Cellular senescence is a fundamental mechanism by which cells remain metabolically active yet cease dividing and undergo distinct phenotypic alterations, including upregulation of p16^Ink4a, profound secretome changes, telomere shortening, and decondensation of pericentromeric satellite DNA. Because senescent cells accumulate in multiple tissues with aging, these cells and the dysfunctional factors they secrete, termed the senescence‐associated secretory phenotype (SASP), are increasingly recognized as promising therapeutic targets to prevent age‐related degenerative pathologies, including osteoporosis. However, the cell type(s) within the bone microenvironment that undergoes senescence with aging in vivo has remained poorly understood, largely because previous studies have focused on senescence in cultured cells. Thus in young (age 6 months) and old (age 24 months) mice, we measured senescence and SASP markers in vivo in highly enriched cell populations, all rapidly isolated from bone/marrow without in vitro culture. In both females and males, p16^Ink4a expression by real‐time quantitative polymerase chain reaction (rt‐qPCR) was significantly higher with aging in B cells, T cells, myeloid cells, osteoblast progenitors, osteoblasts, and osteocytes. Further, in vivo quantification of senescence‐associated distension of satellites (SADS), ie, large‐scale unraveling of pericentromeric satellite DNA, revealed significantly more senescent osteocytes in old compared with young bone cortices (11% versus 2%, p < 0.001). In addition, primary osteocytes from old mice had sixfold more (p < 0.001) telomere dysfunction‐induced foci (TIFs) than osteocytes from young mice. Corresponding with the age‐associated accumulation of senescent osteocytes was significantly higher expression of multiple SASP markers in osteocytes from old versus young mice, several of which also showed dramatic age‐associated upregulation in myeloid cells. These data show that with aging, a subset of cells of various lineages within the bone microenvironment become senescent, although senescent myeloid cells and senescent osteocytes predominantly develop the SASP. Given the critical roles of osteocytes in orchestrating bone remodeling, our findings suggest that senescent osteocytes and their SASP may contribute to age‐related bone loss. © 2016 American Society for Bone and Mineral Research

Osteoclasts are thought to be solely responsible for the removal of bone matrix. However, we show here that osteocytes can also remove bone matrix by reversibly remodeling their perilacunar/canalicular matrix during the reproductive cycle. In contrast, no osteocytic remodeling was observed with experimental unloading despite similar degrees of bone loss. Gene array analysis of osteocytes from lactating animals revealed an elevation of genes known to be utilized by osteoclasts to remove bone, including tartrate‐resistant acid phosphatase (TRAP) and cathepsin K, that returned to virgin levels upon weaning. Infusion of parathyroid hormone–related peptide (PTHrP), known to be elevated during lactation, induced TRAP activity and cathepsin K expression in osteocytes concurrent with osteocytic remodeling. Conversely, animals lacking the parathyroid hormone type 1 receptor (PTHR1) in osteocytes failed to express TRAP or cathepsin K or to remodel their osteocyte perilacunar matrix during lactation. These studies show that osteocytes remove mineralized matrix through molecular mechanisms similar to those utilized by osteoclasts. © 2012 American Society for Bone and Mineral Research.

Exercise has beneficial effects on metabolism and on tissues. The exercise-induced muscle factor β-aminoisobutyric acid (BAIBA) plays a critical role in the browning of white fat and in insulin resistance. Here we show another function for BAIBA, that of a bone-protective factor that prevents osteocyte cell death induced by reactive oxygen species (ROS). l-BAIBA was as or more protective than estrogen or N-acetyl cysteine, signaling through the Mas-Related G Protein-Coupled Receptor Type D (MRGPRD) to prevent the breakdown of mitochondria due to ROS. BAIBA supplied in drinking water prevented bone loss and loss of muscle function in the murine hindlimb unloading model, a model of osteocyte apoptosis. The protective effect of BAIBA was lost with age, not due to loss of the muscle capacity to produce BAIBA but likely to reduced Mrgprd expression with aging. This has implications for understanding the attenuated effect of exercise on bone with aging.

The purpose of this work was to establish a methodology to enable the isolation and study of osteocytes from skeletally mature young (4-month-old) and old (22-month-old) mice. The location of osteo...

