt graft function has also provided important results regarding the role of Na+/K+-ATPase in ischemic renal injury. This study demonstrated that, compared with kidneys with prompt graft function, those with delayed graft function had a significantly greater cytoplasmic concentration of Na+/K+-ATPase and actin-binding proteins — spectrin (also known as fodrin) and ankyrin — that had translocated from the basolateral membrane to the cytoplasm.

Such a translocation of Na+/K+-ATPase from the basolateral membrane to the cytoplasm could explain the decrease in tubular sodium reabsorption that occurs with ARF. An important focus of research is the mechanisms whereby the critical residence of Na+/K+-ATPase in the basolateral membrane (which facilitates vectorial sodium transport) is uncoupled by hypoxia or ischemia.  The actin-binding proteins,

  • spectrin and
  • ankyrin,

serve as substrates for the calcium-activated cysteine protease calpain.

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In vitro studies in proximal tubules have shown a rapid rise in cytosolic calcium concentration during acute hypoxia, which antedates the evidence of tubular injury as assessed by lactic dehydrogenase (LDH) release. There is further evidence to support the importance of the translocation of Na+/K+-ATPase from the basolateral membrane to the cytoplasm during renal ischemia/reperfusion.

Specifically, calpain-mediated breakdown products of the actin-binding protein spectrin occur with renal ischemia. Calpain activity was demonstrated to increase during hypoxia in isolated proximal tubules. Measurement of LDH release following calpain inhibition indicated attenuation of hypoxic damage to proximal tubules. There was no evidence of an increase in cathepsin, a (cysteine protease) in proximal tubules during hypoxia , but there is a calcium-independent pathway for calpain activation during hypoxia.

Calpastatin, an endogenous cellular inhibitor of calpain activation, was shown to be diminished during hypoxia in association with the rise in another cysteine protease, caspase.

This effect of diminished calpastatin activity could be reversed by caspase inhibition. Proteolytic pathways appear to be involved in calpain-mediated proximal tubule cell injury during hypoxia. Calcium activation of phospholipase A has also been shown to contribute to renal tubular injury during ischemia.

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Tubular obstruction during ARF

The existence of proteolytic pathways involving cysteine proteases, namely calpain and caspases, may therefore explain

  • the decrease in proximal tubule sodium reabsorption and
  • increased FENa

secondary to proteolytic uncoupling of Na+/K+-ATPase from its basolateral membrane anchoring proteins.

This tubular perturbation alone, however, does not explain the fall in GFR that leads to nitrogenous-waste retention and thus the rise in BUN and serum creatinine.   Decreased proximal tubule sodium reabsorption may lead to a decreased GFR during ARF. First of all, brush border membranes and cellular debris could provide the substrate for intraluminal obstruction in the highly resistant portion of distal nephrons.

In fact, microdissection of individual nephrons of kidneys from patients with ARF demonstrated obstructing casts in distal tubules and collecting ducts. This observation could explain the dilated proximal tubules that are observed upon renal biopsy of ARF kidneys. The intraluminal casts in ARF kidneys stain prominently for Tamm-Horsfall protein (THP), which is produced in the thick ascending limb. Importantly, THP is secreted into tubular fluid as a monomer but subsequently may become a polymer that forms a gel-like material in the presence of increased luminal Na+ concentration, as occurs in the distal nephron during clinical ARF with the decrease in tubular sodium reabsorption.

Thus, the THP polymeric gel in the distal nephron provides an intraluminal environment for distal cast formation involving viable, apoptotic, and necrotic cells.

The loss of the tubular epithelial cell barrier and/or the tight junctions between viable cells during acute renal ischemia could lead to a leak of glomerular filtrate back into the circulation. (If this occurs and normally non-reabsorbable substances, such as inulin, leak back into the circulation, then a falsely low GFR will be measured as inulin clearance. It should be noted, however, that the degree of extensive tubular damage observed in experimental studies demonstrating tubular fluid backleak is rarely observed with clinical ARF in humans). Moreover, dextran sieving studies in patients with ARF demonstrated that, at best, only a 10% decrease in GFR could be explained by backleak of filtrate. Cadaveric transplanted kidneys with delayed graft function, however, may have severe tubular necrosis, and thus backleak of glomerular filtration may be more important in this setting.

Inflammation and NO

There is now substantial evidence for the involvement of inflammation in the pathogenesis of the decreased GFR associated with acute renal ischemic injury. In this regard, there is experimental evidence that iNOS may contribute to tubular injury during ARF. Hypoxia in isolated proximal tubules has been shown to increase NO release, and there is increased iNOS protein expression in ischemic kidney homogenates. An antisense oligonucleotide was shown to block the upregulation of iNOS and afford functional protection against acute renal ischemia. Moreover, when isolated proximal tubules from iNOS, eNOS, and neuronal NO synthase (nNOS) knockout mice were exposed to hypoxia, only the tubules from the iNOS knockout mice were protected against hypoxia, as assessed by LDH release. The iNOS knockout mice were also shown to have lower mortality during ischemia/reperfusion than wild-type mice.  The scavenging of NO by oxygen radicals produces peroxynitrite causing tubule damage during ischemia. While iNOS may contribute to ischemic injury of renal tubules,  the vascular effect of eNOS in the glomerular afferent arteriole is protective against ischemic injury. In this regard, eNOS knockout mice are more sensitive to endotoxin-related injury than normal mice.

Moreover, the protective role of vascular eNOS may be more important than the deleterious effect of iNOS at the tubule level during renal ischemia.   This is because treatment of mice with the nonspecific NO synthase (NOS) inhibitor L-NAME, which blocks both iNOS and eNOS, worsens renal ischemic injury. NO may downregulate eNOS and is a potent inducer of heme oxygenase-1, which has been shown to be cytoprotective against renal injury. The MAPK pathway also appears to be involved in renal oxidant injury. Activation of extracellular signal–regulated kinase (ERK) or inhibition of JNK ameliorates oxidant injury–induced necrosis in mouse renal proximal tubule cells in vitro. Upregulation of ERK may also be important in the effect of preconditioning whereby early ischemia affords protection against a subsequent ischemia/reperfusion insult. Alterations in cell cycling are also involved in renal ischemic injury. Upregulation of p21, which inhibits cell cycling, appears to allow cellular repair and regeneration, whereas homozygous p21 knockout mice demonstrate enhanced cell necrosis in response to an ischemic insult.

Prolonged duration of the ARF clinical course and the need for dialysis are major factors projecting a poor prognosis. Patients with ARF who require dialysis have a 50–70% mortality rate. Infection and cardiopulmonary complications are the major causes of death in patients with ARF. Excessive fluid administration in patients with established ARF may lead to pulmonary congestion, hypoxia, the need for ventilatory support, pneumonia, and multiorgan dysfunction syndrome, which has an 80–90% mortality rate. Until means to reverse the diminished host defense mechanisms in azotemic patients with clinical ARF are available, every effort should be made to avoid invasive procedures such as the placement of bladder catheters, intravenous lines, and mechanical ventilation. Over and above such supportive care, it may be that combination therapy will be necessary to prevent or attenuate the course of ARF. Such combination therapy must involve agents with potential beneficial effects on vascular tone, tubular obstruction, and inflammation.

