Vitamin K antagonism impairs the bone marrow microenvironment and hematopoiesis.

Vitamin K antagonists (VKAs) have been used in 1% of the world's population for prophylaxis or treatment of thromboembolic events for 64 years. Impairment of osteoblast function and osteoporosis has been described in patients receiving VKAs. Given the involvement of cells of the bone marrow microenvironment (BMM), such as mesenchymal stem cells (MSCs) and macrophages, as well as other factors such as the extracellular matrix for the maintenance of normal hematopoietic stem cells (HSCs), we investigated a possible effect of VKAs on hematopoiesis via the BMM. Using various transplantation and in vitro assays, we show here that VKAs alter parameters of bone physiology and reduce functional HSCs 8-fold. We implicate impairment of the functional, secreted, vitamin K-dependent, γ-carboxylated form of periostin by macrophages and, to a lesser extent, MSCs of the BMM and integrin ß3-AKT signaling in HSCs as at least partly causative of this effect, with VKAs not being directly toxic to HSCs. In patients, VKA use associates with modestly reduced leukocyte and monocyte counts, albeit within the normal reference range. VKAs decrease human HSC engraftment in immunosuppressed mice. Following published examples that alteration of the BMM can lead to hematological malignancies in mice, we describe, without providing a causal link, that the odds of VKA use are higher in patients with vs without a diagnosis of myelodysplastic syndrome (MDS). These results demonstrate that VKA treatment impairs HSC function via impairment of the BMM and the periostin/integrin ß3 axis, possibly associating with increased MDS risk.

Effects of mild hyperbaric oxygen on osteoporosis induced by hindlimb unloading in rats.

INTRODUCTION: Disuse-induced bone loss is caused by a suppression of osteoblastic bone formation and an increase in osteoclastic bone resorption. There are few data available for the effects of environmental conditions, i.e., atmospheric pressure and/or oxygen concentration, on osteoporosis. This study examined the effects of mild hyperbaric oxygen at 1317 hPa with 40% oxygen on unloading-induced osteoporosis. MATERIALS AND METHODS: Eighteen 8-week old male Wistar rats were randomly divided into three groups: the control for 21 days without unloading and mild hyperbaric oxygen (NOR, n = 6), the unloading for 21 days and recovery for 10 days without mild hyperbaric oxygen (HU + NOR, n = 6), and the unloading for 21 days and recovery for 10 days with mild hyperbaric oxygen (HU + MHO, n = 6). RESULTS: The cortical thickness and trabecular bone surface area were decreased in the HU + NOR group compared to the NOR group. There were no differences between the NOR and HU + MHO groups. Osteoclast surface area and Sclerostin (Sost) mRNA expression levels were decreased in the HU + MHO group compared to the HU + NOR group. These results suggested that the loss of the cortical and trabecular bone is inhibited by mild hyperbaric oxygen, because of an inhibition of osteoclasts and enhancement of bone formation with decreased Sost expression. CONCLUSIONS: We conclude that exposure to mild hyperbaric oxygen partially protects from the osteoporosis induced by hindlimb unloading.

Carbonic anhydrase III protects osteocytes from oxidative stress.

Osteocytes are master orchestrators of bone remodeling; they control osteoblast and osteoclast activities both directly via cell-to-cell communication and indirectly via secreted factors, and they are the main postnatal source of sclerostin and RANKL (receptor activator of NF-kB ligand), two regulators of osteoblast and osteoclast function. Despite progress in understanding osteocyte biology and function, much remains to be elucidated. Recently developed osteocytic cell lines-together with new genome editing tools-has allowed a closer look at the biology and molecular makeup of these cells. By using single-cell cloning, we identified genes that are associated with high Sost/sclerostin expression and analyzed their regulation and function. Unbiased transcriptome analysis of high- vs. low-Sost/sclerostin-expressing cells identified known and novel genes. Dmp1 (dentin matrix protein 1), Dkk1 (Dickkopf WNT signaling pathway inhibitor 1), and Phex were among the most up-regulated known genes, whereas Srpx2, Cd200, and carbonic anhydrase III (CAIII) were identified as novel markers of differentiated osteocytes. Aspn, Enpp2, Robo2, Nov, and Serpina3g were among the transcripts that were most significantly suppressed in high-Sost cells. Considering that CAII was recently identified as being regulated by Sost/sclerostin and capable of controlling mineral homeostasis, we focused our attention on CAIII. Here, we report that CAIII is highly expressed in osteocytes, is regulated by parathyroid hormone both in vitro and in vivo, and protects osteocytes from oxidative stress.-Shi, C., Uda, Y., Dedic, C., Azab, E., Sun, N., Hussein, A. I., Petty, C. A., Fulzele, K., Mitterberger-Vogt, M. C., Zwerschke, W., Pereira, R., Wang, K., Divieti Pajevic, P. Carbonic anhydrase III protects osteocytes from oxidative stress.

