SOST mRNA Research Articles

Elevated glucose acts directly on osteocytes to increase sclerostin expression in diabetes

Bone quality in diabetic patients is compromised, leading to weaker bones and increased fracture risk. However, the mechanism by which this occurs in diabetic bone remains to be fully elucidated. We hypothesized that elevated glucose and glucose variation would affect the function of osteocytes, essential regulators of bone homeostasis and quality. To first test this hypothesis, we used the IDG-SW3 osteocyte-like cell line to examine the effects of glucose levels on osteocyte function and viability in vitro. We confirmed our in vitro findings using the in vivo streptozotocin-induced (STZ) diabetic rat model and ex-vivo cultured osteocytes from these rats. IDG-SW3 cells cultured under high glucose conditions displayed significantly increased Sost mRNA(100-fold) and sclerostin protein, a negative regulator of bone formation(5000-fold), compared to cells in control media. mRNA expression of osteoblast markers such as Osx, Ocn and Col1a1 was unaffected by glucose. Factors associated with osteoclast activation were affected by glucose, with Rankl being upregulated by low glucose. Opg was also transiently upregulated by high glucose in mature IDG-SW3 cells. Induction of diabetes in Sprague-Dawley rats via a single dose of STZ (70 mg/kg) resulted in elevated maximum glucose and increased variability compared to control animals (670/796 vs. 102/142 mg/dL). This was accompanied by increased Sost/sclerostin expression in the osteocytes of these animals. These results show that glucose levels directly regulate osteocyte function through sclerostin expression and suggest a potential mechanism for the negative impact of diabetes on bone quality.

Exercise Degrades Bone in Caloric Restriction, Despite Suppression of Marrow Adipose Tissue (MAT).

ABSTRACTMarrow adipose tissue (MAT) and its relevance to skeletal health during caloric restriction (CR) is unknown: It remains unclear whether exercise, which is anabolic to bone in a calorie‐replete state, alters bone or MAT in CR. We hypothesized that response of bone and MAT to exercise in CR differs from the calorie‐replete state. Ten‐week‐old female B6 mice fed a regular diet (RD) or 30% CR diet were allocated to sedentary (RD, CR, n = 10/group) or running exercise (RD‐E, CR‐E, n = 7/group). After 6 weeks, CR mice weighed 20% less than RD, p < 0.001; exercise did not affect weight. Femoral bone volume (BV) via 3D MRI was 20% lower in CR versus RD (p < 0.0001). CR was associated with decreased bone by μCT: Tb.Th was 16% less in CR versus RD, p < 0.003, Ct.Th was 5% less, p < 0.07. In CR‐E, Tb.Th was 40% less than RD‐E, p < 0.0001. Exercise increased Tb.Th in RD (+23% RD‐E versus RD, p < 0.003) but failed to do so in CR. Cortical porosity increased after exercise in CR (+28%, p = 0.04), suggesting exercise during CR is deleterious to bone. In terms of bone fat, metaphyseal MAT/ BV rose 159% in CR versus RD, p = 0.003 via 3D MRI. Exercise decreased MAT/BV by 52% in RD, p < 0.05, and also suppressed MAT in CR (−121%, p = 0.047). Histomorphometric analysis of adipocyte area correlated with MAT by MRI (R2 = 0.6233, p < 0.0001). With respect to bone, TRAP and Sost mRNA were reduced in CR. Intriguingly, the repressed Sost in CR rose with exercise and may underlie the failure of CR‐bone quantity to increase in response to exercise. Notably, CD36, a marker of fatty acid uptake, rose 4088% in CR (p < 0.01 versus RD), suggesting that basal increases in MAT during calorie restriction serve to supply local energy needs and are depleted during exercise with a negative impact on bone. © 2019 The Authors. Journal of Bone and Mineral Research published by American Society for Bone and Mineral Research.

Effect of TNF-α-Induced Sclerostin on Osteocytes during Orthodontic Tooth Movement.

