Osteocytes integrate the responses of bone to mechanical and hormonal stimuli by poorly understood mechanisms. We report here that mice with conditional deletion of the parathyroid hormone (PTH) receptor 1 (Pth1r) in dentin matrix protein 1 (DMP1)-8kb-expressing cells (cKO) exhibit a modest decrease in bone resorption leading to a mild increase in cancellous bone without changes in cortical bone. However, bone resorption in response to endogenous chronic elevation of PTH in growing or adult cKO mice induced by a low calcium diet remained intact, because the increased bone remodeling and bone loss was indistinguishable from that exhibited by control littermates. In contrast, the bone gain and increased bone formation in cancellous and cortical bone induced by daily injections of PTH and the periosteal bone apposition induced by axial ulna loading were markedly reduced in cKO mice compared to controls. Remarkably, however, wild-type (WT) control littermates and transgenic mice overexpressing SOST injected daily with PTH exhibit similar activation of Wnt/β-catenin signaling, increased bone formation, and cancellous and cortical bone gain. Taken together, these findings demonstrate that Pth1r in DMP1-8kb-expressing cells is required to maintain basal levels of bone resorption but is dispensable for the catabolic action of chronic PTH elevation; and it is essential for the anabolic actions of daily PTH injections and mechanical loading. However, downregulation of Sost/sclerostin, previously shown to be required for bone anabolism induced by mechanical loading, is not required for PTH-induced bone gain, showing that other mechanisms downstream of the Pth1r in DMP1-8kb-expressing cells are responsible for the hormonal effect. © 2016 American Society for Bone and Mineral Research.

Endoplasmic reticulum (ER) stress is associated with increased reactive oxygen species (ROS), results from accumulation of misfolded/unfolded proteins, and can trigger apoptosis. ER stress is alleviated by phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), which inhibits protein translation allowing the ER to recover, thus promoting cell viability. We investigated whether osteoblastic cell apoptosis induced by glucocorticoids (GC) is due to induction of ROS/ER stress and whether inhibition of eIF2α dephosphorylation promotes survival opposing the deleterious effects of GC in vitro and in vivo. Apoptosis of osteocytic MLO-Y4 and osteoblastic OB-6 cells induced by dexamethasone was abolished by ROS inhibitors. Like GC, the ER stress inducing agents brefeldin A and tunicamycin induced osteoblastic cell apoptosis. Salubrinal or guanabenz, specific inhibitors of eIF2α dephosphorylation, blocked apoptosis induced by either GC or ER stress inducers. Moreover, GC markedly decreased mineralization in OB-6 cells or primary osteoblasts; and salubrinal or guanabenz increased mineralization and prevented the inhibitory effect of GC. Furthermore, salubrinal (1 mg/kg/day) abolished osteoblast and osteocyte apoptosis in cancellous and cortical bone and partially prevented the loss of BMD at all sites and the decreased vertebral cancellous bone formation induced by treatment with prednisolone for 28 days (1.4 mg/kg/day). We conclude that part of the pro-apoptotic actions of GC on osteoblastic cells are mediated through ER stress, and that inhibition of eIF2α dephosphorylation protects from GC-induced apoptosis of osteoblasts and osteocytes in vitro and in vivo and from the deleterious effects of GC on the skeleton.

Glucocorticoid excess, either endogenous with diseases of the adrenal gland, stress, or aging or when administered for immunosuppression, induces bone and muscle loss, leading to osteopenia and sarcopenia. Muscle weakness increases the propensity for falling, which, combined with the lower bone mass, increases the fracture risk. The mechanisms underlying glucocorticoid-induced bone and muscle atrophy are not completely understood. We have demonstrated that the loss of bone and muscle mass, decreased bone formation, and reduced muscle strength, hallmarks of glucocorticoid excess, are accompanied by upregulation in both tissues in vivo of the atrophy-related genes atrogin1, MuRF1, and MUSA1. These are E3 ubiquitin ligases traditionally considered muscle-specific. Glucocorticoids also upregulated atrophy genes in cultured osteoblastic/osteocytic cells, in ex vivo bone organ cultures, and in muscle organ cultures and C2C12 myoblasts/myotubes. Furthermore, glucocorticoids markedly increased the expression of components of the Notch signaling pathway in muscle in vivo, ex vivo, and in vitro. In contrast, glucocorticoids did not increase Notch signaling in bone or bone cells. Moreover, the increased expression of atrophy-related genes in muscle, but not in bone, and the decreased myotube diameter induced by glucocorticoids were prevented by inhibiting Notch signaling. Thus, glucocorticoids activate different mechanisms in bone and muscle that upregulate atrophy-related genes. However, the role of these genes in the effects of glucocorticoids in bone is unknown. Nevertheless, these findings advance our knowledge of the mechanism of action of glucocorticoids in the musculoskeletal system and provide the basis for novel therapies to prevent glucocorticoid-induced atrophy of bone and muscle.