Osteocyte Signaling Research Articles

Mechanically‐Induced Osteocyte/Th17 Cell Signaling and Subsequent Osteoclast Recruitment

Mechanical loading of bone triggers bone modeling/formation, via osteocyte signaling. However, when mechanical loads of extreme force or repetition create tissue damage, osteocytes stimulate the targeted remodeling of the damaged bone matrix. We have previously reported an up regulation of the pro-inflammatory cytokine GRO/KC, T cell recruiting cytokines (IL-2, MCP-1), and an increase in mRANKL and osteoclast number within fatigue loaded rat ulna compared to non-loaded controls. These findings suggests a relationship between bone fatigue and the subsequent recruitment of both T cells and osteoclasts. We examined a novel, mechanically-induced signaling pathway between osteocytes and Th17 cells, and hypothesized that repetitive fluid shear stress (FSS) would induce osteocyte production of cytokines, IL-6 and TGF-β1, known to stimulate Th17 cell development and their secretion of osteoclastogenic cytokines, IL-17 and sRANKL. MLO-Y4 osteocyte-like cells were exposed to FSS (7±3 dynes and 10±3 dynes, at 1Hz) or Static control. The cells and medium were collected after 30 or 60 minutes of FSS exposure or static conditions. ELISA analysis of MLO-Y4 medium revealed greater IL-6 and TGF-β1 in FSS versus Static groups. MLO-Y4 medium also revealed less sRANKL in FSS versus Static groups. Murine spleen cells were then cultured in T cell supportive conditions and treated with 25% FSS or Static MLO-Y4 medium for 72 hours. ELISA analysis of T cell culture medium revealed greater IL-17 in T cell cultures treated with FSS medium versus Static medium. MLO-Y4 cells were then co-cultured with murine spleen cells in osteoclast supportive conditions and treated with 25% T cell medium (FSS TX or Static TX) for 10 days. ELISA and TRAP stain analysis of osteoclast cultures revealed greater sRANKL and TRAP+ osteoclasts in cultures treated with T cell medium (FSS TX) versus T cell medium (Static TX). To our knowledge these data are the first to support a novel, mechanically-induced osteocyte/Th17 cell signaling pathway. Our data illustrates that mechanically-stimulated MLO-Y4 cells secrete greater amounts of cytokines (IL-6 and TGF-β1) known to induce Th17 cell development, that T cell cultures treated with MLO-Y4 FSS medium produced greater amounts of IL-17, and that the addition of T cell medium (FSS TX) to osteoclast cultures leads to greater osteoclast formation; thus suggesting that repetitive loading of osteocytes potentiates Th17 cell activity and the subsequent recruitment of osteoclasts. Future in vivo studies examining the osteoclast response in relation to our osteocyte/Th17 cell signaling model would provide a new paradigm for targeted bone remodeling in response to mechanical stress. Support or Funding Information NASA Nebraska Space Grant A. Proposed mechanically-induced osteocyte/Th17 cell signaling model. B. Increased TRAP+ osteoclasts in cultures treated with T cell medium (FSS TX: 7+/−3 & 10+/−3) versus T cell medium (Static TX).

Anabolic actions of Notch on mature bone

Notch controls skeletogenesis, but its role in the remodeling of adult bone remains conflicting. In mature mice, the skeleton can become osteopenic or osteosclerotic depending on the time point at which Notch is activated or inactivated. Using adult EGFP reporter mice, we find that Notch expression is localized to osteocytes embedded within bone matrix. Conditional activation of Notch signaling in osteocytes triggers profound bone formation, mainly due to increased mineralization, which rescues both age-associated and ovariectomy-induced bone loss and promotes bone healing following osteotomy. In parallel, mice rendered haploinsufficient in γ-secretase presenilin-1 (Psen1), which inhibits downstream Notch activation, display almost-absent terminal osteoblast differentiation. Consistent with this finding, pharmacologic or genetic disruption of Notch or its ligand Jagged1 inhibits mineralization. We suggest that stimulation of Notch signaling in osteocytes initiates a profound, therapeutically relevant, anabolic response.

Osteocyte-Mediated Parathyroid Hormone (PTH) Signaling Regulates Hematopoietic Stem Cells Under Physiologic and Continuous PTH Exposure

