The down regulation of sclerostin in osteocytes mediates bone formation in response to mechanical cues and parathyroid hormone (PTH). To date, the regulation of sclerostin has been attributed exclusively to the transcriptional downregulation of the Sost gene hours after stimulation. Using mouse models and rodent cell lines, we describe the rapid, minutes-scale post-translational degradation of sclerostin protein by the lysosome following mechanical load and PTH. We present a model, integrating both new and established mechanically- and hormonally-activated effectors into the regulated degradation of sclerostin by lysosomes. Using a mouse forelimb mechanical loading model, we find transient inhibition of lysosomal degradation or the upstream mechano-signaling pathway controlling sclerostin abundance impairs subsequent load-induced bone formation by preventing sclerostin degradation. We also link dysfunctional lysosomes to aberrant sclerostin regulation using human Gaucher disease iPSCs. These results reveal how bone anabolic cues post-translationally regulate sclerostin abundance in osteocytes to regulate bone formation.

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In bone, connexin43 expression in cells of the osteoblast lineage plays an important role in restraining osteoclastogenesis and bone resorption. While there is a consensus around the notion that the anti-osteoclastogenic factor, osteoprotegerin, is a driver of this effect, how connexin43 regulates osteoprotegerin gene expression is unclear. Here, we showed that loss of connexin43 decreased osteoprotegerin gene expression and reduced ERK1/2 activation. Conversely, overexpression of connexin43 increased osteoprotegerin expression and enhanced ERK1/2 activation. This increase in phospho-ERK1/2 is required for connexin43 to induce activity from the osteoprotegerin proximal promoter. Connexin43 increased promoter activity via a specific 200 base pair region of the osteoprotegerin promoter located at −1486 to −1286 with respect to the transcriptional start site, a region which includes four Sp1 binding elements. Further, activation of this promoter region required an intact functional connexin43, as hypomorphic or dominant negative connexin43 mutant constructs, including one with increased hemichannel activity, were unable to stimulate osteoprotegerin expression as strongly as wild type connexin43. Using chromatin immunoprecipitations, we show that connexin43 expression enhanced the recruitment of Sp1, but not Runx2, to the osteoprotegerin proximal promoter. In total, these data show that connexin43-dependent gap junctional communication among osteoblast cells permits efficient ERK1/2 activation. ERK1/2 signaling promoted the recruitment of the potent transcriptional activator, Sp1, to the osteoprotegerin proximal promoter, resulting in robust transcription of antiosteoclastogenic factor, osteoprotegerin.

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