Schrier RW, Wang W, Poole B, and Mitra A. Acute renal failure: definitions, diagnosis, pathogenesis, and therapy. The Journal of Clinical Investigation 2004; 114(1):5-14. http://www.jci.org

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Part IIIb. Additional Related References on NO, oxidative stress and Kidney

Shelgikar PJ, Deshpande KH, Sardeshmukh AS, Katkam RV, Suryakarl AN. Role of oxidants and antioxidants in ARF patients undergoing hemodialysis. Indian J Nephrol 2005;15: 73-76.

Lee JW. Renal Dysfunction in Patients with Chronic Liver Disease. Electrolytes Blood Press 7:42-50, 2009ㆍdoi: 10.5049/EBP.2009.7.2.42.

Saadat H, et al. Endothelial Nitric Oxide Function and Tubular Injury in Premature Infants. Int J App Sci and Technol 2012; 7(6): 77-81. http://www.ijastnet.com.

Amerisan MS. Cardiovascular disease in chronic kidney disease. Indian J Nephrol 2005;15: 1-7.

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Traditional risk factors for CVD in CKD

  • Hypertension
  • Older Age
  • Diabetes Mellitus
  • Male gender
  • High LDL
  • White Race
  • Low HDL
  • Physical inactivity
  • Smoking
  • Menopause
  • LVH

CKD Related CV Risk Factors

  • Blood Pressure
  • ? Homocysteinemia
  • Anemia
  • ? Inflammation
  •   Ca++ x P++
  • ? NO synthesis
  • Na+ Retention
  • ? Lp (a)
  • Hypervolemia
  • ? Insulin Resistance
  • Proteinuria & Hypoalbuminemia
  • Iron over load
  • ? Adeponectin
  • ??Vit. C or E
  • ? 5 Lipoxygenase
  • ROS
  • Genetic factors
  • ADMA (Asymmetric Dimethyl Arginine)

S Vikrant, SC Tiwari. Essential Hypertension – Pathogenesis and Pathophysiology. J Indian Acad Clinical Medicine 2001; 2(3):141-161. Scheme for pathogenesis of salt dependent hypertension.

The hypothesis proposes that early hypertension is episodic and is mediated by a hyperactive sympathetic nervous system or activated renin-angiotensin system.

Cell membrane alterations

Hypotheses linking abnormal ionic fluxes to increased peripheral resistance through increase in cell sodium, calcium, or pH.   The hypertension that is more common in obese people may arise in large part from the insulin resistance and resultant hyperinsulinaemia that results from the increased mass of fat. However, rather unexpectedly, insulin resistance may also be involved in hypertension in non-obese people.

Overall scheme for the mechanisms by which obesity, if predominantly upper body or visceral in location, could promote

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  • diabetes,
  • dyslipidemia and
  • hypertension via hyperinsulinemia.

The explanation for insulin resistance found in as many as half of nonobese hypertensive is not obvious and may involve one or more aspects of insulin’s action

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Proposed mechanisms by which insulin resistance and/or hyperinsulinemia may lead to increased blood pressure.

  1. Enhanced renal sodium and water reabsorption.
  2. Increased blood pressure sensitivity to dietary salt intake
  3. Augmentation of the pressure and
  4. aldosterone responses to AII
  5. Changes in transmembrane electrolyte transport
  • a. Increased intracellular sodium
  • b. Decreased Na+/K+ – ATPase activity
  • c. Increased intracellular Ca2+ pump activity
  • d. Increased intracellular Ca2+ accumulation
  • e. Stimulation of growth factors

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Part IV. New Insights on NO donors

This study investigated the involvement of nitric oxide (NO) into the irradiation-induced increase of cell attachment. These experiments explored the cellular mechanisms of low-power laser therapy. HeLa cells were irradiated with a monochromatic visible-tonear infrared radiation (600–860 nm, 52 J/m2) or with a diode laser (820 nm, 8–120 J/m2) and the number of cells attached to a glass matrix was counted after 30 minute incubation at 37oC. The NO donors

  1. sodium nitroprusside (SNP),
  2. glyceryl trinitrate (GTN), or
  3. sodium nitrite (NaNO2)

were added to the cellular suspension before or after irradiation. The action spectra and the concentration and fluence dependencies obtained were compared and analyzed.

The well-structured action spectrum for the increase of the adhesion of the cells, with maxima at 619, 657, 675, 740, 760, and 820 nm, points to the existence of a photoacceptor responsible for the enhancement of this property (supposedly cytochrome c oxidase, the terminal respiratory chain enzyme), as well as signaling pathways between the cell mitochondria, plasma membrane, and nucleus.

Treating the cellular suspension with SNP before irradiation significantly modifies the action spectrum for the enhancement of the cell attachment property (band maxima at 642, 685, 700, 742, 842, and 856 nm). The action of SNP, GTN, andNaNO2 added before or after irradiation depends on their concentration and radiation fluence.

The NO donors added to the cellular suspension before irradiation eliminate the radiation induced increase in the number of cells attached to the glass matrix, supposedly by way of binding NO to cytochrome c oxidase. NO added to the suspension after irradiation can also inhibit the light-induced signal downstream. Both effects of NO depend on the concentration of the NO donors added.

The results indicate that NO can control the irradiation-activated reactions that increase the attachment of cells.

Karu TI, Pyatibrat LV, and Afanasyeva NI. Cellular Effects of Low Power Laser Therapy Can be Mediated by Nitric Oxide. Lasers Surg. Med 2005; 36:307–314.

IFNa-2b (IFN-a) effect on barrier function of renal tubular epithelium

IFNa treatment can be accompanied by impaired renal function and capillary leak. This study shows IFNa produced dose-dependent and time-dependent decrease in transepithelial resistance (TER) ameliorated by tyrphostin, an inhibitor of phosphotyrosine kinase with increased expression of occludin and E-cadherin. In conclusion, IFNa can directly affect barrier function in renal epithelial cells via ovewrexpression or missorting of the junctional proteins occludin and E-cadherin.

Lechner J, Krall M, Netzer A, Radmayr C, et al. Effects of interferon a-2b on barrier function and junctional complexes of renal proximal tubulat LLC-pK1 cells. Kidney Int 1999; 55:2178-2191.

Ischemia-reperfusion injury

The pathophysiology of acute renal failure (ARF) is complex and not well understood. Numerous models of ARF suggest that oxygen-derived reactive species are important in renal ischemia-reperfusion (I-R) injury, but the nature of the mediators is still controversial. Treatment with oxygen radical scavengers, antioxidants, and iron chelators such as

  • superoxide dismutase,
  • dimethylthiourea,
  • allopurinol, and
  • deferoxamine

are protective in some models, and suggest a role for the hydroxyl radical formation. However, these compounds are not protective in all models of I-R injury, and direct evidence for the generation of hydroxyl radical is absent. Furthermore, these inhibitors have another property in common.

They all directly scavenge or inhibit the formation of peroxynitrite (ONOO−), a highly toxic species derived from nitric oxide (NO) and superoxide. Thus, the protective effects seen with these inhibitors may be due in part to their ability to inhibit ONOO− formation. Even though reactive oxygen species are thought to participate in ischemia-reperfusion (I-R) injury, induction of and production of high levels of  inducible nitric oxide (NO)  also contribute to this injury.

NO combines with superoxide to form the potent oxidant peroxynitrite (ONOO−). NO and ONOO− were investigated in a rat model of renal I-R injury using the selective iNOS inhibitor L-N6-(1-iminoethyl)lysine (L-NIL).

I-R surgery significantly increased plasma creatinine levels to 1.9 ± 0.3 mg/dl (P < .05) and caused renal cortical necrosis. L-NIL administration (3 mg/kg) in animals subjected to I-R significantly decreased plasma creatinine levels to 1.2 ± 0.10 mg/dl (P < .05 compared with I-R) and reduced tubular damage.

