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Genomics-based cure for diabetes on-the-way

Posted in Chemical Biology and its relations to Metabolic Disease, Chemical Genetics, Genome Biology, Reproductive Andrology, Embryology, Genomic Endocrinology, Preimplantation Genetic Diagnosis and Reproductive Genomics, tagged Adox, bivalent modification, cell reprogramming, chip, Diabetes, endocrine cells, epigenetic, ES cells, FACS, glucagon, glucose, histone acetylation, histone methylation, histone modification, homeostasis, Insulin, Islets of Langerhans, α cells, β cells, long noncoding RNA, monovalent modification, pancreatic islets, pluripotent cells, qRT-PCR, RNA sequencing, Therapy, transcriptional, transcriptome, type 1 diabetes, type 2 diabetes on March 4, 2013| 5 Comments »

Reporter: Ritu Saxena, Ph.D.

Diabetes currently affects more than 336 million people worldwide, with healthcare costs by diabetes and its complications of up to $612 million per day in the US alone. The islets of Langerhans, miniature endocrine organs within the pancreas, are essential regulators of blood glucose homeostasis and play a key role in the pathogenesis of diabetes. Islets of Langerhans are composed of several types of endocrine cells. The α- and β-cells are the most abundant and also the most important in that they secrete hormones (glucagon and insulin, respectively) crucial for glucose homeostasis (Bosco D, et al, Diabetes, May 2010;59(5):1202-10).

Diabetes is a ‘bihormonal’ disease, involving both insulin deficiency and excess glucagon. For decades, insulin deficiency was considered to be the sole reason for diabetes; however, recent studies emphasize excess glucagon as an important part of diabetes etiology. Thus, insulin-secreting β cells and glucagon-secreting α cells maintain physiological blood glucose levels, and their malfunction drives diabetes development. Increasing the number of insulin-producing β cells while decreasing the number of glucagon-producing α cells, either in vitro in donor pancreatic islets before transplantation into type 1 diabetics or in vivo in type 2 diabetics, is a promising therapeutic avenue. A huge leap has been taken in this direction by the researchers at the University of Pennsylvania (Philadelphia, PA) in collaboration with Oregon Health and Science University (Portland, OR), USA by demonstrating that α to β cell reprogramming could be promoted by manipulating the histone methylation signature of human pancreatic islets. In fact, the treatment of cultured pancreatic islets with a histone methyltransferase inhibitor leads to colocalization of both glucagon and insulin and glucagon and insulin promoter factor 1 (PDX1) in human islets and colocalization of both glucagon and insulin in mouse islets. The research findings were published in the Journal of Clinical Investigation.

Study design: First step was to study and analyze the epigenetic and transcriptional landscape of human pancreatic human pancreatic α, β, and exocrine cells using ChIP and RNA sequencing. Study design for determination of the transcriptome and differential histone marks included the dispersion and FACS to of human islets to obtain cell populations highly enriched for α, β, and exocrine (duct and acinar) cells. Then, chromatin was prepared for ChIP analysis using antibodies for histone modifications, H3K4me3 (represents gene activation) and H3K27me3 (represents gene repression). RNA-Sequencing analysis was then performed to determine mRNA and lncRNA. Sample purity was confirmed using qRT-PCR of insulin and glucagon expression levels of the individual α and β cell population revealing high sample purity.

Results:

