E3-ubiquitin ligase NEDD4 enhances bone formation by removing TGFβ1-induced pSMAD1 in immature osteoblast

Neural precursor cell expressed developmentally downregulated protein 4 (NEDD4) is an E3 ubiquitin ligase that regulates animal growth and development. To investigate the role of NEDD4 in skeletogenesis in vivo, we established immature osteoblast-specific 2.3-kb Collagen Type I Alpha 1 chain (Col1α1) promoter-driven Nedd4 transgenic (Nedd4-TG, Col1α1-Nedd4Tg/+) mice and conditional knockout (Nedd4-cKO, Col1α1-Cre;Nedd4fl/fl) mice. The Nedd4-TG mice displayed enhanced bone mass accrual and upregulated gene expression of osteogenic markers in bone. In addition, bone formation was decreased in the Nedd4-cKO mice compared to that in their littermates. The proliferation of primary osteoblasts isolated from calvaria and the number and surface area of tibial osteoblasts were higher in the Nedd4-TG mice than those in their littermates. Throughout the osteoblast differentiation, the expression of Nedd4 and Tgfb1 were high at early stage of osteoblast maturation, but decreased at the later stage when Bmp2 expression level is high. TGFβ1 signaling was consolidated by degradation of pSMAD1, which was transiently induced by TGFβ1, in NEDD4-overexpressing osteoblasts. Furthermore, pERK1/2 signaling was enhanced in osteoblast from TG mice than those in their littermates. These results suggest that NEDD4 enhances osteoblast proliferation by removing pSMAD1 activated by TGFβ1, and potentiating pSMAD2 and pERK1/2 pathways at early stage of bone formation.