ONOO− formation was evaluated by detecting 3-nitrotyrosine-protein adducts (3NTyPAs), a stable biomarker of ONOO− formation.   The kidneys from I-R animals had increased levels of 3NTyPAs compared with control animals   L-NIL-treated rats (3 mg/kg) subjected to I-R showed decreased levels of 3NTyPAs.

These results suggests that iNOS-generated NO mediates damage in I-R injury possibly through ONOO− formation.

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In summary,

  1. 3-nitrotyrosine-protein adducts were detected in renal tubules after I-R injury.
  2. Selective inhibition of iNOS by L-NIL decreased injury, improved renal function, and decreased apparent ONOO− formation.
  3. Reactive nitrogen species should be considered potential therapeutic targets in the prevention and treatment of renal I-R injury.

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Walker LM, Walker PD, Imam SZ, et al. Evidence for Peroxynitrite Formation in Renal Ischemia-Reperfusion Injury: Studies with the Inducible Nitric Oxide Synthase InhibitorL-N6-(1-Iminoethyl)lysine1. 2000.

Role of TNFa independent of iNOS Renal failure is a frequent complication of sepsis, mediated by renal vasoconstrictors and vasodilators. Endotoxin induces several proinflammatory cytokines, among which tumor necrosis factor (TNF) is thought to be of major importance. Tumor necrosis factor (TNF) has been suggested to be a factor in the acute renal failure in sepsis or endotoxemia. Passive immunization by anti-TNFa prevented development of septic shock in animal experiments.The development of ARF involves excessive intrarenal vasoconstriction. Involvement of nitric oxide (NO), generated by inducible NO synthase (iNOS), is still a factor in the pathogenesis of endotoxin-induced renal failure. TNF-a leads to a decrease in glomerular filtration rate (GFR).

This study tested the hypothesis that the role of TNF-a in endotoxic shock related ARF is mediated by iNOS-derived NO.   An injection of lipopolysaccharide (LPS) constituent of gram-negative bacteria to wild-type mice resulted in a 70% decrease in glomerular filtration rate (GFR) and in a 40% reduction in renal plasma flow (RPF) 16 hours after the injection.   The results occurred independent of hypotension, morphological changes, apoptosis, and leukocyte accumulation. In mice pretreated with TNFsRp55, only a 30% decrease in GFR was observed without a significant change in RPF as compared with controls. Pretreatment with TNKsRp55 on renal function Wild-type mice were pretreated with TNFsRp55(10 mg/kg IP)  for one hour before the administration of 5 mg/kg intraperitoneal endotoxin. GFR and RPF were determined 16 hours thereafter. Data are expressed as mean 6, SEM, N 5 6. *P , 0.05 vs. Control; §P , 0.05 vs. LPS, by ANOVA.

The serum NO concentration was significantly lower in endotoxemic wild-type mice pretreated with TNFsRp55, as compared with untreated endotoxemic wild-type mice. In LPS-injected iNOS knockout mice and wild-type mice treated with a selective iNOS inhibitor, 1400W, the development of renal failure was similar to that in wild-type mice. As in wild-type mice,TNFsRp55 significantly attenuated the decrease in GFR (a 33% decline, as compared with 75% without TNFsRp55) without a significant change in RPF in iNOS knockout mice given LPS. These results demonstrate a role of TNF in the early renal dysfunction (16 h) in a septic mouse model independent of iNOS,

  • hypotension,
  • apoptosis,
  • leukocyte accumulation,and
  • morphological alterations,

thus suggesting renal hypoperfusion secondary to an imbalance between, as yet to be defined renal vasoconstrictors and vasodilators.

Knotek M, Rogachev B, Wang W,….., Edelstein CL, Dinarello CA, and Schrier RW. Endotoxemic renal failure in mice: Role of tumor necrosis factor independent of inducible nitric oxide synthase. Kidney International 2001; 59:2243–2249

Ischemic acute renal failure

Inflammation plays a major role in the pathophysiology of acute renal failure resulting from ischemia. This review discusses the contribution of

  • endothelial
  • epithelial cells and
  • leukocytes

to this inflammatory response. The roles of cytokines/chemokines in the injury and recovery phase are reviewed. The protection of mouse kidney prior to exposure to ischemia or urinary tract obstruction is  a potential model to  search for pharmacologic agents to protect the kidney against injury by inflammatory mediators produced by tubular epithelial cells and activated leukocytes in renal ischemia/reperfusion (I/R) injury. Tubular epithelia produce

  • TNF-a,
  • IL-1,
  • IL-6,
  • IL-8,
  • TGF-b,
  • MCP-1,
  • ENA-78,
  • RANTES, and
  • fractalkines,

whereas leukocytes produce

  • TNF-a,
  • IL-1,
  • IL-8,
  • MCP-1,
  • ROS, and
  • eicosanoids.

The release of these chemokines and cytokines serve as effectors for a positive feedback pathway enhancing inflammation and cell injury, the cycle of tubular epithelial cell injury and repair following renal ischemia/reperfusion.   Tubular epithelia are typically cuboidal in shape and apically-basally polarized; the Na+/K+-ATPase localizes to basolateral plasma membranes, whereas cell adhesion molecules, such as integrins localize basally. In response to ischemia reperfusion,

  • the Na+/K+-ATPase appears apically, and
  • integrins are detected on lateral and basal plasma membranes.

Some of the injured epithelial cells undergo necrosis and/or apoptosis detaching from the underlying basement membrane into the tubular space where they contribute to tubular occlusion. Viable cells that remain attached, dedifferentiate, spread, and migrate to repopulate the denuded basement membrane. With cell proliferation, cell-cell and cell-matrix contacts are restored, and the epithelium redifferentiates and repolarizes, forming a functional, normal epithelium Inflammation is a significant component of renal I/R injury, playing a considerable role in its pathophysiology.

Although significant progress has been made in defining the major components of this process, the complex cross-talk between endothelial cells, inflammatory cells, and the injured epithelium with each generating and often responding to cytokines and chemokines is not well understood. In addition, we have not yet taken full advantage of the large body of data on inflammation in other organ systems.

Furthermore, preconditioning the kidney to afford protection to subsequent bouts of ischemia may serve as a useful model challenging us to therapeutically mimic endogenous mechanisms of protection.

Understanding the inflammatory response prevalent in ischemic kidney injury will facilitate identification of molecular targets for therapeutic intervention.

Bonventre JV and Zuk A. Ischemic acute renal failure: An inflammatory disease? Forefronts in Nephrology 2002;.. :480-485

Gene expression profiles in renal proximal tubules In kidney disease renal proximal tubular epithelial cells (RPTEC) actively contribute to the progression of tubulointerstitial fibrosisby mediating both

  • an inflammatory response and
  • via epithelial-to-mesenchymal transition.

Using laser capture microdissection we specifically isolated RPTEC from cryosections of the healthy parts of kidneys removed owing to renal cell carcinoma and from kidney biopsies from patients with proteinuric nephropathies. RNA was extracted and hybridized to complementary DNA microarrays after linear RNA amplification. Statistical analysis identified 168 unique genes with known gene ontology association, which separated patients from controls. Besides distinct alterations in signal-transduction pathways (e.g. Wnt signalling), functional annotation revealed a significant upregulation of genes involved in

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  • cell proliferation and cell cycle control (like insulin-like growth factor 1 or cell division cycle 34),
  • cell differentiation (e.g. bone morphogenetic protein 7),
  • immune response,
  • intracellular transport and
  • metabolism

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in RPTEC from patients.