Long noncoding transcripts: Long noncoding RNA molecules have been implicated as important developmental regulators, cell lineage allocators, and contributors to disease development. The authors discovered 12 cell–specific and 5 α cell–specific noncoding (lnc) transcripts, indicative of the valuable research resource represented from transcriptome data. Recently discovered lncRNA molecules in islets are regulated during development and dysregulated in type 2 diabetic islets.
Monovalent histone modification landscapes shared among three cell types: Monovalent H3K4me3-enriched regions, indicative of gene activation, were identified and compared in α, β, and exocrine cells. Strikingly, the vast majority of monovalently H3K4me3-marked genes were shared among the 3 pancreatic cell lineages (83%–95%), reflecting both their related function in protein secretion and common embryonic descent. Similarly, a high degree of overlap was observed in H3K27me3 modification patterns in all the three cell types (73%–83%).
Bivalent histone modifications (H3K4me3 and H3K27me3) were high in α cells: Bernstein colleagues observed bivalent marks to be common in undifferentiated cells, such as ES cells and pluripotent progenitor cells, and in most cases, one of the histone modification marks was lost during differentiation, accompanying lineage specification (Bernstein BE, et al, Cell, 21 Apr 2006; 125(2):315-26). α cells exhibited many more genes bivalently marked, followed by β cells and exocrine cells. Bivalent state was remarkably similar to that of hESC, suggesting a more plastic epigenomic state for α cells.
Monovalent histone modifications were high in β cells: Thousands of the genes that were in bivalent state in α cells were in a monovalent state, carrying only the activating or repressing mark.
Inhibition of histone methyltransferases led to partial cell-fate conversion: Adenosine dialdehye (Adox), a drug that interferes with histone methylation and decreases H3K27me3, when administered in human islet tissue, led to decrease of H3K27me3 enrichment at the 3 gene loci that are originally expressed bivalently in α cells and monovalently in β cells: MAFA, PDX1 and ARX. Adox resulted in the occasional cooccurrence of glucagon and insulin granules within the same islet cell, which was not observed in untreated islets. Thus, inhibition of histone methyltransferases leads to partial endocrine cell-fate conversion.

Conclusion: α cells have been reprogrammed into β cell fate in various mouse models. The reason, as proposed by the authors, might be the presence of more bivalently marked genes that confers a more plastic epigenomic state of the cells that probably drives them to the β cell fate. Therefore, using epigenomic information of different cell types in pancreatic islets and harnessing it for subsequent manipulation of their epigenetic signature could be utilized to reprogram cells and hence provide a path for diabetes therapy.

Source: Bramswig NC, et al, Epigenomic plasticity enables human pancreatic α to β cell reprogramming. J Clin Invest, 22 Feb 2013. pii: 66514.

Related reading on Pharmaceutical Intelligence:

Junk DNA codes for valuable miRNAs: non-coding DNA controls Diabetes

Therapeutic Targets for Diabetes and Related Metabolic Disorders

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CRACKING THE CODE OF HUMAN LIFE: Recent Advances in Genomic Analysis and Disease – Part IIC

2013 Genomics: The Era Beyond the Sequencing of the Human Genome: Francis Collins, Craig Venter, Eric Lander, et al.

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Osteocytes: A Special Issue in Bone

Posted in Biological Networks, Gene Regulation and Evolution, Bone Disease and Musculoskeletal Disease, Cell Biology, Signaling & Cell Circuits, Chemical Biology and its relations to Metabolic Disease, Disease Biology, Small Molecules in Development of Therapeutic Drugs, tagged Apoptosis, Autophagy, Cell Biology, CKD, FGF23, gap junctions, homeostasis, lacunae, mechanical strain, metabolic bone disease, osteoclastogenesis, osteoclasts, osteocytes, primary cilia, PTH, vitamin D on February 6, 2013| 1 Comment »

Reporter: Aviral Vatsa PhD, MBBS

Osteocytes are the professional mechanosensors of bone. They modulate bone remodelling in accordance with external mechanical loads by orchestrating the activity of one forming osteoblasts and bone resorbing osteoclasts. Osteocytes are at the heart of bone metabolism. They constitute >95% of bone cells. They are terminally differentiated cells and reside in the hard mineralised matrix of bone, thus making it difficult to study them in situ. However, recent developments in imaging and tissue processing have made it possible to study osteocytes in their natural milieu. Moreover, increasing number of studies have highlighted the fact that a multifaceted approach from various domains of science such as biomechanics, cell biology, bioengineering, biophysics, biomaterials, computational modelling, endocrinology, and orthopaedics is essential to further our understanding of the intricate processes involved in bone remodelling and the central role of osteocytes in maintaining bone mass and architecture.

