CYLD, a mechanosensitive deubiquitinase, regulates TGFβ signaling in load-induced bone formation

Many signaling pathways involved in bone homeostasis also participate in the anabolic response of bone to mechanical loading. For example, TGF beta signaling coordinates the maintenance of bone mass and bone quality through its effects on osteoblasts, osteoclasts, and osteocytes. TGF beta signaling is also essential for the mechanosensitive formation of new bone. However, the mechanosensitive mechanisms controlling TGF beta signaling in osteocytes remain to be determined, particularly those that integrate TGF beta signaling with other early responses to mechanical stimulation. Here, we used an in vivo mouse hindlimb loading model to identify mechanosensitive molecules in the TGF beta pathway, and MLO-Y4 cells to evaluate their interactions with the prostaglandin E2 (PGE2) pathway, which is well-known for its rapid response to mechanical stimulation and its role in bone anabolism. Although mRNA levels for several TGF beta ligands, receptors, and effectors were unchanged, the level of phosphorylated Smad2/3 (pSmad2/3) was reduced in tibial bone as early as 3 h after early mechanical stimulation. We found that PGE2 and its receptor, EP2, repress pSmad2/3 levels and transactivation of Serpine1 in osteocytes. PGE2 and EP2 control the level of pSmad2/3 through a proteasome-dependent mechanism that relies on the deubiquitinase CYLD. CYLD protein levels were also reduced in the tibiae within 3 h of mechanical loading. Using CYLD-deficient mice, we found that CYLD is required for the rapid load-mediated repression of pSmad2/3 and for load-induced bone formation. These data introduce CYLD as a mechanosensitive deubiquitinase that participates in the prostaglandin-dependent repression of TGF beta signaling in osteocytes.