The study also revealed differential expression of a number of genes responsible for cell adhesion (like BH-protocadherin) with a marked downregulation of most of these transcripts. In summary, the results obtained from RPTEC revealed a differential regulation of genes, which are likely to be involved in either pro-fibrotic or tubulo-protective mechanisms in proteinuric patients at an early stage of kidney disease.

Rudnicki M, Eder S, Perco P, Enrich J, et al. Gene expression profiles of human proximal tubular epithelial cells in proteinuric nephropathies. Kidney International 2006; xx:1-11. Kidney International advance online publication, 20 December 2006; doi:10.1038/sj.ki.5002043. http://www.kidney-international.org

Oxidative stress involved with diabetic nephropathy

Diabetic Nephropathy (DN) poses a major health problem. There is strong evidence for a potential role of the eNOS gene. This case control study investigated the possible role of genetic variants of the endothelial Nitric Oxide Synthase (eNOS) gene and oxidative stress in the pathogenesis of nephropathy in patients with diabetes mellitus. The study included 124 diabetic patients;

  1. 68 of these patients had no diabetic nephropathy (group 1) while
  2. 56 patients exhibited symptoms of diabetic nephropathy (group 2).
  3. Sixty two healthy non-diabetic individuals were also included as a control group.

Blood samples from subjects and controls were analyzed to investigate the eNOS genotypes and to estimate

  • the lipid profile and
  • markers of oxidative stress such as malondialdehyde (MDA) and nitric oxide (NO).

No significant differences were found in the frequency of eNOS genotypes between diabetic patients (either in group 1 or group 2) and controls (p >0.05). Also, no significant differences were found in the frequency of eNOS genotypes between group 1 and group 2 (p >0.05). Both group 1 and group 2 had significantly higher levels of nitrite and MDA when compared with controls (all p = 0.0001). Also group 2 patients had significantly higher levels of nitrite and MDA when compared with group 1 (p = 0.02, p = 0.001 respectively).

The higher serum level of the markers of oxidative stress in diabetic patients particularly those with diabetic nephropathy suggest that oxidative stress and not the eNOS gene polymorphism is involved in the pathogenesis of the diabetic nephropathy in this subset of patients

Badawy A, Elbaz R, Abbas AM, Ahmed Elgendy A, et al. Oxidative stress and not endothelial Nitric Oxide Synthase gene polymorphism involved in diabetic nephropathy. Journal of Diabetes and Endocrinology 2011; 2(3): 29-35.

Metformin in renal ischemia reperfusion

Renal ischemia plays an important role in renal impairment and transplantation. Metformin is a biguanide used in type 2 diabetes, it inhibits hepatic glucose production and increases peripheral insulin sensitivity. While the mode of action of metformin is incompletely understood, it appears to have anti-inflammatory and antioxidant effects involved in its beneficial effects on insulin resistance.   Control, Sham, ischemia/reperfusion (I/R) and Metformin treated I /R groups   A renal I/R injury was done by a left renal pedicle occlusion to induce ischemia for 45 min followed by 60 min of reperfusion with contralateral nephrectomy. Metformin pretreated I/R rats in a dose of 200 mg/kg/day for three weeks before ischemia induction.

  • Nitric oxide (NO),
  • tumor necrosis factor alpha (TNF α) ,
  • catalase (CAT) and
  • reduced glutathione (GSH) activities

were determined in renal tissue, while

  • creatinine clearance (CrCl) ,
  • blood urea nitrogen (BUN) were measured and

5 hour urinary volume and electrolytes were estimated . BUN and CrCl levels in the I/R group were significantly higher than in control rats (p<0.05) table (1).

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Table 1: Creatinine clearance (Cr Cl) and blood urea nitrogen (BUN) levels in control and test groups.
(Mean ± SD)

Groups CrCl   (ml/min) BUN (mg/dl)
Control group 1.30 ±0.11 14.30±0.25
Sham group+ metformin 1.27±0.09 15.70±0.19
I/R group (P1) 1.85±0.25 (<0.001 ) 28.00±0.62 (<0.001)
I/R+ metformin group (P2,P3) 1.55±0.22 (0.001, 0.028) 18.10±1.00 (<0.001, <0.001)
  • P1: Statistical significance between control
    group and saline treated I/R group.
  • P2 Statistical significance between control
    group and Metformin treated I/R group.
  • P3 Statistical significance between saline treated
    I/R group and Metformin treated I/R group

_______________________________________________________________________________________________________________________________________________________

When metformin was administered before I/R, BUN and CrCl levels were still significantly higher than control group but their elevation were significantly lower in comparison to I/R group alone (P<0.05).   TNF α and NO levels were significantly higher in the I/R group than those of the control group (Table 2). Pre-treatment with metformin significantly lowered their levels in comparison to I/R group (P<0.05).

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Table 2: Tumor necrosis factor α (TNF α) and inducible nitric oxide (iNO) levels in control and test groups.
(Mean ± SD)

Groups TNF α (pmol/mg tissue) iNO (nmol/ mg tissue)
Control group 1 7.60 ±5.98 2.54 ± 0.82
Sham group+ metformin 16.70 ±5.50 2.35 ±0.80
I/R group (P1) 54. 00±6.02 (<0.001) 4.50±0.89 (<0.001)
I/R+metformin group (P2,P3) 39 ± 14.01 (<0.001, 0.006) 3.53±0.95 (0.02, 0.03)

 

  • P1: Statistical significance between control group
    and saline treated I/R group.
  • P2 Statistical significance between control group
    and Metformin treated I/R group.
  • P3 Statistical significance between saline treated
    I/R group and Metformin treated I/R group

_________________________________________________________________________________________________________________________________________________________

These results showed significant increase in NO,TNF α, BUN , CrCl and significant decrease in urinary volume , electrolytes, CAT and GSH activities in the I/R group than those in the control group. Metformin decreased significantly NO, TNF α, BUN and CrCl while increased urinary volume, electrolytes, CAT and GSH activities.   Lipid peroxidation is related to I/R induced tissue injury. Production of inducible NO synthase (NOS) under lipid peroxidation and inflammatory conditions results in the induction of NO which react with O2 liberating peroxynitrite (OONO-). NO itself inactivates the antioxidant enzyme system CAT and GSH. Alteration in NO synthesis have been observed in other kidney injuries as nephrotoxicity and acute renal failure induced by endotoxins.

Treatment with iNOS inhibitors improved renal function and decreased peroxynitrite radical which is believed to be responsible for the shedding of proximal convoluted tubules in I/R.   Metformin produced anti-inflammatory renoprotective effect on CrCl and diuresis in renal I/R injury.

Malek HA. The possible mechanism of action of metformin in renal ischemia reperfusion in rats. The Pharma Research Journal 2011; 6(1):42-49.