In this post a variety of reviews from an upcoming special issue on osteocytes in the journal Bone are highlighted that help us add few more pieces of knowledge to the ever growing eclaircissements on the subject.

1. Measurement and estimation of osteocyte mechanical strain

Review Article
Amber Rath Stern, Daniel P. Nicolella

Abstract

Osteocytes are the most abundant cell type in bone and are responsible for sensing mechanical strain and signaling bone (re)modeling, making them the primary mechanosensors within the bone. Under aging and osteoporotic conditions, bone is known to be less responsive to loading (exercise), but it is unclear why. Perhaps, the levels of mechanical strain required to initiate these biological events are not perceived by the osteocytes embedded within the bone tissue. In this review we examine the methods used to measure and estimate the strains experienced by osteocytes in vivo as well as the results of related published experiments. Although the physiological levels of strain experienced by osteocytes in vivo are still under investigation, through computational modeling and laboratory experiments, it has been shown that there is significant amplification of average bone strain at the level of the osteocyte lacunae. It has also been proposed that the material properties of the perilacunar region surrounding the osteocyte can have significant effects of the strain perceived by the embedded osteocyte. These facts have profound implications for studies involving osteoporotic bone where the material properties are known to become stiffer.

2. Glucocorticoids and Osteocyte Autophagy

Review Article
Wei Yao, Weiwei Dai, Jean X. Jiang, Nancy E. Lane

Abstract

Glucocorticoids are used for the treatment of inflammatory and autoimmune diseases. While they are effective therapy, bone loss and incident fracture risk is high. While previous studies have found GC effects on both osteoclasts and oteoblasts, our work has focused on the effects of GCs on osteocytes. Osteocytes exposed to low dose GCs undergo autophagy while osteocytes exposed to high doses of GCs or for a prolonged period of time undergo apoptosis. This paper will review the data to support the role of GCs in osteocyte autophagy.

3. Osteocytes remove and replace perilacunar mineral during reproductive cycles

Review Article
John J. Wysolmerski

Abstract

Lactation is associated with an increased demand for calcium and is accompanied by a remarkable cycle of bone loss and recovery that helps to supply calcium and phosphorus for milk production. Bone loss is the result of increased bone resorption that is due, in part, to increased levels of PTHrP and decreased levels of estrogen. However, the regulation of bone turnover during this time is not fully understood. In the 1960s and 1970s many observations were made to suggest that osteocytes could resorb bone and increase the size of their lacunae. This concept became known as osteocytic osteolysis and studies suggested that it occurred in response to parathyroid hormone and/or an increased systemic demand for calcium. However, this concept fell out of favor in the late 1970s when it was established that osteoclasts were the principal bone-resorbing cells. Given that lactation is associated with increased PTHrP levels and negative calcium balance, we recently examined whether osteocytes contribute to bone loss during this time. Our findings suggest that osteocytes can remodel their perilacunar and pericanalicular matrix and that they participate in the liberation of skeletal calcium stores during reproductive cycles. These findings raise new questions about the role of osteocytes in coordinating bone and mineral metabolism during lactation as well as the recovery of bone mass after weaning. It is also interesting to consider whether osteocyte lacunar and canalicular remodeling contribute more broadly to the maintenance of skeletal and mineral homeostasis.

4. Studying osteocytes within their environment

Review Article
Duncan J. Webster, Philipp Schneider, Sarah L. Dallas, Ralph Müller

Abstract

It is widely hypothesized that osteocytes are the mechano-sensors residing in the bone’s mineralized matrix which control load induced bone adaptation. Owing to their inaccessibility it has proved challenging to generate quantitative in vivo experimental data which supports this hypothesis. Recent advances in in situ imaging, both in non-living and living specimens, have provided new insights into the role of osteocytes in the skeleton. Combined with the retrieval of biochemical information from mechanically stimulated osteocytes using in vivo models, quantitative experimental data is now becoming available which is leading to a more accurate understanding of osteocyte function. With this in mind, here we review i) state of the art ex vivo imaging modalities which are able to precisely capture osteocyte structure in 3D, ii) live cell imaging techniques which are able to track structural morphology and cellular differentiation in both space and time, and iii) in vivo models which when combined with the latest biochemical assays and microfluidic imaging techniques can provide further insight on the biological function of osteocytes.