The bone marrow microenvironment, including osteolineage cells, regulates hematopoietic stem cell (HSC) fate choices. Intermittent pharmacologic treatment of mice with parathyroid hormone, PTH (1-34), indirectly increases HSCs through their niche, as HSCs do not express the PTH receptor (PTH1R). Osteocytes, the most abundant osteolineage cells in bone, are a critical target of the skeletal actions of PTH and coordinate multiple cell types that are components of the HSC niche including osteoblasts, osteoclasts and resident macrophages. While osteocytes express the PTH1R, the role of osteocytes in HSC regulation is unclear. Therefore, we studied the role of osteocyte-mediated PTH regulation of HSCs, using cre recombinase driven by the 8kb-DMP1 promoter to conditionally delete PTH1R in osteocytes (OCyPTHRko mice).OCyPTHRko mice were viable, fertile, did not exhibit any significant skeletal defect as juveniles or at 6 months of age, had no significant difference in serum PTH levels, and had no significant difference in osteoblastic or mesenchymal stem cell numbers compared to WT mice. In juvenile OCyPTH1Rko mice there was a decrease in long-term HSCs as measured by flow cytometric analysis (0.0029 ± 0.00028 vs. 0.0021 ± 0.00021 % of cells, WT vs. OCyPTH1Rko p≤0.05 N≥19 mice/group). OCyPTH1Rko mice had 4 fold lower long-term engraftment capacity as measured by secondary competitive transplantation over 16 weeks (WT vs. OCyPTH1Rko donors, 2-way ANOVA p≤0.001, N≥10 mice/group) that was evident in all hematopoietic lineages. Short-term engraftment however was increased in OCyPTH1Rko mice as measured by primary competitive transplantation (WT vs. OCyPTH1Rko donors, 2-way ANOVA p≤0.01, N≥9 mice/group). These data demonstrate that physiologic PTH signaling in osteocytes regulates the balance of long-term and short-term HSC potential in juvenile, growing mice. Adult OCyPTH1Rko mice also had 5 fold lower long-term engraftment as measured by secondary competitive transplantation over 16 weeks (WT vs. OCyPTH1Rko donors, 2-way ANOVA p≤0.001, N≥15 mice/group). These findings demonstrate a previously unrecognized physiologic role of PTH signaling in HSC regulation. Having demonstrated a role for PTH signaling in HSC homeostasis, we investigated if sustained PTH elevations (as are found in vitamin D deficiency and in hyperparathyroidism) alter HSC function. Therefore, we utilized a murine model of secondary hyperparathyroidism caused by a low calcium (LCa) diet. In juvenile mice placed on the LCa diet immediately upon weaning, serum PTH levels were significantly elevated. Fourteen days on the LCa diet caused a significant reduction in long-term engraftment potential as measured by secondary competitive transplants over 22 weeks (Normal vs. LCa diet donors, 2-way ANOVA p≤0.001, N≥20 mice/group), while there was no decrease in HSCs when adult mice were placed on the LCa diet. These data suggest that sustained PTH signaling decreases microenvironmental support for HSCs in juvenile mice. We utilized the OCyPTHRko mice to study the role of osteocytes in hyperparathyroidism-induced loss of functional HSCs. In juvenile mice the lack of PTH signaling in osteocytes rescued the long-term engraftment defects, suggesting that PTH signaling in osteocytes mediates the loss of long-term HSC support caused by the LCa diet. In further support of a deleterious effect mediated by the PTH1R in osteocytes in the setting of continuous PTH, adult OCyPTH1Rko mice placed on LCa diet had superior long term HSC function.Our findings demonstrate a physiologic role for PTH in HSC regulation and identify osteocytes as a critical constituent of the HSC niche that, either directly or indirectly, contribute to maintenance of the long-term repopulating HSC pool. In addition, we show that continuous exposure to elevated levels of PTH in a model of secondary hyperparathyroidism leads to osteocyte-mediated loss of long-term engraftment potential of HSCs in juvenile mice. We speculate that removing the effect of continuous PTH from osteocytes uncovers additional HSC-supportive effects of continuous PTH, mediated by non-osteocyte HSC niche cellular populations. Together these data establish PTH as a critical regulatory signal in the HSC niche, and show that the relative contributions of niche populations to HSC regulation are modulated by age. DisclosuresCalvi:Fate Therapeutics: Patents & Royalties.

Mapping the osteocytic cell response to fluid flow using RNA-Seq

Bone adaptation to mechanical loading is regulated via signal transduction by mechano-sensing osteocytes. Mineral-embedded osteocytes experience strain-induced interstitial fluid flow and fluid shear stress, and broad shifts in gene expression are key components in the signaling pathways that regulate bone turnover. RNA sequencing analysis, or RNA-Seq, enables more complete characterization of mechano-responsive transcriptome regulation than previously possible. We hypothesized that RNA-Seq of osteocytic MLO-Y4 cells reveals both expected and novel gene transcript regulation in cells previously fluid flowed and analyzed using gene microarrays. MLO-Y4 cells were flowed for 2h with 1Pa oscillating fluid shear stress and post-incubated 2h. RNA-Seq of original samples detected 55 fluid flow-regulated gene transcripts (p-corrected <0.05), the same number previously detected by microarray. However, RNA-Seq demonstrated greater dynamic range, with all 55 transcripts increased >1.5-fold or decreased <0.67-fold whereas 10 of 55 met this cut-off by microarray. Analyses were complimentary in patterns of regulation, though only 6 transcripts were significant in both RNA-Seq and microarray analyses: Cxcl5, Cxcl1, Zc3h12a, Ereg, Slc2a1, and Egln1. As part of a broad inflammatory response inferred by gene ontology analyses, we again observed greatest up-regulation of inflammatory C-X-C motif chemokines, and newly implicated HIF-1α and AMPK signaling pathways. Importantly, we detected both expected fluid flow-sensitive transcripts (e.g. Nos2 [iNOS], Ptgs2 [COX-2], Ccl7) and transcripts not previously identified as flow-sensitive, e.g. Ccl2. We found RNA-Seq advantageous over microarrays because of its greater dynamic range and ability to analyze unbiased estimation of gene expression, informing our understanding of osteocyte signaling.