Possible role of NO donors in ARFThe L-arginine-nitric oxide (NO) pathway has been implicated in many physiological functions in the kidney, including

  • regulation of glomerular hemodynamics,
  • mediation of pressure-natriuresis,
  • maintenance of medullary perfusion,
  • blunting of tubuloglomerular feedback (TGF),
  • inhibition of tubular sodium reabsorption and
  • modulation of renal sympathetic nerve activity

Its net effect in the kidney is to promote natriuresis and diuresis, contributing to adaptation to variations of dietary salt intake and maintenance of normal blood pressure. Nitric oxide has been implicated in many physiologic processes that influence both acute and long-term control of kidney function. Its net effect in the kidney is to promote natriuresis and diuresis, contributing to adaptation to variations of dietary salt intake and maintenance of normal blood pressure. A pretreatment with nitric oxide donors or L-arginine may prevent the ischemic acute renal injury. In chronic kidney diseases, the systolic blood pressure is correlated with the plasma level of asymmetric dimethylarginine, an endogenous inhibitor of nitric oxide synthase. A reduced production and biological action of nitric oxide is associated with an elevation of arterial pressure, and conversely, an exaggerated activity may represent a compensatory mechanism to mitigate the hypertension.

JongUn Lee. Nitric Oxide in the Kidney : Its Physiological Role and Pathophysiological Implications. Electrolyte & Blood Pressure 2008; 6:27-34.

Renal Hypoxia and Dysoxia following Reperfusion

Acute renal failure (ARF) is a common condition which develops in 5% of hospitalized patients. Of the patients who develop ARF, ~10% eventually require renal replacement therapy. Among critical care patients who have acute renal failure and survive, 2%-10% develop terminal renal failure and require long-term dialysis.   The kidneys are particularly susceptible to ischemic injury in many clinical conditions such as renal transplantation, treatment of suprarenal aneurysms, renal artery reconstructions, contrast-agent induced nephropathy, cardiac arrest, and shock. One reason for renal sensitivity to ischemia is that the kidney microvasculature is highly complex and must meet a high energy demand.

Under normal, steady state conditions, the oxygen (O2) supply to the renal tissues is well in excess of oxygen demand.   Under pathological conditions, the delicate balance of oxygen supply versus demand is easily disturbed due to the unique arrangement of the renal microvasculature and its increasing numbers of diffusive shunting pathways.  

The renal microvasculature is serially organized, with almost all descending vasa recta emerging from the efferent arterioles of the juxtamedullary glomeruli. Adequate tissue oxygenation is thus partially dependent on the maintenance of medullary perfusion by adequate cortical perfusion. This, combined with the low amount of medullary blood flow (~10% of total renal blood flow) in the U-shaped microvasculature of the medulla allows O2 shunting between the descending and ascending vasa recta and contributes to the high sensitivity of the medulla and cortico-medullary junction to decreased O2 supply.

Whereas past investigations have focused mainly on tubular injury as the main cause of ischemia-related acute renal failure, increasing evidence implicates alterations in the intra-renal microcirculation pathway and in the O2 handling. Indeed, although acute tubular necrosis (ATN) has classically been believed to be the leading cause of ARF, data from biopsies in patients with ATN have shown few or no changes consistent with tubular necrosis.

The role played by microvascular dysfunction, however, has generated increasing interest. The complex pathophysiology of ischemic ARF includes the inevitable

  • reperfusion phase associated with oxidative stress,
  • cellular dysfunction and
  • altered signal transduction.

During this process, alterations in oxygen transport pathways can result in cellular hypoxia and/or dysoxia. In this context, the distinction between hypoxia and dysoxiais that

  • cellular hypoxia refers to the condition of decreased availability of oxygen due to inadequate convective delivery from the microcirculation.
  • Cellular dysoxia, in contrast, refers to a pathological condition where the ability of mitochondria to perform oxidative phosphorylation is limited, regardless of the amount of available oxygen.

_______________________________________________________________________________________________________________________________________________________

The latter condition is associated with mitochondrial failure and/or activation of alternative pathways for oxygen consumption. Thus, we would expect that an optimal balance between oxygen supply and demand is essential to reducing damage from renal ischemia-reperfusion (I/R) injury. Complex interactions exist between

  • tubular injury,
  • microvascular injury, and
  • inflammation after renal I/R.

On the one hand, insults to the tubule cells promotes the liberation of a number of inflammatory mediators, such as TNF-á, IL-6, TGF-â, and chemotactic cytokines(RANTES, monocyte chemotactic protein-1, ENA-78, Gro-á, and IL-8). On the other hand, chemokine production can promote

  • leukocyte-endothelium interactions and
  • leukocyte activation,

resulting in…..

  • renal blood flow impairment and
  • the expansion of tubular damage
  • impaired renal hemodynamics and
  • electrolyte reabsorption

Adequate medullary tissue oxygenation, in terms of balanced oxygen supply and demand, is dependent on the maintenance of medullary perfusion by adequate cortical perfusion and also on the high rate of O2 consumption required for active electrolyte transport. Furthermore, renal blood flow is closely associated with renal sodium transport, mitochondrial activity and NO-mediated O2 consumption In addition to having a limited O2 supply due to the anatomy of the microcirculation anatomy, the sensitivity of the medulla to hypoxic conditions results from this high O2 consumption.

Renal sodium transport is the main O2-consuming function of the kidney and is closely linked to renal blood flow for sodium transport, particularly in the thick ascending limbs of the loop of Henle and the S3 segments of the proximal tubules. Medullary renal blood flow is also highly dependent on cortical perfusion, with almost all descending vasa recta emerging from the efferent arteriole of juxta medullary glomeruli. A profound reduction in cortical perfusion can disrupt medullary blood flow and lead to an imbalance between O2 supply and O2 consumption. On theother hand, inhibition of tubular reabsorption by diuretics increases medullary pO2 by decreasing the activity of Na+/K+-ATPases and local O2 consumption.

Mitochondrial activity and NO-mediated O2 consumption

The medulla has been found to be the main site of production of NO in the kidney. In addition to the actions described above, NO appears to be a key regulator of renal tubule cell metabolism by inhibiting the activity of the Na+-K+-2Cl- cotransporter and reducing Na+/H+ exchange. Since superoxide (O2-) is required to inhibit solute transport activity, it was assumed that these effects were mediated by peroxynitrite (OONO-). Indeed, mitochondrial nNOS upregulation, together with an increase in NO production, has been shown to increase mitochondrial peroxynitrite generation, which in turn, can induce cytochrome c release and promote apoptosis. NO has also been shown to directly compete with O2 at the mitochondrial level. These findings support the idea that NO acts as an endogenous regulator to match O2 supply to O2 consumption, especially in the renal medulla.   NO reversibly binds to the O2 binding site of cytochrome oxidase, and acts as a potent, rapidMitochondrial activity and NO-mediated O2 consumption, and reversible inhibitor of cytochrome oxidase in competition with molecular O2. This inhibition could be dependent on the O2 level, since the IC50 (the concentration of NO that reduces the specified response by half) decreases with reduction in O2 concentration. The inhibition of electron flux at the cytochrome oxidase level switches the electron transport chain to a reduced state, and consequently leads to depolarization of the mitochondrial membrane potential and electron leakage.

To summarize, while the NO/O2 ratio can act as a regulator of cellular O2 consumption by matching decreases in O2 delivery to decreases in cellular O2 cellular, the inhibitory effect of NO on mitochondrial respiration under hypoxic conditions further impairs cellular aerobic metabolism. This leads to a state of “cytopathic hypoxia,” as described in the sepsis literature.   Only cell-secreted NO competes with O2 and to regulate mitochondrial respiration. In addition to the 3 isoforms (eNOS, iNOS, cnNOS), an α-isoform of neuronal NOS, the mitochondrial isoform (mNOS) located in the inner mitochondrial membrane, has also been shown to regulate mitochondrial respiration. These data support a role for NO in the balanced regulation of renal O2 supply and O2 consumption after renal I/R However, the relationships between the determinants of O2 supply, O2 consumption, and renal function, and their relation to renal damage remain largely unknown.