Osteocytes appear to mobilize calcium within minutes in response to PTH injections; we have previously shown that osteocytes remove their perilacunar matrix during lactation through activation of the PTH type 1 receptor. Mechanisms utilized by osteocytes to mobilize calcium are unknown but we hypothesized that the molecular components may be similar to those used by osteoclasts. Here we show, using IDG‐SW3 cells that ATP6V0D2, an essential component of vacuolar ATPase in osteoclasts, and other genes associated with osteoclastic bone resorption, increase with osteoblast to osteocyte differentiation. Furthermore, PTHrP increases ATP6V0D2 expression and induces proton generation by primary osteocytes, which is blocked by bafilomycin, a vacuolar ATPase inhibitor. These in vitro proton measurements raised the question of osteocyte viability in an acidic environment. Interestingly, osteocytes, showed enhanced viability at pH as low as 5 compared to osteoblasts and fibroblasts in vitro. To study in vivo acidification by osteocytes, virgin and lactating CD1 mice on a low calcium diet were injected with the pH indicator dye, acridine orange, and their osteocyte lacuno‐canalicular system imaged by confocal microscopy. Lower pH was observed in lactating compared to virgin animals. In addition, a novel transgenic mouse line with a topaz variant of green fluorescent protein (GFPtpz)‐tagged collagen α2(I) chain was used. Instead of the expected reduction in GFP‐fluorescence only in the perilacunar matrix, reduced fluorescence was observed in the entire bone matrix of lactating mice. Based on our experiments showing quenching of GFP in vitro, we propose that the observed reduction in GFP fluorescence in lactating mice is due to quenching of GFP by the acidic pH generated by osteocytes. Together these findings provide novel mechanistic insight into how osteocytes remove calcium from their perilacunar/pericanalicular matrices through active acidification of their microenvironment and show that osteocytes, like osteoclasts, are resistant to the negative effects of acid on viability. © 2017 American Society for Bone and Mineral Research.

We examined the effects of osteocyte secreted factors on myogenesis and muscle function. MLO‐Y4 osteocyte‐like cell conditioned media (CM) (10%) increased ex vivo soleus muscle contractile force by ∼25%. MLO‐Y4 and primary osteocyte CM (1% to 10%) stimulated myogenic differentiation of C2C12 myoblasts, but 10% osteoblast CMs did not enhance C2C12 cell differentiation. Because WNT3a and WNT1 are secreted by osteocytes, and the expression level of Wnt3a is increased in MLO‐Y4 cells by fluid flow shear stress, both were compared, showing WNT3a more potent than WNT1 in inducing myogenesis. Treatment of C2C12 myoblasts with WNT3a at concentrations as low as 0.5 ng/mL mirrored the effects of both primary osteocyte and MLO‐Y4 CM by inducing nuclear translocation of β‐catenin with myogenic differentiation, suggesting that Wnts might be potential factors secreted by osteocytes that signal to muscle cells. Knocking down Wnt3a in MLO‐Y4 osteocytes inhibited the effect of CM on C2C12 myogenic differentiation. Sclerostin (100 ng/mL) inhibited both the effects of MLO‐Y4 CM and WNT3a on C2C12 cell differentiation. RT‐PCR array results supported the activation of the Wnt/β‐catenin pathway by MLO‐Y4 CM and WNT3a. These results were confirmed by qPCR, showing upregulation of myogenic markers and two Wnt/β‐catenin downstream genes, Numb and Flh1. We postulated that MLO‐Y4 CM/WNT3a could modulate intracellular calcium homeostasis as the trigger mechanism for the enhanced myogenesis and contractile force. MLO‐Y4 CM and WNT3a increased caffeine‐induced Ca²⁺ release from the sarcoplasmic reticulum (SR) of C2C12 myotubes and the expression of genes directly associated with intracellular Ca²⁺ signaling and homeostasis. Together, these data show that in vitro and ex vivo, osteocytes can stimulate myogenesis and enhance muscle contractile function and suggest that Wnts could be mediators of bone to muscle signaling, likely via modulation of intracellular Ca²⁺ signaling and the Wnt/ β‐Catenin pathway. © 2017 American Society for Bone and Mineral Research