Osteocyte Mechanobiology.

PURPOSE OF REVIEW: Over the past decades, osteocytes have emerged as mechano-sensors of bone and master regulators of bone homeostasis. This article summarizes latest research and progress made in understanding osteocyte mechanobiology and critically reviews tools currently available to study these cells. RECENT FINDINGS: Whereas increased mechanical forces promote bone formation, decrease loading is always associated with bone loss and skeletal fragility. Recent studies identified cilia, integrins, calcium channels, and G-protein coupled receptors as important sensors of mechanical forces and Ca2+ and cAMP signaling as key effectors. Among transcripts regulated by mechanical forces, sclerostin and RANKL have emerged as potential therapeutic targets for disuse-induced bone loss. In this paper, we review the mechanisms by which osteocytes perceive and transduce mechanical cues and the models available to study mechano-transduction. Future directions of the field are also discussed.

The Wnt Inhibitor Sclerostin Is Up-regulated by Mechanical Unloading in Osteocytes in Vitro.

Although bone responds to its mechanical environment, the cellular and molecular mechanisms underlying the response of the skeleton to mechanical unloading are not completely understood. Osteocytes are the most abundant but least understood cells in bones and are thought to be responsible for sensing stresses and strains in bone. Sclerostin, a product of the SOST gene, is produced postnatally primarily by osteocytes and is a negative regulator of bone formation. Recent studies show that SOST is mechanically regulated at both the mRNA and protein levels. During prolonged bed rest and immobilization, circulating sclerostin increases both in humans and in animal models, and its increase is associated with a decrease in parathyroid hormone. To investigate whether SOST/sclerostin up-regulation in mechanical unloading is a cell-autonomous response or a hormonal response to decreased parathyroid hormone levels, we subjected osteocytes to an in vitro unloading environment achieved by the NASA rotating wall vessel system. To perform these studies, we generated a novel osteocytic cell line (Ocy454) that produces high levels of SOST/sclerostin at early time points and in the absence of differentiation factors. Importantly, these osteocytes recapitulated the in vivo response to mechanical unloading with increased expression of SOST (3.4 ± 1.9-fold, p < 0.001), sclerostin (4.7 ± 0.1-fold, p < 0.001), and the receptor activator of nuclear factor κΒ ligand (RANKL)/osteoprotegerin (OPG) (2.5 ± 0.7-fold, p < 0.001) ratio. These data demonstrate for the first time a cell-autonomous increase in SOST/sclerostin and RANKL/OPG ratio in the setting of unloading. Thus, targeted osteocyte therapies could hold promise as novel osteoporosis and disuse-induced bone loss treatments by directly modulating the mechanosensing cells in bone.

Parathyroid hormone (PTH)/PTH-related peptide type 1 receptor (PPR) signaling in osteocytes regulates anabolic and catabolic skeletal responses to PTH.