Sustained endothelial activation Ischemic renal failure leads to persistent endothelial activation, mainly in the form of endothelium-leukocyte interactions and the activation of adhesion molecules. This persistent activation can compromise renal blood flow, prevent the recovery of adequate tissue oxygenation, and jeopardize tubular cell survival despite the initial recovery of renal tubular function. A 30-50% reduction in microvascular density was seen 40 weeks after renal ischemic injury in a rat model. Vascular rarefaction has been proposed to induce chronic hypoxia resulting in tubulointerstitial fibrosis via the molecular activation of fibrogenic factors such as transforming growth factor (TGF)-β, collagen, and fibronectin, all of which may play an important role in the progression of chronic renal disease.

Adaptation to hypoxia Over the last decade, the role of hypoxia-inducible factors (HIFs) in O2 supply and adaptation to hypoxic conditions has found increasing support. HIFs are O2-sensitive transcription factors involved in O2-dependent gene regulation that mediate cellular adaptation to O2 deprivation and tissue protection under hypoxic conditions in the kidney.   NO generation can promote HIF-1α accumulation in a cGMP-independent manner. However, Hagen et al. (2003) showed that NO may reduce the activation of HIF in hypoxia via the inhibitory effect of NO on cytochrome oxidase.

Therefore, it seems that NO has pleiotropic effects on HIF expression, with various responses related to different pathways. HIF-1α upregulates a number of factors implicated in cytoprotection, including angiogenic growth factors, such as vascular endothelial growth factors (VEGF), endothelial progenitor cell recruitment via the endothelial expression of SDF-1, heme-oxygenase-1 (HO-1), and erythropoietin (EPO), and vasomotor regulation.

HO-1 produces carbon monoxide (a potent vasodilator) while degrading heme, which may preserve tissue blood flow during reperfusion. Thus, it has been suggested that the induction of HO-1 can protect the kidney from ischemic damage by decreasing oxidative damage and NO generation.

Finally, in addition to its anti-apoptotic properties, EPO may protect the kidney from ischemic damage by restoring the renal microcirculation by stimulating the mobilization and differentiation of progenitor cells toward an endothelial phenotype and by inducing NO release from eNOS.

Pharmacological interventions

Use of pharmacological interventions which act at the microcirculatory level may be a successful strategy to overcome ischemia-induced vascular damage and prevent ARF. Activated protein C (APC), an endogenous vitamin K-dependent serine protease with multiple biological activities, may meet these criteria. Along with antithrombotic and profibrinolytic properties, APC can reduce the chemotaxis and interactions of leukocytes with activated endothelium.

However, renal dysfunction was not improved in the largest study published so far. In addition, APC has been discontinued by Lilly for the use intended in severe sepsis. Moreover, neither drugs with renal vasodilatory effects (i.e., dopamine, fenoldopam, endothelin receptors blockers, adenosine antagonists) nor agents that decrease renal oxygen consumption (i.e., loop diuretics) have been shown to protect the kidney from ischemic damage. We have to bear in mind that a magic bullet to treat the highly complex condition of which is renal I/R is not in sight.

We can expect that understanding the balance between O2 delivery and O2 consumption, as well as the function of O2-consuming pathways (i.e., mitochondrial function, reactive oxygen species generation) will be central to this treatment strategy.

Take home point

The deleterious effects of NO are thought to be associated with the NO generated by the induction of iNOS and its contribution to oxidative stress both resulting in vascular dysfunction and tissue damage. Ischemic injury also leads to structural damage to the endothelium and leukocyte infiltration. Consequently, renal tissue hypoxia is proposed to promote the initial tubular damage, leading to acute organ dysfunction.   Comment: I express great appreciation for refeering to this work, which does provide enormous new insights into hypoxia-induced acute renal failure, and ties together the anatomy, physiology, and gene regulation through signaling pathways.

Ince C, Legrand M, Mik E , Johannes T, Payen D. Renal Hypoxia and Dysoxia following Reperfusion of the Ischemic Kidney. Molecular Medicine (Proof) 2008; pp36. http://www.molmed.org

Nitric oxide and non-hemodynamic functions of the kidney

One of the major scientific advances in the past decade in understanding of the renal function and disease is the prolific growth of literature incriminating nitric oxide (NO) in renal physiology and pathophysiology. NO was first shown to be identical with endothelial derived relaxing factor (EDRF) in 1987 and this was followed by a rapid flurry of information defining the significance of NO in not only vascular physiology and hemodynamics but also in neurotransmission, inflammation and immune defense systems. Although most actions of NO are mediated by cyclic guanosine monophosphate (cGMP) signaling, S-nitrosylation of cysteine residues in target proteins constitutes another well defined non-cGMP dependent mechanism of NO effects. Recent years have witnessed a phenomenal scientific interest in the vascular biology, particularly the relevance of nitric oxide (NO) in cardiovascular and renal physiology and pathophysiology. Although hemodynamic actions of NO received initial attention, a variety of non-hemodynamic actions are now known to be mediated by NO in the normal kidney, which include

  • tubular transport of electrolyte and water,
  • maintenance of acid-base homeostasis,
  • modulation of glomerular and interstitial functions,
  • renin-angiotensin activation and
  • regulation of immune defense mechanism in the kidney.

____________________________________________________________________________________________________________________________________________________________

Table 1 : Functions of NO in the kidney

  • 1. Renal macrovascular and microvascular dilatation (afferent > efferent)
  • 2. Regulation of mitochondrial respiration.
  • 3. Modulation renal medullary blood flow
  • 4. Stimulation of fluid, sodium and HCO3 – reabsorption in the proximal tubule
  • 5. Stimulation of renal acidification in proximal tubule by stimulation of NHE activity
  • 6. Inhibition of Na+, Cl- and HCO3 – reabsorption in the mTALH
  • 7. Inhibition of Na+ conductance in the CCD
  • 8. Inhibition of H+-ATPase in CCD

_____________________________________________________________________________________________________________________________________________________________

One of the renal regulatory mechanisms related to maintenance of arterial blood pressure involves the phenomenon of pressure-natriuresis in response to elevation of arterial pressure. This effect implies inhibition of tubular sodium reabsorption resulting in natriuresis, in an effort to lower arterial pressure. Experimental evidence from indicates that intra-renal NO modulates pressure natriuresis.

Furthermore many studies have confirmed the role of intra renal NO in mediating tubulo-glomerular feedback (TGF). In vivo micropuncture studies have shown that NO derived from nNOS in macula densa specifically inhibits the TGF responses leading to renal afferent arteriolar vasoconstriction in response to sodium reabsorption in the distal tubule. Other recent studies support the inhibitory role of NO from eNOS and iNOS in mTALH segment on TGF effects.

Recent observations in vascular biology have yielded new information that endothelial dysfunction early in the course might contribute to the pathophysiology of acute renal failure.  Structural and functional changes in the vascular endothelium are demonstrable in early ischemic renal failure. Altered NO production and /or decreased bioavailability of NO comprise the endothelial function in acute renal failure.

Several studies have indicated imbalance of NOS activity with enhanced expression and activity of iNOS and decreased eNOS in ischemic kidneys.

The imbalance results from enhanced iNOS activity and attenuated eNOS activity in the kidney.  