Osteocytes are abundant cells in bone, which contribute to bone maintenance. Osteocytes express receptor activator of nuclear factor kappa-B ligand (RANKL) and regulate osteoclast formation. Orthodontic tooth movement (OTM) occurs by osteoclast resorption of alveolar bone. Osteocyte-derived RANKL is critical in bone resorption during OTM. Additionally, tumor necrosis factor-α (TNF-α) is important in osteoclastogenesis during OTM. Sclerostin has been reported to enhance RANKL expression in the MLO-Y4 osteocyte-like cell line. This study investigated the effect of TNF-α on sclerostin expression in osteocytes during OTM. In vitro analysis of primary osteocytes, which were isolated from DMP1-Topaz mice by sorting the Topaz variant of GFP-positive cells, revealed that SOST mRNA expression was increased when osteocytes were cultured with TNF-α and that RANKL mRNA expression was increased when osteocytes were cultured with sclerostin. Moreover, the number of TRAP-positive cells was increased in osteocytes and osteoclast precursors cocultured with sclerostin. In vivo analysis of mouse calvariae that had been subcutaneously injected with phosphate-buffered saline (PBS) or TNF-α revealed that the number of TRAP-positive cells and the percentage of sclerostin-positive osteocytes were higher in the TNF-α group than in the PBS group. Furthermore, the level of SOST mRNA was increased by TNF-α. As an OTM model, a Ni-Ti closed-coil spring connecting the upper incisors and upper-left first molar was placed to move the first molar to the mesial direction in wild-type (WT) mice and TNF receptor 1- and 2-deficient (TNFRsKO) mice. After 6 days of OTM, the percentage of sclerostin-positive osteocytes on the compression side of the first molar in TNFRsKO mice was lower than that in WT mice. In this study, TNF-α increased sclerostin expression in osteocytes, and sclerostin enhanced RANKL expression in osteocytes. Thus, TNF-α may play an important role in sclerostin expression in osteocytes and enhance osteoclast formation during OTM.

FGF2 crosstalk with Wnt signaling in mediating the anabolic action of PTH on bone formation

The mechanisms of the anabolic effect of parathyroid hormone (PTH) in bone are not fully defined. The bone anabolic effects of PTH require fibroblast growth factor 2 (FGF2) as well as Wnt signaling and FGF2 modulates Wnt signaling in osteoblasts. In vivo PTH administration differentially modulated Wnt signaling in bones of wild type (WT) and in mice that Fgf2 was knocked out (Fgf2KO). PTH increased Wnt10b mRNA and protein in WT but not in KO mice. Wnt antagonist SOST mRNA and protein was significantly higher in KO group. However, PTH decreased Sost mRNA significantly in WT as well as in Fgf2KO mice, but to a lesser extent in Fgf2KO. Dickhopf 2 (DKK2) is critical for osteoblast mineralization. PTH increased Dkk2 mRNA in WT mice but the response was impaired in Fgf2KO mice. PTH significantly increased Lrp5 mRNA and phosphorylation of Lrp6 in WT but the increase was markedly attenuated in Fgf2KO mice. PTH increased β-catenin expression and Wnt/β-catenin transcriptional activity significantly in WT but not in Fgf2KO mice. These data suggest that the impaired bone anabolic response to PTH in Fgf2KO mice is partially mediated by attenuated Wnt signaling.

Mechanical Loading Differentially Affects Osteocytes in Fibulae from Lactating Mice Compared to Osteocytes in Virgin Mice: Possible Role for Lacuna Size

Hormonal changes during lactation are associated with profound changes in bone cell biology, such as osteocytic osteolysis, resulting in larger lacunae. Larger lacuna shape theoretically enhances the transmission of mechanical signals to osteocytes. We aimed to provide experimental evidence supporting this theory by comparing the mechanoresponse of osteocytes in the bone of lactating mice, which have enlarged lacunae due to osteocytic osteolysis, with the response of osteocytes in bone from age-matched virgin mice. The osteocyte mechanoresponse was measured in excised fibulae that were cultured in hormone-free medium for 24 h and cyclically loaded for 10 min (sinusoidal compressive load, 3000 µε, 5 Hz) by quantifying loading-related changes in Sost mRNA expression (qPCR) and sclerostin and β-catenin protein expression (immunohistochemistry). Loading decreased Sost expression by ~ threefold in fibulae of lactating mice. The loading-induced decrease in sclerostin protein expression by osteocytes was larger in lactating mice (55% decrease ± 14 (± SD), n = 8) than virgin mice (33% decrease ± 15, n = 7). Mechanical loading upregulated β-catenin expression in osteocytes in lactating mice by 3.5-fold (± 0.2, n = 6) which is significantly (p < 0.01) higher than the 1.6-fold increase in β-catenin expression by osteocytes in fibulae from virgin mice (± 0.12, n = 4). These results suggest that osteocytes in fibulae from lactating mice with large lacunae may respond stronger to mechanical loading than those from virgin mice. This could indicate that osteocytes residing in larger lacuna show a stronger response to mechanical loading.