5. Osteocyte apoptosis

Review Article
Robert L. Jilka, Brendon Noble, Robert S. Weinstein

Abstract

Apoptotic death of osteocytes was recognized over 15 years ago, but its significance for bone homeostasis has remained elusive. A new paradigm has emerged that invokes osteocyte apoptosis as a critical event in the recruitment of osteoclasts to a specific site in response to skeletal unloading, fatigue damage, estrogen deficiency and perhaps in other states where bone must be removed. This is accomplished by yet to be defined signals emanating from dying osteocytes, which stimulate neighboring viable osteocytes to produce osteoclastogenic cytokines. The osteocyte apoptosis caused by chronic glucocorticoid administration does not increase osteoclasts; however, it does negatively impact maintenance of bone hydration, vascularity, and strength.

6. Emerging role of primary cilia as mechanosensors in osteocytes

Review Article
An M. Nguyen, Christopher R. Jacobs

Abstract

The primary cilium is a solitary, immotile microtubule-based extension present on nearly every mammalian cell. This organelle has established mechanosensory roles in several contexts including kidney, liver, and the embryonic node. Mechanical load deflects the cilium, triggering biochemical responses. Defects in cilium function have been associated with numerous human diseases. Recent research has implicated the primary cilium as a mechanosensor in bone. In this review, we discuss the cilium, the growing evidence for its mechanosensory role in bone, and areas of future study.

7. Mechanosensation and transduction in osteocytes

Review Article
Jenneke Klein-Nulend, Astrid D. Bakker, Rommel G. Bacabac, Aviral Vatsa, Sheldon Weinbaum

Abstract

The human skeleton is a miracle of engineering, combining both toughness and light weight. It does so because bones possess cellular mechanisms wherein external mechanical loads are sensed. These mechanical loads are transformed into biological signals, which ultimately direct bone formation and/or bone resorption. Osteocytes, since they are ubiquitous in the mineralized matrix, are the cells that sense mechanical loads and transduce the mechanical signals into a chemical response. The osteocytes then release signaling molecules, which orchestrate the recruitment and activity of osteoblasts or osteoclasts, resulting in the adaptation of bone mass and structure. In this review, we highlight current insights in bone adaptation to external mechanical loading, with an emphasis on how a mechanical load placed on whole bones is translated and amplified into a mechanical signal that is subsequently sensed by the osteocytes.

8. The osteocyte in CKD: New concepts regarding the role of FGF23 in mineral metabolism and systemic complications

Review Article
Katherine Wesseling-Perry, Harald Jüppner

Abstract

The identification of elevated circulating levels of the osteocytic protein fibroblast growth factor 23 (FGF23) in patients with chronic kidney disease (CKD), along with recent data linking these values to the pathogenesis of secondary hyperparathyroidism and to systemic complications, has changed the approach to the pathophysiology and treatment of disordered bone and mineral metabolism in renal failure. It now appears that osteocyte biology is altered very early in the course of CKD and these changes have implications for bone biology, as well as for progressive cardiovascular and renal disease. Since circulating FGF23 values are influenced by therapies used to treat secondary hyperparathyroidism, the effects of different therapeutic paradigms on FGF23 have important implications for mineral metabolism as well as for morbidity and mortality. Further studies are critically needed to identify the initial trigger for abnormalities of skeletal mineralization and turnover as well as the potential effects that current therapeutic options may have on osteocyte biology.

