Background: Recent studies have suggested osteocytes as key players in mechanosensation and skeletal metabolism. Results: Simulated microgravity induces an autonomous up-regulation of SOST/sclerostin and RANKL/OPG in a novel osteocytic cell line, Ocy454. Conclusion: Mechanical loading regulates intrinsic osteocyte responses in concert with hormonal and cytokine inputs. Significance: Learning how osteocytes sense mechanical loads would enable novel interventions to prevent disuse-induced bone loss.

Background: Osteocytes, the most abundant cells in adult bone, express PPR. Results: Mice with constitutive PPR deletion in osteocytes demonstrate blunted anabolic and catabolic bone responses and the inability to recruit osteoblasts and osteoclasts upon PTH administration. Conclusion: PPR in osteocytes is needed for a full skeletal response to PTH administration. Significance: PPR signaling in osteocytes is necessary for PTH-driven anabolic effects during osteoporosis therapy.

Cells of the osteoblast lineage are increasingly identified as participants in whole-body metabolism by primarily targeting pancreatic insulin secretion or consuming energy. Osteocytes, the most abundant bone cells, secrete a Wnt-signaling inhibitor called sclerostin. Here we examined three mouse models expressing high sclerostin levels, achieved through constitutive or inducible loss of the stimulatory subunit of G-proteins (Gsα in mature osteoblasts and/or osteocytes). These mice showed progressive loss of white adipose tissue (WAT) with tendency toward increased energy expenditure but no changes in glucose or insulin metabolism. Interestingly beige adipocytes were increased extensively in both gonadal and inguinal WAT and had reduced canonical β-catenin signaling. To determine if sclerostin directly contributes to the increased beige adipogenesis, we engineered an osteocytic cell line lacking Gsα which has high sclerostin secretion. Conditioned media from these cells significantly increased expression of UCP1 in primary adipocytes, and this effect was partially reduced after depletion of sclerostin from the conditioned media. Similarly, treatment of Gsα-deficient animals with sclerostin-neutralizing antibody partially reduced the increased UCP1 expression in WAT. Moreover, direct treatment of sclerostin to wild-type mice significantly increased UCP1 expression in WAT. These results show that osteocytes and/or osteoblasts secrete factors regulating beige adipogenesis, at least in part, through the Wnt-signaling inhibitor sclerostin. Further studies are needed to assess metabolic effects of sclerostin on adipocytes and other metabolic tissues.

Osteocytes are master orchestrators of bone remodeling; they control osteoblast and osteoclast activities both directly cell-to-cell communication and indirectly 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- 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 and , 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.

Calcium ion concentration ([Ca]) in the systemic extracellular fluid, ECF-[Ca], is maintained around a genetically predetermined set-point, which combines the operational level of the kidney and bone/ECF interfaces. The ECF-[Ca] is maintained within a narrow oscillation range by the regulatory action of Parathyroid Hormone (PTH), Calcitonin, FGF-23, and 1,25(OH)D. This model implies two correction mechanisms, i.e. tubular Ca reabsorption and osteoclast Ca resorption. Although their alterations have an effect on the ECF-[Ca] maintenance, they cannot fully account for rapid correction of the continuing perturbations of plasma [Ca], which occur daily in life. The existence of Ca fluxes at quiescent bone surfaces fulfills the role of a short-term error correction mechanism in Ca homeostasis. To explore the hypothesis that PTH regulates the cell system responsible for the fast Ca fluxes at the bone/ECF interface, we have performed direct real-time measurements of Ca fluxes at the surface of ex-vivo metatarsal bones maintained in physiological conditions mimicking ECF, and exposed to PTH. To further characterize whether the PTH receptor on osteocytes is a critical component of the minute-to-minute ECF-[Ca] regulation, metatarsal bones from mice lacking the PTH receptor in these cells were tested ex vivo for rapid Ca exchange. We performed direct real-time measurements of Ca fluxes and concentration gradients by a scanning ion-selective electrode technique (SIET). To validate ex vivo measurements, we also evaluated acute calcemic response to PTH in vivo in mice lacking PTH receptors in osteocytes vs littermate controls. Our data demonstrated that Ca fluxes at the bone-ECF interface in excised bones as well as acute calcemic response in the short-term were unaffected by PTH exposure and its signaling through its receptor in osteocytes. Rapid minute-to-minute regulation of the ECF-[Ca] was found to be independent of PTH actions on osteocytes. Similarly, mice lacking PTH receptor in osteocytes, responded to PTH challenge with similar calcemic increases.

The stimulatory subunit of G-protein, Gsα, acts as a secondary messenger of G-protein coupled receptors (GPCRs) that primarily activates cAMP-induced signaling. GPCRs, such as the parathyroid receptor (PTHR), are critical regulators of bone formation as shown by number of genetic manipulation studies targeting early osteoblast lineage cells. In this study, we have examined the role of Gsa in osteocytes, the terminally differentiated and most abundant cells of the osteoblast lineage. Mice lacking the stimulatory subunit of G-proteins (Gsα) in osteocytes (DMP1-GsαKO) have significant decrease of both trabecular and cortical bone, as assessed by μCT. Histomorphometric analysis showed that the osteopenia was mostly driven by more than 90% decrease in osteoblast numbers and activity whereas osteoclasts were only slightly decreased. The decrease in osteoblast number was associated with a striking lack of endocortical osteoblasts. We have previously shown that loss of the stimulatory subunit of G-proteins (Gsα) in osteocytes in vitro or in vivo has high expression of sclerostin. To determine if the increased sclerostin levels contributed to the decreased endosteal bone lining cells and osteopenia, we treated wild-type mice with recombinant sclerostin and the DMP1-GsαKO mice with anti-sclerostin antibody. Treatment of wild-type mice with 100mg/Kg sclerostin for 3-weeks significantly reduced the numbers of bone lining cells and led to osteopenia. Next, the DMP1-GsαKO and control littermates were treated with the anti-sclerostin antibody (25 mg/kg, 2 times per week) for 4-weeks. Upon the antibody treatment, the endocortical osteoblasts reappeared in the DMP1-GsαKO mice to a comparable level to that of the vehicle treated control littermates. In control mice, E11/gp38 positive osteocytes were observed in parallel with the endocortical osteoblasts with higher dendrite density towards the endocortical osteoblasts. In DMP1-GsαKO mice, E11/gp38 positive osteocytes were lacking dendrites and were randomly scattered throughout the bone matrix. After treatment with anti-sclerostin antibody, DMP1-GsαKO mice showed increased E11/gp38 positive osteocytes near the endosteal bone surface and endosteal osteoblasts. The anti-sclerostin antibody treatment proportionally increased the bone volume but it could not completely rescue the osteopenia in the DMP1-GsαKO mice. Taken together, this data suggests that Gsα signaling in osteocytes leads to osteopenia driven, at least in part, by increased secretion of sclerostin.