Metformin protects bone mass in ultra-high-molecular-weight polyethylene particle-induced osteolysis by regulating osteocyte secretion.

Metformin, an anti-hyperglycemic agent used for type 2 diabetes, has recently been found to have more effects apart from glucose regulation. We found that, in ultra-high-molecular-weight polyethylene particle-induced osteolysis mouse models, metformin had bone protect property and reduced the negative regulator of bone formation sclerostin (SOST) and Dickkopf-related protein 1 (DKK1), and increased osteoprotegerin (OPG) secretion and the ratio of OPG/Receptor Activator for Nuclear Factor-κB Ligand (RANKL). In vitro, we established a 3D co-culture system in which metformin affects osteoblasts and osteoclasts through mature osteocytes secretion. Metformin (50μM) significantly decreased SOST and DKK1 mRNA expression, stimulating alkaline phosphatase activity and proliferation of osteoblast, and increased OPG secretion and the ratio of OPG/RANKL, inhibiting osteoclastogenesis. Moreover, the effect on OPG was reversed by adenosine 5'-monophosphate-activated protein kinase inhibitor, Compound C. Our finding suggests that metformin induces differentiation and mineralization of osteoblasts, while inhibits osteoclastogenesis via mature osteocytes secretion. Therefore, the drug might be beneficial for not only diabetes but also in other bone disorders by acting on mature osteocytes.

Sclerostin and DKK1 Inhibition Preserves and Augments Alveolar Bone Volume and Architecture in Rats with Alveolar Bone Loss

Alveolar bone is a mechanosensitive tissue that provides structural support for teeth. Alveolar bone loss is common with aging, menopause, tooth loss, and periodontitis and can lead to additional tooth loss, reduced denture fixation, and challenges in placing dental implants. The current studies suggest that sclerostin and DKK1, which are established osteocyte-derived inhibitors of bone formation, contribute to alveolar bone loss associated with estrogen ablation and edentulism in rats. Estrogen-deficient ovariectomized rats showed significant mandibular bone loss that was reversed by systemic administration of sclerostin antibody (SAB) alone and in combination with DKK1 antibody (DAB). Osteocytes in the dentate and edentulous rat maxilla expressed Sost (sclerostin) and Dkk1 (DKK1) mRNA, and molar extraction appeared to acutely increase DKK1 expression. In a chronic rat maxillary molar extraction model, systemic SAB administration augmented the volume and height of atrophic alveolar ridges, effects that were enhanced by coadministering DAB. SAB and SAB+DAB also fully reversed bone loss that developed in the opposing mandible as a result of hypo-occlusion. In both treatment studies, alveolar bone augmentation with SAB or SAB+DAB was accompanied by increased bone mass in the postcranial skeleton. Jaw bone biomechanics showed that intact sclerostin-deficient mice exhibited stronger and denser mandibles as compared with wild-type controls. These studies show that sclerostin inhibition, with and without DKK1 coinhibition, augmented alveolar bone volume and architecture in rats with alveolar bone loss. These noninvasive approaches may have utility for the conservative augmentation of alveolar bone.

CYR61/CCN1 Regulates Sclerostin Levels and Bone Maintenance.