Parathyroid hormone (PTH) is the only Food and Drug Administration-approved anabolic agent to treat osteoporosis; however, the cellular targets of PTH action in bone remain controversial. PTH modulates bone turnover by binding to the PTH/PTH-related peptide (PTHrP) type 1 receptor (PPR), a G-protein-coupled receptor highly expressed in bone and kidneys. Osteocytes, the most abundant cells in adult bone, also express PPR. However, the physiological relevance of PPR signaling in osteocytes remains to be elucidated. Toward this goal, we generated mice with PPR deletion in osteocytes (Ocy-PPRKO). Skeletal analysis of these mice revealed a significant increase in bone mineral density and trabecular and cortical bone parameters. Osteoblast activities were reduced in these animals, as demonstrated by decreased collagen type I α1 mRNA and receptor activator of NF-κB ligand (RANKL) expression. Importantly, when subjected to an anabolic or catabolic PTH regimen, Ocy-PPRKO animals demonstrated blunted skeletal responses. PTH failed to suppress SOST/Sclerostin or induce RANKL expression in Ocy-PPRKO animals compared with controls. In vitro, osteoclastogenesis was significantly impaired in Ocy-PPRKO upon PTH administration, indicating that osteocytes control osteoclast formation through a PPR-mediated mechanism. Taken together, these data indicate that PPR signaling in osteocytes is required for bone remodeling, and receptor signaling in osteocytes is needed for anabolic and catabolic skeletal responses.

Protectin DX as a therapeutic strategy against frailty in mice.

Frailty in aging is driven by the dysregulation of multiple biological pathways. Protectin DX (PDX) is a docosahexaenoic acid (DHA)-derived molecule that alleviates many chronic inflammatory disorders, but its potential effects on frailty remain unknown. Our goal is to identify age-related impairments in metabolic systems and to evaluate the therapeutic potential of PDX on frailty, physical performance, and health parameters. A set of 22-month-old C57BL/6 male and female mice were assigned to vehicle (Old) or PDX daily gavage treatment for 9 weeks, whereas 6-month-old (Adult) mice received only vehicle. Forelimb and hindlimb strength, endurance, voluntary wheel activity and walking speed determined physical performance and were combined with a frailty index score and body weight loss to determine frailty status. Our data shows that old vehicle-treated mice from both sexes had body weight loss paralleling visceromegaly, and Old females also had impaired insulin clearance as compared to the Adult group. Aging was associated with physical performance decline together with higher odds of frailty development. There was also age-driven mesangial expansion and glomerular hypertrophy as well as bone mineral density loss. All of the in vivo and in vitro impairments observed with aging co-occurred with upregulation of inflammatory pathways and Myc signaling as well as downregulation of genes related to adipogenesis and oxidative phosphorylation in liver. PDX attenuated the age-driven physical performance (strength, exhaustion, walking speed) decline, promoted robustness, prevented bone losses and partially reversed changes in hepatic expression of Myc targets and metabolic genes. In conclusion, our data provides evidence of the beneficial therapeutic effect of PDX against features of frailty in mice. Further studies are warranted to investigate the mechanisms of action and the potential for human translation.

Impaired 1,25 dihydroxyvitamin D3 action and hypophosphatemia underlie the altered lacuno-canalicular remodeling observed in the Hyp mouse model of XLH.