Bone's responses to mechanical loading are impaired in type 1 diabetes

Diabetes adversely impacts many organ systems including the skeleton. Clinical trials have revealed a startling elevation in fracture risk in diabetic patients. Bone fractures can be life threatening: nearly 1 in 6 hip fracture patients die within one year. Because physical exercise is proven to improve bone properties and reduce fracture risk in non-diabetic subjects, we tested its efficacy in type 1 diabetes. We hypothesized that diabetic bone's response to anabolic mechanical loading would be attenuated, partially due to impaired mechanosensing of osteocytes under hyperglycemia. Heterozygous C57BL/6-Ins2Akita/J (Akita) male and female diabetic mice and their age- and gender-matched wild-type (WT) C57BL/6J controls (7-month-old, N=5–7 mice/group) were subjected to unilateral axial ulnar loading with a peak strain of 3500με at 2Hz and 3min/day for 5days. The Akita female mice, which exhibited a relatively normal body weight and a mild 40% elevation of blood glucose level, responded with increased bone formation (+6.5% in Ct.B.Ar, and 4 to 36-fold increase in Ec.BFR/BS and Ps.BFR/BS), and the loading effects, in terms of changes of static and dynamic indices, did not differ between Akita and WT females (p≥0.1). However, loading-induced anabolic effects were greatly diminished in Akita males, which exhibited reduced body weight, severe hyperglycemia (+230%), diminished bone formation (ΔCt.B.Ar: 0.003 vs. 0.030mm2, p=0.005), and suppressed periosteal bone appositions (ΔPs.BFR/BS, p=0.02). Hyperglycemia (25mM glucose) was further found to impair the flow-induced intracellular calcium signaling in MLO-Y4 osteocytes, and significantly inhibited the flow-induced downstream responses including reduction in apoptosis and sRANKL secretion and PGE2 release. These results, along with previous findings showing adverse effects of hyperglycemia on osteoblasts and mesenchymal stem cells, suggest that failure to maintain normal glucose levels may impair bone's responses to mechanical loading in diabetics.

The dependences of osteocyte network on bone compartment, age, and disease.

Osteocytes, the most abundant bone cells, form an interconnected network in the lacunar-canalicular pore system (LCS) buried within the mineralized matrix, which allows osteocytes to obtain nutrients from the blood supply, sense external mechanical signals, and communicate among themselves and with other cells on bone surfaces. In this study, we examined key features of the LCS network including the topological parameter and the detailed structure of individual connections and their variations in cortical and cancellous compartments, at different ages, and in two disease conditions with altered mechanosensing (perlecan deficiency and diabetes). LCS network showed both topological stability, in terms of conservation of connectivity among osteocyte lacunae (similar to the “nodes” in a computer network), and considerable variability the pericellular annular fluid gap surrounding lacunae and canaliculi (similar to the “bandwidth” of individual links in a computer network). Age, in the range of our study (15–32 weeks), affected only the pericellular fluid annulus in cortical bone but not in cancellous bone. Diabetes impacted the spacing of the lacunae, while the perlecan deficiency had a profound influence on the pericellular fluid annulus. The LCS network features play important roles in osteocyte signaling and regulation of bone growth and adaptation.

In vivo mechanical loading rapidly activates β-catenin signaling in osteocytes through a prostaglandin mediated mechanism