CYR61/CCN1 is a matricellular protein that resides in the extracellular matrix, but serves regulatory rather than structural roles. CYR61/CCN1 is found in mineralized tissues and has been shown to influence bone healing in vivo and osteogenic differentiation in vitro. In this study we generated Cyr61 bone-specific knockout mice to examine the physiological role of CYR61/CCN1 in bone development and maintenance in vivo. Extensive analysis of Cyr61 conditional knockout mice showed a significant decrease in both trabecular and cortical bone mass as compared to WT littermates. Our data suggest that CYR61/CCN1 exerts its effects on mature osteoblast/osteocyte function to modulate bone mass. Specifically, changes were observed in osteocyte/osteoblast expression of RankL, VegfA, and Sost. The increase in RankL expression was correlated with a significant increase in osteoclast number; decreased VegfA expression was correlated with a significant decrease in bone vasculature; increased Sost expression was associated with decreased Wnt signaling, as revealed by decreased Axin2 expression and increased adiposity in the bone marrow. Although the decreased number of vascular elements in bone likely contributes to the low bone mass phenotype in Cyr61 conditional knockout mice, this cannot explain the observed increase in osteoclasts and the decrease in Wnt signaling. We conducted in vitro assays using UMR-106 osteosarcoma cells to explore the role CYR61/CCN1 plays in modulating Sost mRNA and protein expression in osteocytes and osteoblasts. Overexpression of CYR61/CCN1 can suppress Sost expression in both control and Cyr61 knockout cells, and blocking Sost with siRNA can rescue Wnt responsiveness in Cyr61 knockout cells in vitro. Overall, our data suggest that CYR61/CCN1 modulates mature osteoblast and osteocyte function to regulate bone mass through angiogenic effects as well as by modulating Wnt signaling, at least in part through the Wnt antagonist Sost. © 2018 American Society for Bone and Mineral Research.

Low-1 level mechanical vibration improves bone microstructure, tissue mechanical properties and porous titanium implant osseointegration by promoting anabolic response in type 1 diabetic rabbits

Type 1 diabetes mellitus (T1DM) is associated with reduced bone mass, increased fracture risk, and impaired bone defect regeneration potential. These skeletal complications are becoming important clinical challenges due to the rapidly increasing T1DM population, which necessitates developing effective treatment for T1DM-associated osteopenia/osteoporosis and bone trauma. This study aims to investigate the effects of whole-body vibration (WBV), an easy and non-invasive biophysical method, on bone microstructure, tissue-level mechanical properties and porous titanium (pTi) osseointegration in alloxan-diabetic rabbits. Six non-diabetic and twelve alloxan-treated diabetic rabbits were equally assigned to the Control, DM, and DM with WBV stimulation (WBV) groups. A cylindrical drill-hole defect was established on the left femoral lateral condyle of all rabbits and filled with a novel non-toxic Ti2448 pTi. Rabbits in the WBV group were exposed to 1h/day WBV (0.3g, 30Hz) for 8weeks. After sacrifice, the left femoral condyles were harvested for histological, histomorphometric and nanoindentation analyses. The femoral sample with 2-cm height above the defect was used for qRT-PCR analysis. The right distal femora were scanned with μCT. We found that all alloxan-treated rabbits exhibited hyperglycemia throughout the experimental period. WBV inhibited the deterioration of cancellous and cortical bone architecture and tissue-level mechanical properties via μCT, histological and nanoindentation examinations. T1DM-induced reduction of bone formation was inhibited by WBV, as evidenced by elevated serum OCN and increased mineral apposition rate (MAR), whereas no alteration was observed in bone resorption marker TRACP5b. WBV also stimulated more adequate ingrowths of mineralized bone tissue into pTi pore spaces, and improved peri-implant bone tissue-level mechanical properties and MAR in T1DM bone defects. WBV mitigated the reductions in femoral BMP2, OCN, Wnt3a, Lrp6, and β-catenin and inhibited Sost mRNA expression but did not alter RANKL or RANK gene expression in T1DM rabbits. Our findings demonstrated that WBV improved bone architecture, tissue-level mechanical properties, and pTi osseointegration by promoting canonical Wnt signaling-mediated skeletal anabolic response. This study not only advances our understanding of T1DM skeletal sensitivity in response to external mechanical cues but also offers new treatment alternatives for T1DM-associated osteopenia/osteoporosis and osseous defects in an economic and highly efficient manner.

Parathyroid Hormone (PTH) Induces Autophagy to Protect Osteocyte Cell Survival from Dexamethasone Damage.