Osteocytes remodel the perilacunar matrix and canaliculi. X-linked hypophosphatemia (XLH) is characterized by elevated serum levels of fibroblast growth factor 23 (FGF23), leading to decreased 1,25 dihydroxyvitamin D3 (1,25D) production and hypophosphatemia. Bones from mice with XLH (Hyp) have enlarged osteocyte lacunae, enhanced osteocyte expression of genes of bone remodeling, and impaired canalicular structure. The altered lacuno-canalicular (LCN) phenotype is improved with 1,25D or anti-FGF23 antibody treatment, pointing to roles for 1,25D and/or phosphate in regulating this process. To address whether impaired 1,25D action results in LCN alterations, the LCN phenotype was characterized in mice lacking the vitamin D receptor (VDR) in osteocytes (VDRf/f;DMP1Cre+). Mice lacking the sodium phosphate transporter NPT2a (NPT2aKO) have hypophosphatemia and high serum 1,25D levels, therefore the LCN phenotype was characterized in these mice to determine if increased 1,25D compensates for hypophosphatemia in regulating LCN remodeling. Unlike Hyp mice, neither VDRf/f;DMP1Cre+ nor NPT2aKO mice have dramatic alterations in cortical microarchitecture, allowing for dissecting 1,25D and phosphate specific effects on LCN remodeling in tibial cortices. Histomorphometric analyses demonstrate that, like Hyp mice, tibiae and calvariae in VDRf/f;DMP1Cre+ and NPT2aKO mice have enlarged osteocyte lacunae (tibiae: 0.15±0.02µm2(VDRf/f;DMP1Cre-) vs 0.19±0.02µm2(VDRf/f;DMP1Cre+), 0.12±0.02µm2(WT) vs 0.18±0.0µm2(NPT2aKO), calvariae: 0.09±0.02µm2(VDRf/f;DMP1Cre-) vs 0.11±0.02µm2(VDRf/f;DMP1Cre+), 0.08±0.02µm2(WT) vs 0.13±0.02µm2(NPT2aKO), p<0.05 all comparisons) and increased immunoreactivity of bone resorption marker Cathepsin K (Ctsk). The osteocyte enriched RNA isolated from tibiae in VDRf/f;DMP1Cre+ and NPT2aKO mice have enhanced expression of matrix resorption genes that are classically expressed by osteoclasts (Ctsk, Acp5, Atp6v0d2, Nhedc2). Treatment of Ocy454 osteocytes with 1,25D or phosphate inhibits the expression of these genes. Like Hyp mice, VDRf/f;DMP1Cre+ and NPT2aKO mice have impaired canalicular organization in tibia and calvaria. These studies demonstrate that hypophosphatemia and osteocyte-specific 1,25D actions regulate LCN remodeling. Impaired 1,25D action and low phosphate levels contribute to the abnormal LCN phenotype observed in XLH.

Emerging evidence that adaptive bone formation inhibition by non-steroidal anti-inflammatory drugs increases stress fracture risk.

There is mounting evidence suggesting that the commonly used analgesics, non-steroidal anti-inflammatory drugs (NSAIDs), may inhibit new bone formation with physical training and increase risk of stress fractures in physically active populations. Stress fractures are thought to occur when bones are subjected to repetitive mechanical loading, which can lead to a cycle of tissue microdamage, repair, and continued mechanical loading until fracture. Adaptive bone formation, particularly on the periosteal surface of long bones, is a concurrent adaptive response of bone to heightened mechanical loading that can improve the fatigue resistance of the skeletal structure, and therefore may play a critical role in offsetting the risk of stress fracture. Reports from animal studies suggest that NSAID administration may suppress this important adaptive response to mechanical loading. These observations have implications for populations such as endurance athletes and military recruits who are at risk of stress fracture and whose use of NSAIDs is widespread. However, results from human trials evaluating exercise and bone adaptation with NSAID consumption have been less conclusive. In this review, we identify knowledge gaps that must be addressed to further support NSAID-related guidelines intended for at-risk populations and individuals.

Parathyroid hormone signaling in mature osteoblasts/osteocytes protects mice from age-related bone loss.

Aging is accompanied by osteopenia, characterized by reduced bone formation and increased bone resorption. Osteocytes, the terminally differentiated osteoblasts, are regulators of bone homeostasis, and parathyroid hormone (PTH) receptor (PPR) signaling in mature osteoblasts/osteocytes is essential for PTH-driven anabolic and catabolic skeletal responses. However, the role of PPR signaling in those cells during aging has not been investigated. The aim of this study was to analyze the role of PTH signaling in mature osteoblasts/osteocytes during aging. Mice lacking PPR in osteocyte (Dmp1-PPRKO) display an age-dependent osteopenia characterized by a significant decrease in osteoblast activity and increase in osteoclast number and activity. At the molecular level, the absence of PPR signaling in mature osteoblasts/osteocytes is associated with an increase in serum sclerostin and a significant increase in osteocytes expressing 4-hydroxy-2-nonenals, a marker of oxidative stress. In Dmp1-PPRKO mice there was an age-dependent increase in p16Ink4a/Cdkn2a expression, whereas it was unchanged in controls. In vitro studies demonstrated that PTH protects osteocytes from oxidative stress-induced cell death. In summary, we reported that PPR signaling in osteocytes is important for protecting the skeleton from age-induced bone loss by restraining osteoclast's activity and protecting osteocytes from oxidative stresses.

