Mechanically induced Ca2+ oscillations in osteocytes release extracellular vesicles and enhance bone formation

Abstract

The vast osteocytic network is believed to orchestrate bone metabolic activity in response to mechanical stimuli through production of sclerostin, RANKL, and osteoprotegerin (OPG). However, the mechanisms of osteocyte mechanotransduction remain poorly understood. We’ve previously shown that osteocyte mechanosensitivity is encoded through unique intracellular calcium (Ca2+) dynamics. Here, by simultaneously monitoring Ca2+ and actin dynamics in single cells exposed to fluid shear flow, we detected actin network contractions immediately upon onset of flow-induced Ca2+ transients, which were facilitated by smooth muscle myosin and further confirmed in native osteocytes ex vivo. Actomyosin contractions have been linked to the secretion of extracellular vesicles (EVs), and our studies demonstrate that mechanical stimulation upregulates EV production in osteocytes through immunostaining for the secretory vesicle marker Lysosomal-associated membrane protein 1 (LAMP1) and quantifying EV release in conditioned medium, both of which are blunted when Ca2+ signaling was inhibited by neomycin. Axial tibia compression was used to induce anabolic bone formation responses in mice, revealing upregulated LAMP1 and expected downregulation of sclerostin in vivo. This load-related increase in LAMP1 expression was inhibited in neomycin-injected mice compared to vehicle. Micro-computed tomography revealed significant load-related increases in both trabecular bone volume fraction and cortical thickness after two weeks of loading, which were blunted by neomycin treatment. In summary, we found mechanical stimulation of osteocytes activates Ca2+-dependent contractions and enhances the production and release of EVs containing bone regulatory proteins. Further, blocking Ca2+ signaling significantly attenuates adaptation to mechanical loading in vivo, suggesting a critical role for Ca2+-mediated signaling in bone adaptation., People gain bone in response to exercise and lose it during prolonged bedrest; now we’re closer to understanding how this happens., Bone cells called osteocytes act as mechanical sensors, responding to changes in force by regulating the activity of bone-forming osteoblasts and bone- resorbing osteoclasts. X. Edward Guo at Columbia University in New York and colleagues had previously shown that osteocytes exhibit oscillations in intracellular calcium in response to mechanical stimulation, but the downstream effects of this had been unclear. Using multiple approaches, they have now shown that the cytoskeleton contracts in response to these oscillations, in turn triggering the production and release of extracellular vesicles containing bone-regulatory proteins., When calcium signaling was blocked, vesicle production and release was blunted, and mice failed to show the normal increase in bone formation in response to mechanical loading.