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 ...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 (Ca^(2+) ) dynamics. Here, by simultaneously monitoring Ca^(2+) and actin dynamics in single cells exposed to fluid shear flow, we detected actin network contractions immediately upon onset of flow-induced Ca^(2+) 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 Ca^(2+) 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 Ca^(2+) -dependent contractions and enhances the production and release of EVs containing bone regulatory proteins. Further, blocking Ca^(2+) signaling significantly attenuates adaptation to mechanical loading in vivo, suggesting a critical role for Ca^(2+) -mediated signaling in bone adaptation.展开更多
Objective To test whether in the absence of actin, actin-binding proteins such as caldesmon, calponin, and tropomyosin interact with the myosin of unphosphorylation, Ca 2+ -dependent phosphorylation (CDP), and Ca 2+ -...Objective To test whether in the absence of actin, actin-binding proteins such as caldesmon, calponin, and tropomyosin interact with the myosin of unphosphorylation, Ca 2+ -dependent phosphorylation (CDP), and Ca 2+ -independent phosphorylati-on (CIP) and stimulate myosin Mg 2+ -ATPase activities. Methods Mg 2+ -ATPase activities were measured to evaluate the effects of caldesmon, calponin, and tropomyosin on the myosin in unphosphorylation, CDP by myosin light chain kinase (MLCK), and CIP by MLCK. Results (1) At different incubation-time, i.e., 5, 10, 20, 40, and 60 minutes, the highest Mg 2+ -ATPase activity was ob-served when myosin was in the state of CDP, the medium was CIP of myosin, and the lowest was the unphosphorylated myosin. (2) In the absence of caldesmon, calponin, and tropomyosin, the Mg 2+ -ATPase activities from high to low were in the following order: CDP, CIP, and unphosphorylated myosin. However, in the presence of caldesmon, calponin, and tropo-myosin, the order of relative value of Mg 2+ -ATPase activities from high to low was unphosphorylated, CIP, and CDP of myosin respectively compared to the corresponding controls. Conclusions The results propose that caldesmon, calponin, and tropomyosin are capable of stimulating Mg 2+ -ATPase activity of smooth muscle myosin in Ca 2+ -independent manner, since Ca 2+ is not obligating for the stimulating effects of the three proteins. The common characteristic of the three proteins is that when myosin activities are low, their activations are relatively strong and this property might be involved in smooth muscle tension keeping.展开更多
基金supported by NIH R01 AR052461 and NIH R01 AR069148supported by a NSF Graduate Research Fellowship. A. E. M.supported by training grant T32 AR059038
文摘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 (Ca^(2+) ) dynamics. Here, by simultaneously monitoring Ca^(2+) and actin dynamics in single cells exposed to fluid shear flow, we detected actin network contractions immediately upon onset of flow-induced Ca^(2+) 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 Ca^(2+) 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 Ca^(2+) -dependent contractions and enhances the production and release of EVs containing bone regulatory proteins. Further, blocking Ca^(2+) signaling significantly attenuates adaptation to mechanical loading in vivo, suggesting a critical role for Ca^(2+) -mediated signaling in bone adaptation.
基金Supported by the National Natural Science Foundation of China ( 30070203).
文摘Objective To test whether in the absence of actin, actin-binding proteins such as caldesmon, calponin, and tropomyosin interact with the myosin of unphosphorylation, Ca 2+ -dependent phosphorylation (CDP), and Ca 2+ -independent phosphorylati-on (CIP) and stimulate myosin Mg 2+ -ATPase activities. Methods Mg 2+ -ATPase activities were measured to evaluate the effects of caldesmon, calponin, and tropomyosin on the myosin in unphosphorylation, CDP by myosin light chain kinase (MLCK), and CIP by MLCK. Results (1) At different incubation-time, i.e., 5, 10, 20, 40, and 60 minutes, the highest Mg 2+ -ATPase activity was ob-served when myosin was in the state of CDP, the medium was CIP of myosin, and the lowest was the unphosphorylated myosin. (2) In the absence of caldesmon, calponin, and tropomyosin, the Mg 2+ -ATPase activities from high to low were in the following order: CDP, CIP, and unphosphorylated myosin. However, in the presence of caldesmon, calponin, and tropo-myosin, the order of relative value of Mg 2+ -ATPase activities from high to low was unphosphorylated, CIP, and CDP of myosin respectively compared to the corresponding controls. Conclusions The results propose that caldesmon, calponin, and tropomyosin are capable of stimulating Mg 2+ -ATPase activity of smooth muscle myosin in Ca 2+ -independent manner, since Ca 2+ is not obligating for the stimulating effects of the three proteins. The common characteristic of the three proteins is that when myosin activities are low, their activations are relatively strong and this property might be involved in smooth muscle tension keeping.