A FAK/HDAC5 signaling axis controls osteocyte mechanotransduction.

Osteocytes, cells ensconced within mineralized bone matrix, are the primary skeletal mechanosensors. Osteocytes sense mechanical cues by changes in fluid flow shear stress (FFSS) across their dendritic projections. Loading-induced reductions of osteocytic Sclerostin (encoded by Sost) expression stimulates new bone formation. However, the molecular steps linking mechanotransduction and Sost suppression remain unknown. Here, we report that class IIa histone deacetylases (HDAC4 and HDAC5) are required for loading-induced Sost suppression and bone formation. FFSS signaling drives class IIa HDAC nuclear translocation through a signaling pathway involving direct HDAC5 tyrosine 642 phosphorylation by focal adhesion kinase (FAK), a HDAC5 post-translational modification that controls its subcellular localization. Osteocyte cell adhesion supports FAK tyrosine phosphorylation, and FFSS triggers FAK dephosphorylation. Pharmacologic FAK catalytic inhibition reduces Sost mRNA expression in vitro and in vivo. These studies demonstrate a role for HDAC5 as a transducer of matrix-derived cues to regulate cell type-specific gene expression.

Glycerol-3-phosphate is an FGF23 regulator derived from the injured kidney.

Fibroblast growth factor 23 (FGF23) is a bone-derived hormone that controls blood phosphate levels by increasing renal phosphate excretion and reducing 1,25-dihydroxyvitamin D3 [1,25(OH)2D] production. Disorders of FGF23 homeostasis are associated with significant morbidity and mortality, but a fundamental understanding of what regulates FGF23 production is lacking. Because the kidney is the major end organ of FGF23 action, we hypothesized that it releases a factor that regulates FGF23 synthesis. Using aptamer-based proteomics and liquid chromatography-mass spectrometry-based (LC-MS-based) metabolomics, we profiled more than 1600 molecules in renal venous plasma obtained from human subjects. Renal vein glycerol-3-phosphate (G-3-P) had the strongest correlation with circulating FGF23. In mice, exogenous G-3-P stimulated bone and bone marrow FGF23 production through local G-3-P acyltransferase-mediated (GPAT-mediated) lysophosphatidic acid (LPA) synthesis. Further, the stimulatory effect of G-3-P and LPA on FGF23 required LPA receptor 1 (LPAR1). Acute kidney injury (AKI), which increases FGF23 levels, rapidly increased circulating G-3-P in humans and mice, and the effect of AKI on FGF23 was abrogated by GPAT inhibition or Lpar1 deletion. Together, our findings establish a role for kidney-derived G-3-P in mineral metabolism and outline potential targets to modulate FGF23 production during kidney injury.

Impaired Gastric Hormone Regulation of Osteoblasts and Lysyl Oxidase Drives Bone Disease in Diabetes Mellitus.

Diabetic bone disease is a complication of type I and type II diabetes, both of which are increasing in the United States and elsewhere. Increased hip and foot fracture rates do not correlate well with changes in bone mineral density (BMD), whereas studies support the importance of collagen structure to bone strength. Extracellular lysyl oxidase (LOX) catalyzes the oxidative deamination of hydroxylysine and lysine residues in collagens resulting in aldehydes that subsequently form critically important biosynthetic crosslinks that stabilize functional collagens. Although LOX-dependent biosynthetic crosslinks in bone collagen are deficient in diabetic bone, the expression and regulation of bone LOXs in diabetes have not been comprehensively studied. Here, we found that LOX is profoundly downregulated in bone in diabetes. Moreover, we have identified a novel metabolic regulatory relationship that is dysregulated in diabetes using mouse models. Data indicate that the incretin (gastric hormone) known as glucose-dependent insulinotropic polypeptide (GIP) that is anabolic to osteoblasts strongly upregulates LOX, and that this regulation is disrupted in the streptozotocin-induced model of diabetes in mice. In vivo and in vitro studies support that diabetes results in elevated circulating peripheral dopamine, likely also derived from the gut, and is responsible for blocking GIP signaling and LOX levels in osteoblasts. Moreover, peripheral administration of the dopamine D2 receptor antagonist amisulpride to diabetic mice restored trabecular bone structure to near normal and partially reversed downregulation of LOX. Taken together our data identifies a novel metabolic relationship between the gut-derived hormone GIP and bone-derived LOX, and points to the importance of LOX dysregulation in the pathology of diabetic bone disease. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.