The response of the skeleton to loading appears to be mediated through the activation of the Wnt/β-catenin signaling pathway and osteocytes have long been postulated to be the primary mechanosensory cells in bone. To examine the kinetics of the mechanoresponse of bone and cell types involved in vivo, we performed forearm loading of 17-week-old female TOPGAL mice. β-catenin signaling was observed only in embedded osteocytes, not osteoblasts, at 1h post-loading, spreading to additional osteocytes and finally to cells on the bone surface by 24h. This early activation at 1h appeared to be independent of receptor (Lrp5/6) mediated activation as it occurred in the presence of the inhibitors sclerostin and/or Dkk1. The COX-2 inhibitor, Carprofen, blocked the activation of β-catenin signaling and decline in sclerostin positive osteocytes post-loading implying an important role for prostaglandin. In vitro, PI3K/Akt activation was shown to be required for β-catenin nuclear translocation downstream from prostaglandin in MLO-Y4 osteocyte-like cells supporting this mechanism. Downstream targets of β-catenin signaling, sclerostin and Dkk1, were also examined and found to be significantly downregulated in osteocytes in vivo at 24h post-loading. The pattern of initially activated osteocytes appeared random and in order to understand this heterogeneous expression, a novel finite element model of the strain field in the ulna was developed, which predicts highly variable local magnitudes of strain experienced by osteocytes. In summary, both in vivo and in vitro models show the rapid activation of β-catenin in response to load through the early release of prostaglandin and that strain fields in the bone are extremely heterogeneous resulting in heterogeneous activation of the β-catenin pathway in osteocytes in vivo.

Notch- and TNFα-Activated Signaling Pathways Mediate Osteocyte Apoptosis Triggered By Multiple Myeloma Cells

Osteocytes comprise 95% of all bone cells and are central regulators of bone homeostasis and skeletal integrity. However the role of osteocytes in MM bone disease is unknown. We have previously shown that interactions with multiple myeloma cells have a profound effect on osteocytic gene expression, increasing Sost and RANKL transcripts and decreasing OPG. More recently, we and others have shown that myeloma cells increase the prevalence of osteocyte apoptosis, which might contribute to MM induced bone disease by increasing resorption in specific areas of bone. However, the mechanisms responsible for and the potential for targeting osteocyte apoptosis in myeloma are unknown. Osteocytes extensively communicate with each other and with cells on the bone surface and in the marrow, through cytoplasmic connections that run within canaliculi, which allows direct cell-to-cell contact and the distribution of secreted molecules among all bone and marrow cells including myeloma cells. Therefore, to explore the mechanism underlying osteocyte apoptosis induced by myeloma cells we used a co-culture system that allows both cell-to-cell contact and exchange of soluble factors between osteocytic MLO-A5 cells and the human JJN3 MM cell line or primary CD138+ cells isolated from MM patients. Osteocyte apoptosis was quantified by trypan blue uptake and chromatin condensation/nuclear fragmentation in the absence or presence of the caspase3 specific inhibitor DEVD. We found that osteocyte apoptosis was increased 2-3 fold when osteocytes were co-cultured with JJN3 cells compared to osteocytes cultured alone. Apoptosis occurred within 8h of co-culture and gradually increased for up to 48h. Osteocyte apoptosis was completely inhibited by DEVD. Importantly, co-culture with primary CD138+ MM cells from 5 different patients also increased osteocyte apoptosis, which was also blocked by DEVD. We next determined if direct cell-to-cell contact was required for MM cells to induce osteocyte apoptosis and whether Notch signaling, a signaling pathway with profound effects on the skeleton activated by such interactions, was involved. We found that osteocytes co-cultured with JJN3 cells exhibited 3-7 fold higher levels of expression of the Notch target genes Hes1 and Hey1, detected as early as 4h and maintained up to 48h of co-culture. The pharmacological specific Notch inhibitor GSIXX completely blocked osteocyte apoptosis induced by either JJN3 cells or primary CD138+ MM cells measured at 8h and 24h. Moreover, cultures of osteocytes grown on plates coated with the Notch ligand Delta 1 fused to IgG2 exhibited 2-5 times higher levels of apoptosis compared to osteocytes cultured on IgG2 control, and this effect was inhibited by GSIXX. In addition, overexpression of the Notch intracellular domains 1 or 2, known to activate Notch signaling, increased osteocyte apoptosis by 2 fold. These findings demonstrate that rapid activation Notch signaling in osteocytes triggered by direct cell-to-cell contact with myeloma cells induces osteocyte apoptosis. Interestingly, Notch inhibition by GSIXX only partially prevented osteocyte apoptosis induced by JJN3 cells measured at 48h, suggesting the involvement of other mechanisms. To further investigate this finding, we measured soluble levels of TNFα in JJN3 cultures since TNFα is a recognized inducer of osteocyte apoptosis. The levels of TNFα secreted by JJN3 cells increased 5 fold (from 2 to 9 pg/ml) during 4 to 48h of culture. Conditioned medium (CM) from JJN3 cells cultured alone for 48h increased osteocyte apoptosis, and this effect was blocked by DEVD and by a neutralizing anti-human TNFα antibody, but not by GSIXX. Moreover, combination of GSIXX and anti-TNFα antibody completely inhibited osteocyte apoptosis induced by co-culture with JJN3 cells, while each agent added separately only partially inhibited osteocyte apoptosis measured at 48h. These results demonstrate that direct interactions with MM cells induces caspase3-dependent osteocyte apoptosis, triggered by rapid activation of Notch signaling through cell-cell contact and is maintained by accumulation of MM-derived TNFα. Our findings suggest both Notch and TNFα signaling pathways are potential targets to reverse or prevent myeloma induced osteocyte apoptosis. DisclosuresNo relevant conflicts of interest to declare.