BackgroundGlucocorticoids (GC) have direct adverse effects on osteocytes, the most abundant bone cell type, and play an important role in osteonecrosis of the femoral head (ONFH). Teriparatide has been reported to be an effective treatment for ONFH. However, the underlying mechanism is unclear.Material/MethodsAn osteocyte cell line, MLO-Y4, was used under various doses of dexamethasone (Dex) with or without rhPTH (1–34). Cell viability, autophagy, and apoptosis markers and osteocyte characteristic mRNAs were investigated to better understand this phenomenon.ResultsInduction of apoptosis by Dex was increased in a time- and dose-dependent manner in MLO-Y4 cells. Autophagy markers (LC3-II and Beclin-1) were increased at the low dose of Dex (10−7 or 10−6 M) and decreased at the high dose (10−5 M). In MOL-Y4 cells, rhPTH (1–34) was shown to be protective against Dex-induced apoptosis. The upregulation of LC3-II and Beclin-1 and decreased level of Caspase-3 was observed in the rhPTH (1–34)-treated group compared with the Dex-only-treated group. Furthermore, the changes induced by Dex in osteocytes, such as increased SOST, RANKL, and DMP-1 mRNA level and decreased Destrin mRNA level, were reversed by rhPTH (1–34). A similar result was found in osteocyte-specific proteins sclerostin expression encoded by SOST mRNA, which acted as a bone formation inhibitor.ConclusionsThe self-activation of autophagy may be a protective mechanism against apoptosis induced by Dex. The protection effect of rhPTH (1–34) for GC-induced ONFH thus results, at least in part, from enhanced autophagy.

Thirty days of spaceflight does not alter murine calvariae structure despite increased Sost expression

Previously our laboratory documented increases in calvaria bone volume and thickness in mice exposed to 15days of spaceflight aboard the NASA Shuttle mission STS-131. However, the tissues were not processed for gene expression studies to determine what bone formation pathways might contribute to these structural adaptations. Therefore, this study was designed to investigate both the structural and molecular changes in mice calvariae after a longer duration of spaceflight. The primary purpose was to determine the calvaria bone volume and thickness of mice exposed to 30days of spaceflight using micro-computed tomography for comparison with our previous findings. Because sclerostin, the secreted glycoprotein of the Sost gene, is a potent inhibitor of bone formation, our second aim was to quantify Sost mRNA expression using quantitative PCR. Calvariae were obtained from six mice aboard the Russian 30-day Bion-M1 biosatellite and seven ground controls. In mice exposed to 30days of spaceflight, calvaria bone structure was not significantly different from that of their controls (bone volume was about 5% lower in spaceflight mice, p=0.534). However, Sost mRNA expression was 16-fold (16.4±0.4, p<0.001) greater in the spaceflight group than that in the ground control group. Therefore, bone formation may have been suppressed in mice exposed to 30days of spaceflight. Genetic responsiveness (e.g. sex or strain of animals) or in-flight environmental conditions other than microgravity (e.g. pCO2 levels) may have elicited different bone adaptations in STS-131 and Bion-M1 mice. Although structural results were not significant, this study provides biochemical evidence that calvaria mechanotransduction pathways may be altered during spaceflight, which could reflect vascular and interstitial fluid adaptations in non-weight bearing bones. Future studies are warranted to elucidate the processes that mediate these effects and the factors responsible for discordant calvaria bone adaptations between STS-131 and Bion-M1 mice.

Intermittent compressive stress regulates Notch target gene expression via transforming growth factor-β signaling in murine pre-osteoblast cell line

ObjectiveDifferent mechanical stimuli regulate behaviors of various cell types, including osteoblasts, osteocytes, and periodontal ligament fibroblasts. Notch signaling participates in the mechanical stress-regulated cell responses. The present study investigated the regulation of Notch target gene and sclerostin (Sost) expression in murine pre-osteoblast cell line (MC3T3-E1) under intermittent compressive stress. MethodsMC3T3-E1 were subjected to the intermittent compressive force under the computerized controlled machine. In some experiments, cells were pretreated with chemical inhibitors for Notch and transforming growth factor (TGF)-β signaling prior to mechanical stimuli. To evaluate role of Notch signaling in MC3T3-E1 cells under unloaded condition, cells were seeded on indirect immobilized Notch ligand (Jagged1). Gene expression was determined using real-time quantitative polymerase chain reaction. ResultsThe intermittent compressive stress significantly upregulated Notch target gene expression (Hes Family BHLH transcription factor 1; Hes1 and Hairy/enhancer-of-split related with YRPW motif protein1; Hey1). The intermittent stress-induced Hes1 and Hey1 mRNA expression could be inhibited by a γ-secretase inhibitor (DAPT) or a TGF-β superfamily type I activing receptor-like kinase receptors inhibitor (SB431542). The results imply that intermittent compressive stress regulates Notch signaling via TGF-β pathway. Further, the intermittent compressive stress reduced Sost mRNA expression and this phenomenon could be rescued by a DAPT pretreatment, implying the involvement of Notch signaling. However, activation of Notch signaling under the unloaded condition resulted in the increase of Sost expression and the reduction of osteogenic marker genes. ConclusionsThese results imply the involvement of Notch signaling in the homeostasis maintaining of osteogenic cells under mechanical stress stimuli.