Many experimental studies support a contributory role for NO in glomerulonephritis (GN). Evidence from recent studies pointed out that NO may be involved in peroxynitrite formation, pro-inflammatory chemokines and signaling pathways in addition to direct glomerular effects that promote albumin permeability in GN. Although originally macrophages and other leukocytes were first considered as the source renal NO production in GN, it is now clear iNOS derived NO from glomerular mesangial cells are the primary source of NO in GN.

In most pathological states, the role of NO is dependent by the stage of the disease, the nitric oxide synthase (NOS) isoform involved and the presence or absence of other modifying intrarenal factors. Additionally NO may have a dual role in several disease states of the kidney such as acute renal failure, inflammatory nephritides, diabetic nephropathy and transplant rejection.

A rapidly growing body of evidence supports a critical role for NO in tubulointerstitial nephritis (TIN). In the rat model of autoimmune TIN, Gabbai et al. demonstrated increased iNOS expression in the kidney and NO metabolites in urine and plasma. However the effects of iNOS on renal damage in TIN seem to have a biphasic effect- since iNOS specific inhibitors (eg. L-Nil) are renoprotective in the acute phase while they actually accelerated the renal damage in the chronic phase.

Thus chronic NOS inhibition is used to induce chronic tubulointerstitial injury and fibrosis along with mild glomerulosclerosis and hypertension.

Major pathways of L-arginine metabolism.

L-arginine may be metabolized by the urea cycle enzyme arginase to L-ornithine and urea by arginine decarboxylase to agmatine and CO2 or by NOS to nitric oxide (NO) and L-citrulline.

Adapted from Klahr S: Can L-arginine manipulation reduce renal disease? Semin Nephrol 1999; 61:304-309.

It is obvious that kidney is not only a major source of arginine and nitric oxide but NO plays an important role in the water and electrolyte balance and acid-base physiology and many other homeostatic functions in the kidney. Unfortunately we are far from a precise understanding of the significance of NO alterations in various disease states primarily due to conflicting data from the existing literature.

Therapeutic potential for manipulation of L-arginine- nitric oxide axis in renal disease states has been discussed. More studies are required to elucidate the abnormalities in NO metabolism in renal diseases and to confirm the therapeutic potential of L-arginine.

Sharma SP. Nitric oxide and the kidney. Indian J Nephrol 2004;14: 77-84

Inhibition of Constitutive Nitric Oxide Synthase

Excess NO generation plays a major role in the hypotension and systemic vasodilatation characteristic of sepsis. Yet the kidney response to sepsis is characterized by vasoconstriction resulting in renal dysfunction. We have examined the roles of inducible nitric oxide synthase (iNOS) and endothelial NOS (eNOS) on the renal effects of lipopolysaccharide administration by comparing the effects of specific iNOS inhibition, L-N6-(1-iminoethyl)lysine (L-NIL), and 2,4-diamino-6-hydroxy-pyrimidine vs. nonspecific NOS inhibitors (nitro-L-arginine-methylester). cGMP responses to carbamylcholine (CCh) (stimulated, basal) and sodium nitroprusside in isolated glomeruli were used as indices of eNOS and guanylate cyclase (GC) activity, respectively. LPS significantly decreased blood pressure and GFR (P =0.05) and inhibited the cGMP response to CCh.

GC activity was reciprocally increased. L-NIL and 2,4-diamino-6-hydroxy-pyrimidine administration prevented the decrease in GFR, restored the normal response to CCh, and GC activity was normalized. In vitro application of L-NIL also restored CCh responses in LPS glomeruli. Neuronal NOS inhibitors verified that CCh responses reflected eNOS activity.

L-NAME, a nonspecific inhibitor, worsened GFR, a reduction that was functional and not related to glomerular thrombosis, and eliminated the CCh response. No differences were observed in eNOS mRNA expression among the experimental groups. Selective iNOS inhibition prevents reductions in GFR, whereas nonselective inhibition of NOS further decreases GFR.

These findings suggest that the decrease in GFR after LPS is due to local inhibition of eNOS by iNOS, possibly via NO autoinhibition.

Schwartz D, Mendonca M, Schwartz I, Xia Y, et al. Inhibition of Constitutive Nitric Oxide Synthase (NOS) by Nitric Oxide Generated by Inducible NOS after Lipopolysaccharide Administration Provokes Renal Dysfunction in Rats. J. Clin. Invest. 1997; 100:439–448.

Salt-Sensitivity and Hypertension Renin-angiotensin system (RAS) plays a key role in the regulation of renal function, volume of extracellular fluid and blood pressure. The activation of RAS also induces oxidative stress, particularly superoxide anion (O2-) formation.

Although the involvement of O2 – production in the pathology of many diseases is known for long, recent studies also strongly suggest its physiological regulatory function of many organs including the kidney. However, a marked accumulation of O2- in the kidney alters normal regulation of renal function and may contribute to the development of salt-sensitivity and hypertension.

In the kidney, O2- acts as vasoconstrictor and enhances tubular sodium reabsoption. Nitric oxide (NO), another important radical that exhibits opposite effects than O2 -, is also involved in the regulation of kidney function. O2- rapidly interacts with NO and thus, when O2- production increases, it diminishes the bioavailability of NO leading to the impairment of organ function. As the activation of RAS, particularly the enhanced production of angiotensin II, can induce both O2- and NO generation, it has been suggested that physiological interactions of

  • RAS,
  • NO and
  • O2-

provide a coordinated regulation of kidney function.   The imbalance of these interactions is critically linked to the pathophysiology of salt-sensitivity and hypertension.

Kopkan L, Červenka L. Renal Interactions of Renin-Angiotensin System, Nitric Oxide and Superoxide Anion: Implications in the Pathophysiology of Salt-Sensitivity and Hypertension. Physiol. Res. 2009; 58 (Suppl. 2): S55-S67.

Epicrisis

In this review I attempted to evaluate complex and still incomplete and conflicting conclusions from many studies. I thus broke the report into three major portions:

___________________________________________________________________________________________________________________________________________________________

  • 1 The kidney and its anatomy, physiology, and ontogeny.
  • 2 The pathological disease variation affecting the kidney
  • a: a tie in to eNON and iNos, nitric oxide, cGMP and glutaminase – in acute renal failure, hypertension, chronic renal failure, dialysis the pathology of acute tubular necrosis, glomerular function, efferent arteriolar and kidney medullary circulatory impairment, and cast formation related to Tamm Horsfall protein
  • b :The role of NO, eNOS and iNOS in disorders of the lund alveolar cell and subendothelial matrix, and of liver disease also affecting the kidney, and the heart. c Additional references
  • 3.     a Acute renal failure, oxidate stress, ischemia-reperfusion injury, tubulointerstitial chronic inflammation
  • 3       b Additional references 4. Nitric oxide donors – opportunities for therapeutic targeting? As we see this in as full a context as possible, it is hard to distinguish the cart from the horse.

___________________________________________________________________________________________________________________________________________________________

We know that there is an unquestionable role of NO, and a competing balance to be achieved between eNOS, iNOS, an effect on tubular water and ion-cation reabsorptrion, a role of TNFa, and consequently an important role in essential/malignant hypertension, with the size of the effect related to the stage of disorder, the amount of interstitial fibrosis, the remaining nephron population, the hypertonicity of the medulla, the vasodilation of the medullary circulation, and the renin-angiotensin-aldosterone system. Substantial data and multiple patients with many factors per patient would be need to extract the best model using a supercomputer.

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Author and Curator: Ritu Saxena, Ph.D.