9. Vitamin D signaling in osteocytes: Effects on bone and mineral homeostasis

Review Article
Liesbet Lieben, Geert Carmeliet

Abstract

The active form of vitamin D [1,25(OH)₂D] is an important regulator of calcium and bone homeostasis, as evidenced by the consequences of 1,25(OH)₂D inactivity in man and mice, which include hypocalcemia, hypophosphatemia, secondary hyperparathyroidism and bone abnormalities. The recent generation of tissue-specific (intestine, osteoblast/osteocyte, chondrocyte) vitamin D receptor (Vdr) null mice has provided mechanistic insight in the cell-specific actions of 1,25(OH)₂D and their contribution to the integrative physiology of VDR signaling that controls bone and mineral metabolism. These studies have demonstrated that even with normal dietary calcium intake, 1,25(OH)₂D is crucial to maintain normal calcium and bone homeostasis and accomplishes this through this primarily through stimulation of intestinal calcium transport. When, moreover, insufficient calcium is acquired from the diet (severe dietary calcium restriction, lack of intestinal VDR activity), 1,25(OH)₂D levels will increase and will directly act on osteoblasts and osteocytes to enhance bone resorption and to suppress bone matrix mineralization. Although this system is essential to maintain normal calcium levels in blood during a negative calcium balance, the consequences for bone are disastrous and generate an increased fracture risk. These findings evidently demonstrate that preservation of serum calcium levels has priority over skeletal integrity. Since vitamin D supplementation is an essential part of anti-osteoporotic therapy, mechanistic insight in vitamin D actions is required to define the optimal therapeutic regimen, taking into account the amount of dietary calcium supply, in order to maximize the targeted outcome and to avoid side-effects. We will review the current understanding concerning the functions of osteoblastic/osteocytic VDR signaling which not only include the regulation of bone metabolism, but also comprise the control of calcium and phosphate homeostasis via fibroblast growth factor (FGF) 23 secretion and the maintenance of the hematopoeitic stem cell (HSC) niche, with special focus on the experimental data obtained from systemic and osteoblast/osteocyte-specific Vdr null mice.

10. In vitro and in vivo approaches to study osteocyte biology

Review Article
Ivo Kalajzic, Brya G. Matthews, Elena Torreggiani, Marie A. Harris, Paola Divieti Pajevic, Stephen E. Harris

Abstract

Osteocytes, the most abundant cell population of the bone lineage, have been a major focus in the bone research field in recent years. This population of cells that resides within mineralized matrix is now thought to be the mechanosensory cell in bone and plays major roles in the regulation of bone formation and resorption. Studies of osteocytes had been impaired by their location, resulting in numerous attempts to isolate primary osteocytes and to generate cell lines representative of the osteocytic phenotype. Progress has been achieved in recent years by utilizing in vivo genetic technology and generation of osteocyte directed transgenic and gene deficiency mouse models.

We will provide an overview of the current in vitro and in vivo models utilized to study osteocyte biology. We discuss generation of osteocyte-like cell lines and isolation of primary osteocytes and summarize studies that have utilized these cellular models to understand the functional role of osteocytes. Approaches that attempt to selectively identify and isolate osteocytes using fluorescent protein reporters driven by regulatory elements of genes that are highly expressed in osteocytes will be discussed.

In addition, recent in vivo studies utilizing overexpression or conditional deletion of various genes using dentin matrix protein (Dmp1) directed Cre recombinase are outlined. In conclusion, evaluation of the benefits and deficiencies of currently used cell lines/genetic models in understanding osteocyte biology underlines the current progress in this field. The future efforts will be directed towards developing novel in vitro and in vivo models that would additionally facilitate in understanding the multiple roles of osteocytes.