The differential effect of socio-economic status on body mass index among Aboriginal children & adolescents

11 -hydroxysteroid dehydrogenase type 2. Sevenweek-old male tg mice and their wild type (WT) littermates (n = 6—9/group) were fed ad libitum a control diet (14.3% energy from fat, 25.5% from protein) or an isoenergetic high-fat diet (HFD; 43.0% energy from fat, 25.5% from protein) for 18 weeks. Body weight and food intake were measured weekly. Serum corticosterone levels were quantified after 10 weeks of feeding, and body composition and bone parameters were assessed at endpoint. As animals were fed an isoenergetic diet, changes in body weight and body composition did not differ between the four groups. Both WT and tg mice fed the HFD had higher serum corticosterone levels than WT or tg control mice (pooled measures: 386± 35 nM for HFD vs. 285± 36 nM for control diet; p = 0.054). Corticosterone levels in WT and tg mice fed the same diets (either HFD or control) were similar. At endpoint, WT mice fed a HFD had significantly lower BMD than both WT mice receiving the control diet (0.058± 0.001 g/cm2 vs. 0.064± 0.0007 g/cm2; p < 0.005) and tg animals fed the HFD (0.058± 0.001 g/cm2 vs. 0.063± 0.001 g/cm2; p < 0.005). Similarly, WT mice on HFD exhibited lower BMC than WT control mice (0.64± 0.02 g vs. 0.74± 0.02 g; p < 0.005) and tg mice receiving the HFD (0.64± 0.02 g vs. 0.72± 0.02 g; p < 0.005). Neither BMD nor BMC differed significantly between tg control and tg HFD mice. We conclude that high dietary fat intake negatively affects bone mass via glucocorticoid signalling in osteoblasts and osteocytes.

Osteocyte control of bone remodeling: is sclerostin a key molecular coordinator of the balanced bone resorption-formation cycles?

Osteocytes, entrapped within a newly mineralized bone matrix, possess a unique cellular identity due to a specialized morphology and a molecular signature. These features endow them to serve as a bone response mechanism for mechanical stress in their microenvironment. Sclerostin, a primarily osteocyte product, is widely considered as a mechanotranduction key molecule whose expression is suppressed by mechanical loading, or it is induced by unloading. This review presents a model suggesting that sclerostin is major mediator for integrating mechanical, local, and hormonal signals, sensed by the osteocytes, in controlling the remodeling apparatus. This central role is achieved through interplay between two opposing mechanisms: (1) unloading-induced high sclerostin levels, which antagonize Wnt-canonical-β-catenin signaling in osteocytes and osteoblasts, permitting simultaneously Wnt-noncanonical and/or other pathways in osteocytes and osteoclasts, directed at bone resorption; (2) mechanical loading results in low sclerostin levels, activation of Wnt-canonical signaling, and bone formation. Therefore, adaptive bone remodeling occurring at a distinct bone compartment is orchestrated by altered sclerostin levels, which regulate the expression of the other osteocyte-specific proteins, such as RANKL, OPG, and proteins encoded by "mineralization-related genes" (DMP1, PHEX, and probably FGF23). For example, under specific terms, sclerostin regulates differential RANKL and OPG production, and creates a dynamic RANKL/OPG ratio, leading either to bone formation or resorption. It also controls the expression of PHEX, DMP1, and most likely FGF23, leading to either bone matrix mineralization or its inhibition. Such opposing up- or down-regulation of remodeling phases allows osteocytes to function as an "external unit", ensuring transition from bone resorption to bone formation.Mini Abstract: The osteocyte network plays a central role in directing bone response either to mechanical loading, or to unloading, leading correspondingly to bone formation or resorption. This review shows a key role of the osteocyte-produced sclerostin as a major mediator of the molecular mechanisms involved in the process of adaptive bone remodeling.

Neurofibromin inactivation impairs osteocyte development in Nf1Prx1 and Nf1Col1 mouse models