 

Introduction

Nitric oxide (NO) is a lipophilic, highly diffusible and short-lived molecule that acts as a physiological messenger and has been known to regulate a variety of important physiological responses including vasodilation, respiration, cell migration, immune response and apoptosis. Jordi Muntané et al

NO is synthesized by the Nitric Oxide synthase (NOS) enzyme and the enzyme is encoded in three different forms in mammals: neuronal NOS (nNOS or NOS-1), inducible NOS (iNOS or NOS-2), and endothelial NOS (eNOS or NOS-3). The three isoforms, although similar in structure and catalytic function, differ in the way their activity and synthesis in controlled inside a cell. NOS-2, for example is induced in response to inflammatory stimuli, while NOS-1 and NOS-3 are constitutively expressed.

Regulation by Nitric oxide

NO is a versatile signaling molecule and the net effect of NO on gene regulation is variable and ranges from activation to inhibition of transcription.

The intracellular localization is relevant for the activity of NOS. Infact, NOSs are subject to specific targeting to subcellular compartments (plasma membrane, Golgi, cytosol, nucleus and mitochondria) and that this trafficking is crucial for NO production and specific post-translational modifications of target proteins.

Role of Nitric oxide in Cancer

One in four cases of cancer worldwide are a result of chronic inflammation. An inflammatory response causes high levels of activated macrophages. Macrophage activation, in turn, leads to the induction of iNOS gene that results in the generation of large amount of NO. The expression of iNOS induced by inflammatory stimuli coupled with the constitutive expression of nNOS and eNOS may contribute to increased cancer risk. NO can have varied roles in the tumor environment influencing DNA repair, cell cycle, and apoptosis. It can result in antagonistic actions including DNA damage and protection from cytotoxicity, inhibiting and stimulation cell proliferation, and being both anti-apoptotic and pro-apoptotic. Genotoxicity due to high levels of NO could be through direct modification of DNA (nitrosative deamination of nucleic acid bases, transition and/or transversion of nucleic acids, alkylation and DNA strand breakage) and inhibition of DNA repair enzymes (such as alkyltransferase and DNA ligase) through direct or indirect mechanisms. The Multiple actions of NO are probably the result of its chemical (post-translational modifications) and biological heterogeneity (cellular production, consumption and responses). Post-translational modifications of proteins by nitration, nitrosation, phosphorylation, acetylation or polyADP-ribosylation could lead to an increase in the cancer risk. This process can drive carcinogenesis by altering targets and pathways that are crucial for cancer progression much faster than would otherwise occur in healthy tissue.

NO can have several effects even within the tumor microenvironment where it could originate from several cell types including cancer cells, host cells, tumor endothelial cells. Tumor-derived NO could have several functional roles. It can affect cancer progression by augmenting cancer cell proliferation and invasiveness. Infact, it has been proposed that NO promotes tumor growth by regulating blood flow and maintaining the vasodilated tumor microenvironment. NO can stimulate angiogenesis and can also promote metastasis by increasing vascular permeability and upregulating matrix metalloproteinases (MMPs). MMPs have been associated with several functions including cell proliferation, migration, adhesion, differentiation, angiogenesis and so on. Recently, it was reported that metastatic tumor-released NO might impair the immune system, which enables them to escape the immunosurveillance mechanism of cells. Molecular regulation of tumour angiogenesis by nitric oxide.

S-nitrosylation and Cancer

The most prominent and recognized NO reaction with thiols groups of cysteine residues is called S-nitrosylation or S-nitrosation, which leads to the formation of more stable nitrosothiols. High concentrations of intracellular NO can result in high concentrations of S-nitrosylated proteins and dysregulated S-nitrosylation has been implicated in cancer. Oxidative and nitrosative stress is sensed and closely associated with transcriptional regulation of multiple target genes.

Following are a few proteins that are modified via NO and modification of these proteins, in turn, has been known to play direct or indirect roles in cancer.

NO mediated aberrant proteins in Cancer

Bcl2

Bcl-2 is an important anti-apoptotic protein. It works by inhibiting mitochondrial Cytochrome C that is released in response to apoptotic stimuli. In a variety of tumors, Bcl-2 has been shown to be upregulated, and it has additionally been implicated with cancer chemo-resistance through dysregulation of apoptosis. NO exposure causes S-nitrosylation at the two cysteine residues – Cys158 and Cys229 that prevents ubiquitin-proteasomal pathway mediated degradation of the protein. Once prevented from degradation, the protein attenuates its anti-apoptotic effects in cancer progression. The S-nitrosylation based modification of Bcl-2 has been observed to be relevant in drug treatment studies (for eg. Cisplatin). Thus, the impairment of S-nitrosylated Bcl-2 proteins might serve as an effective therapeutic target to decrease cancer-drug resistance.

p53

p53 has been well documented as a tumor suppressor protein and acts as a major player in response to DNA damage and other genomic alterations within the cell. The activation of p53 can lead to cell cycle arrest and DNA repair, however, in case of irrepairable DNA damage, p53 can lead to apoptosis. Nuclear p53 accumulation has been related to NO-mediated anti-tumoral properties. High concentration of NO has been found to cause conformational changes in p53 resulting in biological dysfunction.. In RAW264.7, a murine macrophage cell line, NO donors induce p53 accumulation and apoptosis through JNK-1/2.

HIF-1a

Hypoxia-inducible factor 1 (HIF1) is a heterodimeric transcription factor that is predominantly active under hypoxic conditions because the HIF-1a subunit is rapidly degraded in normoxic conditions by proteasomal degradation. It regulates the transciption of several genes including those involved in angiogenesis, cell cycle, cell metabolism, and apoptosis. Hypoxic conditions within the tumor can lead to overexpression of HIF-1a. Similar to hypoxia-mediated stress, nitrosative stress can stabilize HIF-1a. NO derivatives have also been shown to participate in hypoxia signaling. Resistance to radiotherapy has been traced back to NO-mediated HIF-1a in solid tumors in some cases.

PTEN

Phosphatase and tensin homolog deleted on chromosome ten (PTEN), is again a tumor suppressor protein. It is a phosphatase and has been implicated in many human cancers. PTEN is a crucial negative regulator of PI3K/Akt signaling pathway. Over-activation of PI3K/Akt mediated signaling pathway is known to play a major role in tumorigenesis and angiogenesis. S-nitrosylation of PTEN, that could be a result of NO stress, inhibits PTEN. Inhibition of PTEN phosphatase activity, in turn, leads to promotion of angiogenesis.

C-Src

C-src belongs to the Src family of protein tyrosine kinases and has been implicated in the promotion of cancer cell invasion and metastasis. It was demonstrated that S-nitrosylation of c-Src at cysteine 498 enhanced its kinase activity, thus, resulting in the enhancement of cancer cell invasion and metastasis.

Reference:

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Wang Z. Protein S-nitrosylation and cancer. Cancer Lett. 2012 Jul 28;320(2):123-9. http://www.ncbi.nlm.nih.gov/pubmed/22425962

Ziche M and Morbidelli L. Molecular regulation of tumour angiogenesis by nitric oxide. Eur Cytokine Netw. 2009 Dec;20(4):164-70.http://www.ncbi.nlm.nih.gov/pubmed/20167555

Jaiswal M, et al. Nitric oxide in gastrointestinal epithelial cell carcinogenesis: linking inflammation to oncogenesis. Am J Physiol Gastrointest Liver Physiol. 2001 Sep;281(3):G626-34. http://www.ncbi.nlm.nih.gov/pubmed/11518674

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