11. Gap junction and hemichannel functions in osteocytes

Review Article
Alayna E. Loiselle, Jean X. Jiang, Henry J. Donahue

Abstract

Cell-to-cell and cell-to-matrix communication in bone cells mediated by gap junctions and hemichannels, respectively, maintains bone homeostasis. Gap junctional communication between cells permits the passage of small molecules including calcium and cyclic AMP. This cell-to-cell communication occurs between bone cells including osteoblasts, osteoclasts and osteocytes, and is important in both bone formation and bone resorption. Connexin (Cx) 43 is the predominant gap junction protein in bone cells, and facilitates the communication of cellular signals either through docking of gap junctions between two cells, or through the formation of un-paired hemichannels. Systemic deletion of Cx43 results in perinatal lethality, so conditional deletion models are necessary to study the postnatal role of gap junctions in bone. These models provide the opportunity to determine the role of gap junctions in specific bone cells, notably the osteocyte. In this review, we summarize the key roles that gap junctions and hemichannels in osteocytes play in bone cell response to many stimuli including mechanical loading, intracellular and extracellular stimuli, such as parathyroid hormone, PGE₂, plasma calcium levels and pH, as well as in maintaining osteocyte survival.

12. Effects of PTH on osteocyte function

Review Article
Teresita Bellido, Vaibhav Saini, Paola Divieti Pajevic

Abstract

Osteocytes are ideally positioned to detect and respond to mechanical and hormonal stimuli and to coordinate the function of osteoblasts and osteoclasts. However, evidence supporting the involvement of osteocytes in specific aspects of skeletal biology has been limited mainly due to the lack of suitable experimental approaches. Few crucial advances in the field in the past several years have markedly increased our understanding of the function of osteocytes. The development of osteocytic cell lines initiated a plethora of in vitro studies that have provided insights into the unique biology of osteocytes and continue to generate novel hypotheses. Genetic approaches using promoter fragments that direct gene expression to osteocytes allowed the generation of mice with gain or loss of function of particular genes revealing their role in osteocyte function. Furthermore, evidence that Sost/sclerostin is expressed primarily in osteocytes and inhibits bone formation by osteoblasts, fueled research attempting to identify regulators of this gene as well as other osteocyte products that impact the function of osteoblasts and osteoclasts. The discovery that parathyroid hormone (PTH), a central regulator of bone homeostasis, inhibits sclerostin expression generated a cascade of studies that revealed that osteocytes are crucial target cells of the actions of PTH. This review highlights these investigations and discusses their significance for advancing our understanding of the mechanisms by which osteocytes regulate bone homeostasis and for developing therapies for bone diseases targeting osteocytes.

13. For whom the bell tolls: Distress signals from long-lived osteocytes and the pathogenesis of metabolic bone diseases

Review Article
Stavros C. Manolagas, A. Michael Parfitt

Abstract

Osteocytes are long-lived and far more numerous than the short-lived osteoblasts and osteoclasts. Immured within the lacunar–canalicular system and mineralized matrix, osteocytes are ideally located throughout the bone to detect the need for, and accordingly choreograph, the bone regeneration process by independently controlling rate limiting steps of bone resorption and formation. Consistent with this role, emerging evidence indicates that signals arising from apoptotic and old/or dysfunctional osteocytes are seminal culprits in the pathogenesis of involutional, post-menopausal, steroid-, and immobilization-induced osteoporosis. Osteocyte-originated signals may also contribute to the increased bone fragility associated with bone matrix disorders like osteogenesis imperfecta, and perhaps the rapid reversal of bone turnover above baseline following discontinuation of anti-resorptive treatments, like denosumab.

14. Osteocyte control of osteoclastogenesis

Review Article
Charles A. O’Brien, Tomoki Nakashima, Hiroshi Takayanagi

Abstract

Multiple lines of evidence support the idea that osteocytes act as mechanosensors in bone and that they control bone formation, in part, by expressing the Wnt antagonist sclerostin. However, the role of osteocytes in the control of bone resorption has been less clear. Recent studies have demonstrated that osteocytes are the major source of the cytokine RANKL involved in osteoclast formation in cancellous bone. The goal of this review is to discuss these and other studies that reveal mechanisms whereby osteocytes control osteoclast formation and thus bone resorption.

References