Neurofibromin has been identified as a critical regulator of osteoblast differentiation. Osteoblast specific inactivation of neurofibromin in mice results in a high bone mass phenotype and hyperosteoidosis. Here, we show that inactivation of the Nf1 gene also impairs osteocyte development. We analyzed cortical bone tissue in two conditional mouse models, Nf1Prx1 and Nf1Col1, for morphological and molecular effects. Backscattered electron microscopy revealed significantly enlarged osteocyte lacunae in Nf1Prx1 and Nf1Col1 mice (level E2: ctrl=1.90±0.52%, Nf1Prx1=3.40±0.95%; ctrl 1.60±0.47%, Nf1Col1 2.46±0.91%). Moreover, the osteocyte lacunae appeared misshaped in Nf1Prx1 and Nf1Col1 mice as indicated by increased Feret ratios. Strongest osteocyte and dendritic network disorganization was observed in proximity of muscle attachment sites in Nf1Prx1 humeri. In contrast to control cells, Nf1Prx1 osteocytes contained abundant cytosolic vacuoles and accumulated immature organic matrix within the perilacunar space, a phenotype reminiscent of the hyperosteoidosis shown Nf1 deficient mice. Cortical bone lysates further revealed approx. twofold upregulated MAPK signalling in osteocytes of Nf1Prx1 mice. This was associated with transcriptional downregulation of collagens and genes involved in mechanical sensing in Nf1Prx1 and Nf1Col1 bone tissue. In contrast, matrix gla protein (MGP), phosphate regulating endopeptidase homolog, X-linked (PHEX), and genes involved in lipid metabolism were upregulated. In line with previously described hyperactivation of Nf1 deficient osteoblasts, systemic plasma levels of the bone formation markers osteocalcin (OCN) and procollagen typ I N-propeptide (PINP) were approx. twofold increased in Nf1Prx1 mice. Histochemical and molecular analysis ascertained that osteocytes in Nf1Prx1 cortical bone were viable and did not undergo apoptosis or autophagy. We conclude that loss of neurofibromin is not only critical for osteoblasts but also hinders normal osteocyte development. These findings expand the effect of neurofibromin onto yet another cell type where it is likely involved in the regulation of mechanical sensing, bone matrix composition and mechanical resistance of bone tissue.

The osteoblast to osteocyte transition: epigenetic changes and response to the vitamin D3 hormone.

Osteocytes are derived from osteoblast lineage cells that become progressively embedded in mineralized bone. Development of the osteocytogenic cell line IDG-SW3 has enabled a temporal and mechanistic investigation of this process. Through RNA-sequencing analyses, we show that although substantial changes in gene expression occur during the osteoblast to osteocyte transition, the majority of the transcriptome remains qualitatively osteoblast like. Genes either up-regulated or expressed uniquely in the osteocyte include local and systemic factors such as Sost and Fgf23 as well as genes implicated in neuronal, muscle, vascular, or regulatory function. As assessed by chromatin immunoprecipitation coupled to high-throughput sequencing, numerous changes in epigenetic histone modifications also occur during osteocytogenesis; these are largely qualitative rather than quantitative. Specific epigenetic changes correlate with altered gene expression patterns that are observed during the transition. These genomic changes likely influence the highly restricted transcriptomic response to 1,25(OH)(2)D(3) that occurs during differentiation. VDR binding in osteocytes revealed an extensive cistrome co-occupied by retinoid X receptor and located predominantly at sites distal to regulated genes. Although sites of VDR binding were apparent near many 1,25(OH)(2)D(3)-regulated genes, the expression of others adjacent to VDR-binding sites were unaffected; lack of VDR binding was particularly prevalent at down-regulated genes. Interestingly, 1,25(OH)(2)D(3) was found to induce the Boc and Cdon coreceptors that are active in hedgehog signaling in osteocytes. We conclude that osteocytogenesis is accompanied by changes in gene expression that may be driven by both genetic and epigenetic components. These changes are likely responsible for the osteocyte phenotype and may contribute to reduced sensitivity to 1,25(OH)(2)D(3).

Enhanced prostacyclin formation and Wnt signaling in sclerostin deficient osteocytes and bone

We show that prostacyclin production is increased in bone and osteocytes from sclerostin (Sost) knockout mice which have greatly increased bone mass. The addition of prostacyclin or a prostacyclin analog to bone forming osteoblasts enhances differentiation and matrix mineralization of osteoblasts. The increase in prostacyclin synthesis is linked to increases in β-catenin concentrations and activity as shown by enhanced binding of lymphoid enhancer factor, Lef1, to promoter elements within the prostacyclin synthase promoter. Blockade of Wnt signaling reduces prostacyclin production in osteocytes. Increased prostacyclin production by osteocytes from sclerostin deficient mice could potentially contribute to the increased bone formation seen in this condition.