TG-interacting factor 1 (Tgif1)-deficiency attenuates bone remodeling and blunts the anabolic response to parathyroid hormone.

Osteoporosis is caused by increased bone resorption and decreased bone formation. Intermittent administration of a fragment of Parathyroid hormone (PTH) activates osteoblast-mediated bone formation and is used in patients with severe osteoporosis. However, the mechanisms by which PTH elicits its anabolic effect are not fully elucidated. Here we show that the absence of the homeodomain protein TG-interacting factor 1 (Tgif1) impairs osteoblast differentiation and activity, leading to a reduced bone formation. Deletion of Tgif1 in osteoblasts and osteocytes decreases bone resorption due to an increased secretion of Semaphorin 3E (Sema3E), an osteoclast-inhibiting factor. Tgif1 is a PTH target gene and PTH treatment failed to increase bone formation and bone mass in Tgif1-deficient mice. Thus, our study identifies Tgif1 as a novel regulator of bone remodeling and an essential component of the PTH anabolic action. These insights contribute to a better understanding of bone metabolism and the anabolic function of PTH.

Osteocyte secreted factors inhibit skeletal muscle differentiation.

It is generally accepted that bone and muscle possess the capacity to act in an autocrine, paracrine, or endocrine manner, with a growing body of evidence that suggests muscle can secrete muscle specific cytokines or "myokines", which influence bone metabolism. However, there has been little investigation into the identity of bone specific cytokines that modulate skeletal muscle differentiation and function. This study aimed to elucidate the influence of osteocytes on muscle progenitor cells in vitro and to identify potential bone specific cytokines or "osteokines". We treated C2C12 myoblasts with media collected from differentiated osteocytes (Ocy454 cells) grown in 3D, either under static or fluid flow culture conditions (2 dynes/cm2). C2C12 differentiation was significantly inhibited with a 75% reduction in the number of myofibers formed. mRNA analysis revealed a significant reduction in the expression of myogenic regulatory genes. Cytokine array analysis on the conditioned media demonstrated that osteocytes produce a significant number of cytokines "osteokines" capable of inhibiting myogenesis. Furthermore, we demonstrated that when osteocytes are mechanically activated they induce a greater inhibitory effect on myogenesis compared to a static state. Lastly, we identified the downregulation of numerous cytokines, including Il-6, Il-13, Il-1ß, MIP-1α, and Cxcl9, involved in myogenesis, which may lead to future investigation of the role "osteokines" play in musculoskeletal health and pathology.

PTH Signaling in Osteoprogenitors Is Essential for B-Lymphocyte Differentiation and Mobilization.

Cells of the osteoblast lineage provide critical support for B lymphopoiesis in the bone marrow (BM). Parathyroid hormone (PTH) signaling in osteoblastic cells through its receptor (PPR) is an important regulator of hematopoietic stem cells; however, its role in regulation of B lymphopoiesis is not clear. Here we demonstrate that deletion of PPR in osteoprogenitors results in a significant loss of trabecular and cortical bone. PPR signaling in osteoprogenitors, but not in mature osteoblasts or osteocytes, is critical for B-cell precursor differentiation via IL-7 production. Interestingly, despite a severe reduction in B-cell progenitors in BM, mature B-lymphocytes were increased 3.5-fold in the BM of mice lacking PPR in osteoprogenitors. This retention of mature IgD(+) B cells in the BM was associated with increased expression of vascular cell adhesion molecule 1 (VCAM1) by PPR-deficient osteoprogenitors, and treatment with VCAM1 neutralizing antibody increased mobilization of B lymphocytes from mutant BM. Our results demonstrate that PPR signaling in early osteoblasts is necessary for B-cell differentiation via IL-7 secretion and for B-lymphocyte mobilization via VCAM1.

