Loss of Gsα in osteocytes leads to osteopenia due to sclerostin induced suppression of osteoblast activity.

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 hormone 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 Gsα 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 induces 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 100 µg/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.

Calcium fluxes at the bone/plasma interface: Acute effects of parathyroid hormone (PTH) and targeted deletion of PTH/PTH-related peptide (PTHrP) receptor in the osteocytes.

Calcium ion concentration ([Ca2+]) in the systemic extracellular fluid, ECF-[Ca2+], is maintained around a genetically predetermined set-point, which combines the operational level of the kidney and bone/ECF interfaces. The ECF-[Ca2+] is maintained within a narrow oscillation range by the regulatory action of Parathyroid Hormone (PTH), Calcitonin, FGF-23, and 1,25(OH)2D3. This model implies two correction mechanisms, i.e. tubular Ca2+ reabsorption and osteoclast Ca2+ resorption. Although their alterations have an effect on the ECF-[Ca2+] maintenance, they cannot fully account for rapid correction of the continuing perturbations of plasma [Ca2+], which occur daily in life. The existence of Ca2+ fluxes at quiescent bone surfaces fulfills the role of a short-term error correction mechanism in Ca2+ homeostasis. To explore the hypothesis that PTH regulates the cell system responsible for the fast Ca2+ fluxes at the bone/ECF interface, we have performed direct real-time measurements of Ca2+ 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-[Ca2+] regulation, metatarsal bones from mice lacking the PTH receptor in these cells were tested ex vivo for rapid Ca2+ exchange. We performed direct real-time measurements of Ca2+ 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 Ca2+ 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-[Ca2+] 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.

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-Secreted Wnt Signaling Inhibitor Sclerostin Contributes to Beige Adipogenesis in Peripheral Fat Depots.

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 ß-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. © 2016 American Society for Bone and Mineral Research.

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.