Effects of SOST Gene Silencing on Proliferation, Apoptosis, Invasion, and Migration of Human Osteosarcoma Cells Through the Wnt/β-Catenin Signaling Pathway.

Our study explored the effects of SOST gene silencing on the proliferation, apoptosis, invasion, and migration of human osteosarcoma cells through Wnt/β-catenin signaling pathway. Fresh tissues were obtained from 108 patients with osteosarcoma and 46 patients with osteochondroma. Human osteosarcoma cells (MG-63, U2-OS, HOS, and Saos-2) and normal osteoblast (hFoB1.19) were selected and cultured. Osteosarcoma cells were grouped randomly into the blank group, the scrambled control group, and the SOST-siRNA group. Cell proliferation was determined by MTT assay. Cell cycle and apoptosis were tested by flow cytometry. Transwell and scratch test were performed to determine cell invasion and migration. The qRT-PCR and Western blotting were used to detect mRNA and protein expression level of sclerostin, Wnt1, β-catenin, C-Myc, Cyclin D1, and MMP-7. The activity of caspase-3 was assessed by immunocytochemistry. Alkaline phosphatase (ALP) activity was measured using P-nitrophenylphosphate as a substrate. Low SOST mRNA and sclerostin protein expression levels were observed in osteosarcoma tissues and cells. Compared with the blank and scrambled control groups, sclerostin expression, apoptotic cells, ALP activity, and caspase-3 activity were down-regulated, while the proliferation, invasion, and migration abilities of osteosarcoma cells were evidently enhanced in the SOST-siRNA group. After SOST gene silencing, the mRNA and protein expression levels of Wnt1, β-catenin, C-Myc, Cyclin D1, and MMP-7 in osteosarcoma cells and β-catenin protein expression levels in the nucleus and cytoplasm were significantly elevated. SOST gene silencing promotes the proliferation, invasion, and migration, and inhibits apoptosis of osteosarcoma cells by activating Wnt/β-catenin signaling pathway.

Conditional deletion of IGF-I in osteocytes unexpectedly accelerates bony union of the fracture gap in mice

This study evaluated the effects of deficient IGF-I expression in osteocytes on fracture healing. Transgenic mice with conditional knockout (cKO) of Igf1 in osteocytes were generated by crossing Dmp1-Cre mice with Igf1 flox mice. Fractures were created on the mid-shaft of tibia of 12-week-old male cKO mice and wild-type (WT) littermates by three-point bending. At 21 and 28days post-fracture healing, the increases in cortical bone mineral density, mineral content, bone area, and thickness, as well as sub-cortical bone mineral content at the fracture site were each greater in cKO calluses than in WT calluses. There were 85% decrease in the cartilage area and >2-fold increase in the number of osteoclasts in cKO calluses at 14days post-fracture, suggesting a more rapid remodeling of endochondral bone. The upregulation of mRNA levels of osteoblast marker genes (cbfa1, alp, Opn, and Ocn) was greater in cKO calluses than in WT calluses. μ-CT analysis suggested an accelerated bony union of the fracture gap in cKO mice. The Sost mRNA level was reduced by 50% and the Bmp2 mRNA level was increased 3-fold in cKO fractures at 14days post-fracture, but the levels of these two mRNAs in WT fractures were unchanged, suggesting that the accelerated fracture repair may in part act through the Wnt and/or BMP signaling. In conclusion, conditional deletion of Igf1 in osteocytes not only did not impair, but unexpectedly enhanced, bony union of the fracture gap. The accelerated bony union was due in part to upregulation of the Wnt and BMP2 signaling in response to deficient osteocyte-derived IGF-I expression, which in turn favors intramembranous over endochondral bone repair.