HDAC5 controls MEF2C-driven sclerostin expression in osteocytes.

Osteocytes secrete paracrine factors that regulate the balance between bone formation and destruction. Among these molecules, sclerostin (encoded by the gene SOST) inhibits osteoblastic bone formation and is an osteoporosis drug target. The molecular mechanisms underlying SOST expression remain largely unexplored. Here, we report that histone deacetylase 5 (HDAC5) negatively regulates sclerostin levels in osteocytes in vitro and in vivo. HDAC5 shRNA increases, whereas HDAC5 overexpression decreases SOST expression in the novel murine Ocy454 osteocytic cell line. HDAC5 knockout mice show increased levels of SOST mRNA, more sclerostin-positive osteocytes, decreased Wnt activity, low trabecular bone density, and reduced bone formation by osteoblasts. In osteocytes, HDAC5 binds and inhibits the function of MEF2C, a crucial transcription factor for SOST expression. Using chromatin immunoprecipitation, we have mapped endogenous MEF2C binding in the SOST gene to a distal intergenic enhancer 45 kB downstream from the transcription start site. HDAC5 deficiency increases SOST enhancer MEF2C chromatin association and H3K27 acetylation and decreases recruitment of corepressors NCoR and HDAC3. HDAC5 associates with and regulates the transcriptional activity of this enhancer, suggesting direct regulation of SOST gene expression by HDAC5 in osteocytes. Finally, increased sclerostin production achieved by HDAC5 shRNA is abrogated by simultaneous knockdown of MEF2C, indicating that MEF2C is a major target of HDAC5 in osteocytes.

Loss of Gsα early in the osteoblast lineage favors adipogenic differentiation of mesenchymal progenitors and committed osteoblast precursors.

In humans, aging and glucocorticoid treatment are associated with reduced bone mass and increased marrow adiposity, suggesting that the differentiation of osteoblasts and adipocytes may be coordinately regulated. Within the bone marrow, both osteoblasts and adipocytes are derived from mesenchymal progenitor cells, but the mechanisms guiding the commitment of mesenchymal progenitors into osteoblast versus adipocyte lineages are not fully defined. The heterotrimeric G protein subunit Gs α activates protein kinase A signaling downstream of several G protein-coupled receptors including the parathyroid hormone receptor, and plays a crucial role in regulating bone mass. Here, we show that targeted ablation of Gs α in early osteoblast precursors, but not in differentiated osteocytes, results in a dramatic increase in bone marrow adipocytes. Mutant mice have reduced numbers of mesenchymal progenitors overall, with an increase in the proportion of progenitors committed to the adipocyte lineage. Furthermore, cells committed to the osteoblast lineage retain adipogenic potential both in vitro and in vivo. These findings have clinical implications for developing therapeutic approaches to direct the commitment of mesenchymal progenitors into the osteoblast lineage.

Effects of PTH on osteocyte function.

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.

Osteocyte biology and space flight.

The last decade has seen an impressive expansion of our understanding of the role of osteocytes in skeletal homeostasis. These amazing cells, deeply embedded into the mineralized matrix, are the key regulators of bone homeostasis and skeletal mechano sensation and transduction. They are the cells that can sense the mechanical forces applied to the bone and then translate these forces into biological responses. They are also ideally positioned to detect and respond to hormonal stimuli and to coordinate the function of osteoblasts and osteoclasts through the production and secretion of molecules such as Sclerostin and RANKL. How osteocytes perceive mechanical forces and translate them into biological responses in still an open question. Novel "in vitro" models as well the opportunity to study these cells under microgravity condition, will allow a closer look at the molecular and cellular mechanisms of mechano transduction. This article highlights novel investigations on osteocytes and discusses their significance in our understanding of skeletal mechano transduction.