In this dissertation, the role of TGFβ signaling is dissected in the context of mechanical loading and mechanotransduction. Using an in vivo mechanical loading regimen, this study is the first to show the mechanosensitive regulation of TGFβ in bone and specifically osteocytes. TGFβ signaling is rapidly repressed within 5 hours of mechanical loading. This was shown using three different techniques including in vivo luciferase Smad2/3 functional assay, immunohistochemistry, and western blotting. TGFβ is required for the anabolic effect of mechanotransduction in bone. Mice with reduced TGFβ signaling due to a dominant negative TβRII allele expressed under the control of an osteoblast/osteocyte specific promoter displayed an impairment in loadinduced bone formation. TGFβ regulates an osteocyte specific gene, SOST, to transduce mechanical stimulus for bone formation. The first study in this dissertation establishes TGFβ as a major pathway that plays a role in load-induced bone formation.
The rapid response of TGFβ signaling to load begs for the question of the mechanistic regulation of Smad2/3 at the early time points of mechanotransduction in bone. The second study in this dissertation focused on determining the mechanism of repression of Smad2/3 in osteocytes. We approached this question using an in vivo loading regimen and an in vitro fluid flow assay to understand how TGFβ is being regulated. Prostalgandins E2, or PGE2, a pathway that is upregulated within 10 min of mechanical stimulus in osteocytes. We showed that the protein expression of TβRI is dependent on the PGE2 receptor, EP2. Furthermore, this study is the first to show the mechanosensitivity of a deubiquitinase, called CYLD, in osteocytes. CYLD protein expression is reduced within 3 hours after load but not it’s mRNA levels indicating a posttranslational modification step. EP2-mediated repression of TβRI is dependent on CYLD activity. Understanding the signaling mechanism of load-mediated mechanotransduction and the TGFβ pathway in this physiological process could provide new targets for therapeutic intervention in low bone mass diseases such as osteoporosis
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