Variations of SOST mRNA expression in human bone are associated with DNA polymorphism and DNA methylation in the SOST gene

SOST encodes sclerostin, an inhibitor of bone formation that antagonizes canonical Wnt signaling. Variations of SOST expression have an impact on bone mineral density (BMD) and bone strength. We hypothesized that genetic and epigenetic DNA modifications have an impact on SOST gene expression. By analyzing 43 bone samples from women, we found that rs851054 (G/A) is associated with SOST mRNA expression, donors with one or two G allele(s) displaying higher SOST expression (p<0.05). Beside this polymorphism, we also investigated the role of CpG methylation in SOST mRNA expression, and analyzed methylation variation at 13 CpG sites on the 1st exon of SOST in 14 human bone samples. Principal component analysis identified three groups of CpG sites that explained most of the methylation variation. We calculated the percentage of methylation in the main group of CpGs, and showed that higher rates of methylated CpGs are associated with higher SOST mRNA expression. To demonstrate that change in SOST expression might be related to human bone disease, we analyzed 131 patients with osteogenesis imperfecta (OI), a rare disease characterized by low BMD, bone fragility, and marked intra-familial variability of bone phenotypes. We found an association between rs851054 of the SOST promoter and the fracture rate only during childhood (p<0.01). In conclusion, genetic and epigenetic changes contribute to variation in SOST expression in human bone. Our data also indicate that these variations may be related to the severity of OI.

Targeted disruption of BMP signaling through type IA receptor (BMPR1A) in osteocyte suppresses SOST and RANKL, leading to dramatic increase in bone mass, bone mineral density and mechanical strength

Recent studies suggest a critical role of osteocytes in controlling skeletal development and bone remodeling although the molecular mechanism is largely unknown. This study investigated BMP signaling in osteocytes by disrupting Bmpr1a under the Dmp1-promoter. The conditional knockout (cKO) mice displayed a striking osteosclerotic phenotype with increased trabecular bone volume, thickness, number, and mineral density as assessed by X-ray and micro-CT. The bone histomorphometry, H&E, and TRAP staining revealed a dramatic increase in trabecular and cortical bone masses but a sharp reduction in osteoclast number. Moreover, there was an increase in BrdU positive osteocytes (2–5-fold) and osteoid volume (~4-fold) but a decrease in the bone formation rate (~85%) in the cKO bones, indicating a defective mineralization. The SEM analysis revealed poorly formed osteocytes: a sharp increase in cell numbers, a great reduction in cell dendrites, and a remarkable change in the cell distribution pattern. Molecular studies demonstrated a significant decrease in the Sost mRNA levels in bone (>95%), and the SOST protein levels in serum (~85%) and bone matrices. There was a significant increase in the β-catenin (>3-fold) mRNA levels as well as its target genes Tcf1 (>6-fold) and Tcf3 (~2-fold) in the cKO bones. We also showed a significant decrease in the RANKL levels of serum proteins (~65%) and bone mRNA (~57%), and a significant increase in the Opg mRNA levels (>20-fold) together with a significant reduction in the Rankl/Opg ratio (>95%), which are responsible for a sharp reduction in the cKO osteoclasts. The values of mechanical strength were higher in cKO femora (i.e. max force, displacement, and work failure). These results suggest that loss of BMP signaling specifically in osteocytes dramatically increases bone mass presumably through simultaneous inhibition of RANKL and SOST, leading to osteoclast inhibition and Wnt activation together. Finally, a working hypothesis is proposed to explain how BMPR1A controls bone remodeling by inhibiting cell proliferation and stimulating differentiation. It is reported that RANKL and SOST are abundantly expressed by osteocytes. Thus, BMP signaling through BMPR1A plays important roles in osteocytes.

Isolation of osteocytes from human trabecular bone

Osteocytes are essential regulators of bone homeostasis. However, they are difficult to study due to their location within the bone mineralised matrix. Although several techniques have been published for the isolation of osteocytes from mouse bone, no such technique has been described for human osteocytes. We have therefore developed a protocol for the isolation of osteocytes from human trabecular bone samples acquired during surgery. The cells were digested from the bone matrix by sequential collagenase and ethylenediaminetetraacetic acid (EDTA) digestions and the cells from later digests displayed characteristic dendritic osteocyte morphology when cultured ex vivo. Furthermore, the cells expressed characteristic osteocyte marker genes, such as E11, dentin matrix protein 1 (DMP1), SOST, matrix extracellular phosphoglycoprotein (MEPE) and phosphate regulating endopeptidase homologue, X-linked (PHEX). In addition, genes associated with osteocyte perilacunar remodelling, including matrix metallopeptidase-13 (MMP13), cathepsin K (CTSK) and carbonic anhydrase 2 (CAR2) were expressed. The cells also responded to parathyroid hormone (PTH) by downregulating SOST mRNA expression and to 1α,25-dihydroxyvitamin D3 (1,25D) by upregulating fibroblast growth factor 23 (FGF23) mRNA expression. Therefore, the cells behave in a similar manner to osteocytes in vivo. These cells represent an important tool in enhancing current knowledge in human osteocyte biology.

