Browsing by Subject "osteoprotegerin"

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    Investigation of the Mechanism of the Gene Regulation of OPG (Osteoprotegerin) by Cx43
    (Office of the Vice Chancellor for Research, 2013-04-05) Hassan, Iraj; Pacheco-Costa, Rafael; Plotkin, Lillian I.
    The main objective is to determine whether the gene regulation of OPG, osteoprotegerin, by Cx43, is at the promoter level. In a recent project, it was found that deletion in Cx43 from osteocytes resulted in a decreased OPG expression. Furthermore, it was found that deletion of Cx43 from osteocytes resulted in enhanced osteoclast differentiation. Osteoprotegerin (OPG), an osteoblast-secreted decoy receptor that modulates osteoclast formation could be directly controlled by Cx43 at the promoter binding sites of p53 and Sp1. Cx43 and OPG in turn are widely up regulated by Wnt, lipid-modified signaling proteins that influence cell proliferation, differentiation, and survival. The activation of Wnt signaling results in the binding of the transcription factor Tcf in gene promoters, which leads to increased gene expression. The investigation was carried out using reporter constructs in which the activation of the promoter resulted in the transcription of the enzyme luciferase. Luciferase activity, in turn, can be measured using a commercially available substrate that emits luminescence when luciferase is present. OPG-Luc and Tcf-Luc were grown in E. coli and purified using a kit from Qiagen. Transfection of OPG 1 and Tcf-Luc reporter constructs on MLO-Y4 osteocyte cells deleted for Cx43 (Cx43shRNA) and Cx37 (Cx37shRNA) was conducted after seeding of the cells a day in advance. For each cell line, regular and Lithium Chloride (to mimic the effects of Wnt) induced medium was used, and cells were cultured for 24h. From the assay, it was deemed that luciferase activity was higher in Wnt induced cells. OPG is a target of Wnt signaling downstream of the transcription factor Tcf. We therefore also measure Tcf-mediated transcription using a Tcf-luciferase construct. Expression of OPG-Luc and Tcf-Luc was higher in cell lines that are not silenced for Cx43 and Cx37. According to ANOVA test, the results did reach statistical significance. However, future trials will be conducted to mimic the results.
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    Mechanically induced Ca2+ oscillations in osteocytes release extracellular vesicles and enhance bone formation
    (Nature Publishing group, 2018-03-19) Morrell, Andrea E.; Brown, Genevieve N.; Robinson, Samuel T.; Sattler, Rachel L.; Baik, Andrew D.; Zhen, Gehua; Cao, Xu; Bonewald, Lynda F.; Jin, Weiyang; Kam, Lance C.; Guo, X. Edward; Medicine, School of Medicine
    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.