Vitamin D hormone regulates the expression of hedgehog coreceptor genes Boc and Cdon in osteocytes (946.4)

Osteocytes are terminally differentiated cells descended from mesenchymal stem cells that are embedded in bone. Osteocytes are known to play an essential role in maintaining bone health, acting as mechanical sensors in bone: translating mechanical load into biochemical signals. Osteocytes have diverse regulatory functions that extend beyond bone, such as lipid regulation and immune system development. These cells are regulated by a number of hormones and cofactors, including the active form of vitamin D, 1,25 dihydroxyvitamin D3 (1,25D3). 1,25D3 binds and activates the vitamin D receptor (VDR) and this complex serves as a transcriptional regulator of various genes. Previously, chromatin immuno‐precipitation coupled to high throughput sequencing (ChIP‐seq) revealed that VDR binds to regions upstream and downstream of the genes coding for Cell adhesion molecule‐related/down‐regulate by oncogenes protein (Cdon) and Brother of Cdon (Boc). These two genes encode cell surface receptor proteins in the Hedgehog signaling pathway, that play an important role in animal tissue development. To better understand 1,25D3’s role in regulating Boc and Cdon, MC3T3‐E1 cells were transfected with luciferase reporter plasmids containing regions of VDRE occupancy identified by ChIP‐seq. These results revealed that these regions of Boc and Cdon mediate up‐regulation by 1,25D3. Specific vitamin D response elements (VDREs) within the enhancer regions were then identified by in silico analyses and selectively mutated. Analysis of these VDRE mutated luciferase reporters revealed an elimination of 1,25D3 activity. In summary, we have shown 1,25D3’s novel role in the up‐regulation of Boc and Cdon and identified the specific VDREs that are responsible for the 1,25D3 response of these genes in osteocytes. Further studies are underway to define the impact of 1,25D3 on Hedgehog signaling in osteocytes. These results contribute to an understanding of 1,25D3’s role in osteocyte and bone health and development.Grant Funding Source: Supported by NIH DK072281

Vitamin D endocrine system and osteocytes

The physiological role of the osteocyte, the most numerous of the three bone cell types, was significantly underestimated until recently. It is now known that they not only coordinate bone remodeling but also have an endocrine function as part of the regulatory network for calcium and phosphate homeostasis. Vitamin D and osteocytes interact in numerous ways to accomplish these activities. The major source of active vitamin D (1,25(OH)2D3) is the kidney but there is evidence that osteocytes can produce it as well. Renal 1,25(OH)2D3 regulates osteocyte production of fibroblast growth factor 23 (FGF23), a powerful phosphaturic factor with far-reaching physiological effects. The function of 1,25(OH)2D3 produced by osteocytes themselves is poorly understood and is an area of active research. Osteocytes affect local bone remodeling by producing regulatory factors for osteoblasts and osteoclasts in response to mechanical loading and to endocrine signals such as serum 1,25(OH)2D3. In addition, 1,25(OH)2D3 may inhibit mineralization in osteocyte lacunae. Whether 1,25(OH)2D3 has a role in osteocytic perilacunar remodeling is currently unknown. This short review presents the current state of our knowledge about the physiologically and clinically significant roles of vitamin D signaling in osteocytes.

IL-6 Alters Osteocyte Signaling toward Osteoblasts but Not Osteoclasts

Mechanosensitive osteocytes regulate bone mass in adults. Interleukin 6 (IL-6), such as present during orthodontic tooth movement, also strongly affects bone mass, but little is known about the effect of IL-6 on osteocyte function. Therefore we aimed to determine in vitro whether IL-6 affects osteocyte mechanosensitivity, and osteocyte regulation of osteoclastogenesis and osteoblast differentiation. MLO-Y4 osteocytes were incubated with/without IL-6 (1 or 10 pg/mL) for 24 hr. Subsequently, osteocytes were subjected to mechanical loading by pulsating fluid flow (PFF) for 1 hr. Mouse osteoclast precursors were cultured for 7 days on top of IL-6-treated osteocytes. Conditioned medium from osteocytes treated with/without IL-6 was added to MC3T3-E1 pre-osteoblasts for 14 days. Exogenous IL-6 (10 pg/mL) did not alter the osteocyte response to PFF. PFF significantly enhanced IL-6 production by osteocytes. IL-6 enhanced Rankl expression but reduced caspase 3/7 activity by osteocytes, and therefore did not affect osteocyte-stimulated osteoclastogenesis. Conditioned medium from IL-6-treated osteocytes reduced alkaline phosphatase (ALP) activity and Runx2 expression in osteoblasts, but increased expression of the proliferation marker Ki67 and osteocalcin. Our results suggest that IL-6 is produced by shear-loaded osteocytes and that IL-6 may affect bone mass by modulating osteocyte communication toward osteoblasts.