Generation and characterization of two immortalized human osteoblastic cell lines useful for epigenetic studies.

Different model systems using osteoblastic cell lines have been developed to help understand the process of bone formation. Here, we report the establishment of two human osteoblastic cell lines obtained from primary cultures upon transduction of immortalizing genes. The resulting cell lines had no major differences to their parental lines in their gene expression profiles. Similar to primary osteoblastic cells, osteocalcin transcription increased following 1,25-dihydroxyvitamin D3 treatment and the immortalized cells formed a mineralized matrix, as detected by Alizarin Red staining. Moreover, these human cell lines responded by upregulating ALPL gene expression after treatment with the demethylating agent 5-aza-2'-deoxycytidine (AzadC), as shown before for primary osteoblasts. We further demonstrate that these cell lines can differentiate in vivo, using a hydroxyapatite/tricalcium phosphate composite as a scaffold, to produce bone matrix. More importantly, we show that these cells respond to demethylating treatment, as shown by the increase in SOST mRNA levels, the gene encoding sclerostin, upon treatment of the recipient mice with AzadC. This also confirms, in vivo, the role of DNA methylation in the regulation of SOST expression previously shown in vitro. Altogether our results show that these immortalized cell lines constitute a particularly useful model system to obtain further insight into bone homeostasis, and particularly into the epigenetic mechanisms regulating sclerostin production.

Lipid Nanoparticle Delivery of siRNA to Osteocytes Leads to Effective Silencing of SOST and Inhibition of Sclerostin In Vivo.

Sclerostin is a protein secreted by osteocytes that is encoded by the SOST gene; it decreases bone formation by reducing osteoblast differentiation through inhibition of the Wnt signaling pathway. Silencing the SOST gene using RNA interference (RNAi) could therefore be an effective way to treat osteoporosis. Here, we investigate the utility of lipid nanoparticle (LNP) formulations of siRNA to silence the SOST gene in vitro and in vivo. It is shown that primary mouse embryonic fibroblasts (MEF) provide a useful model system in which the SOST gene can be induced by incubation in osteogenic media, allowing development of optimized SOST siRNA for silencing the SOST gene. Incubation of MEF cells with LNP containing optimized SOST siRNA produced significant, prolonged knockdown of the induced SOST gene in vitro, which was associated with an increase in osteogenic markers. Intravenous (i.v.) administration of LNP containing SOST siRNA to mice showed significant accumulation of LNP in osteocytes in compact bone, depletion of SOST mRNA and subsequent reduction of circulating sclerostin protein, establishing the potential utility for LNP siRNA systems to promote bone formation.

Early response of the human SOST gene to stimulation by 1α,25-dihydroxyvitamin D3

The osteocyte expressed gene SOST encodes sclerostin, a potent negative regulator of bone formation and inducer of bone resorption. We have recently demonstrated that the human SOST gene is positively regulated in response to 1α,25-dihydroxyvitamin D3 (1,25D). Responsiveness may be mediated at least in part by a single classical DR3-type vitamin D response element (VDRE). In this study we examined the early responsiveness of the SOST gene to both 1,25D and to parathyroid hormone (PTH), a known repressor of SOST expression, in SaOS2 cells differentiated to an osteocyte-like stage of cell maturation. Both SOST mRNA levels and sclerostin protein levels increased in these cultures as early as 3h post-treatment with 1,25D and declined in response to PTH in the same timeframe. For 1,25D, the level of induced SOST appeared dependent on the extent, to which the degradative enzyme 1,25-dihydroxyvitamin D 24-hydroxylase (CYP24A1) was induced. Together with the observed rapid decrease in SOST/sclerostin levels in response to PTH, endocrine regulation of sclerostin production appears to be an important determinant of sclerostin levels. These findings confirm that the human SOST gene and sclerostin expression can be considered to be directly 1,25D-responsive in osteocytes.