The Osteocyte as an Orchestrator of Bone Remodeling: An Engineer’s Perspective

Bone is adapted to mechanical loading. The trabeculae in cancellous bone and the osteons in cortical bone are aligned to the mechanical loading direction. Our bones are constantly remodeled by bone-resorbing osteoclasts and bone-forming osteoblasts, cooperating in so-called basic multicellular units or BMUs. In cortical bone, osteoclasts dig tunnels through solid bone, while in cancellous bone, they dig trenches across the trabecular surface. Osteoblasts fill these tunnels and trenches, creating osteons and hemi-osteons, respectively. How mechanical forces guide these cells is still uncertain, but mechanosensitive osteocytes are believed to orchestrate bone remodeling by sending signals to the cells at the bone surface. Computer simulations have demonstrated that local remodeling regulated by mechanosensitive osteocytes indeed produces load-aligned trabeculae and osteons. The strains around a BMU resorption cavity are concentrated at the lateral sides, away from the loading axis. Strain-induced osteocyte signals from these regions likely repel osteoclasts, forcing them to resorb bone in the loading direction, and at the same time, such signals could recruit osteoblasts to start bone formation. Thus, mechanosensitive osteocytes likely regulate the steering of and coupling within BMUs. A region of osteocyte death (therefore, lacking osteoclast-repelling signals) near the path of the BMU redirects its course to resorb this region. This may provide a mechanism for damage removal, because osteocyte death is associated with microdamage. BMUs may also function with disuse-induced osteocyte signals that recruit osteoclasts to the relatively unloaded region in front of the BMU and inhibit osteoblastic bone formation by osteoblast-inhibiting signals such as sclerostin when the tunnel or trench is sufficiently filled.

Resorption Controls Bone Anabolism Driven by Parathyroid Hormone (PTH) Receptor Signaling in Osteocytes

The contribution of remodeling-based bone formation coupled to osteoclast activity versus modeling-based bone formation that occurs independently of resorption, to the anabolic effect of PTH remains unclear. We addressed this question using transgenic mice with activated PTH receptor signaling in osteocytes that exhibit increased bone mass and remodeling, recognized skeletal effects of PTH elevation. Direct inhibition of bone formation was accomplished genetically by overexpressing the Wnt antagonist Sost/sclerostin; and resorption-dependent bone formation was inhibited pharmacologically with the bisphosphonate alendronate. We found that bone formation induced by osteocytic PTH receptor signaling on the periosteal surface depends on Wnt signaling but not on resorption. In contrast, bone formation on the endocortical surface results from a combination of Wnt-driven increased osteoblast number and resorption-dependent osteoblast activity. Moreover, elevated osteoclasts and intracortical/calvarial porosity is exacerbated by overexpressing Sost and reversed by blocking resorption. Furthermore, increased cancellous bone is abolished by Wnt inhibition but further increased by blocking resorption. Thus, resorption induced by PTH receptor signaling in osteocytes is critical for full anabolism in cortical bone, but tempers bone gain in cancellous bone. Dissecting underlying mechanisms of PTH receptor signaling would allow targeting actions in different bone compartments, enhancing the therapeutic potential of the pathway.

Notch Signaling in Osteocytes Differentially Regulates Cancellous and Cortical Bone Remodeling

Notch receptors play a role in skeletal development and homeostasis, and Notch activation in undifferentiated and mature osteoblasts causes osteopenia. In contrast, Notch activation in osteocytes increases bone mass, but the mechanisms involved and exact functions of Notch are not known. In this study, Notch1 and -2 were inactivated preferentially in osteocytes by mating Notch1/2 conditional mice, where Notch alleles are flanked by loxP sequences, with transgenics expressing Cre directed by the Dmp1 (dentin matrix protein 1) promoter. Notch1/2 conditional null male and female mice exhibited an increase in trabecular bone volume due to an increase in osteoblasts and decrease in osteoclasts. In male null mice, this was followed by an increase in osteoclast number and normalization of bone volume. To activate Notch preferentially in osteocytes, Dmp1-Cre transgenics were crossed with Rosa(Notch) mice, where a loxP-flanked STOP cassette is placed between the Rosa26 promoter and Notch1 intracellular domain sequences. Dmp1-Cre(+/-);Rosa(Notch) mice exhibited an increase in trabecular bone volume due to decreased bone resorption and an increase in cortical bone due to increased bone formation. Biomechanical and chemical properties were not affected. Osteoprotegerin mRNA was increased, sclerostin and dickkopf1 mRNA were decreased, and Wnt signaling was enhanced in Dmp1-Cre(+/-);Rosa(Notch) femurs. Botulinum toxin A-induced muscle paralysis caused pronounced osteopenia in control mice, but bone mass was preserved in mice harboring the Notch activation in osteocytes. In conclusion, Notch plays a unique role in osteocytes, up-regulates osteoprotegerin and Wnt signaling, and differentially regulates trabecular and cortical bone homeostasis.

JAK-STAT and bone metabolism

Emerging evidences suggest Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway plays an important role in bone development and metabolism. Effects of JAK-STAT pathway on skeletal development are summarized based on skeletal phenotype of individual JAK and STAT gene knockout mouse. Furthermore, STAT3 has more profound effects on bone homeostasis compared with the other STATs. STAT3 mutation causes a disease called Job syndrome, most patients with which have associated craniofacial and skeletal features. Selective inactivation of STAT3 in osteoblasts decreases bone formation and skeletal responsiveness to mechanical loading. Future research includes investigating JAK-STAT signaling in osteoclasts and osteocytes.