Transforming growth factor ß suppresses osteoblast differentiation via the vimentin activating transcription factor 4 (ATF4) axis.

ATF4 is an osteoblast-enriched transcription factor of the leucine zipper family. We recently identified that vimentin, a leucine zipper-containing intermediate filament protein, suppresses ATF4-dependent osteocalcin (Ocn) transcription and osteoblast differentiation. Here we show that TGFß inhibits ATF4-dependent activation of Ocn by up-regulation of vimentin expression. Osteoblasts lacking Atf4 (Atf4(-/-)) were less sensitive than wild-type (WT) cells to the inhibition by TGFß on alkaline phosphatase activity, Ocn transcription and mineralization. Importantly, the anabolic effect of a monoclonal antibody neutralizing active TGFß ligands on bone in WT mice was blunted in Atf4(-/-) mice. These data establish that ATF4 is required for TGFß-related suppression of Ocn transcription and osteoblast differentiation in vitro and in vivo. Interestingly, TGFß did not directly regulate the expression of ATF4; instead, it enhanced the expression of vimentin, a negative regulator of ATF4, at the post-transcriptional level. Accordingly, knockdown of endogenous vimentin in 2T3 osteoblasts abolished the inhibition of Ocn transcription by TGFß, confirming an indirect mechanism by which TGFß acts through vimentin to suppress ATF4-dependent Ocn activation. Furthermore, inhibition of PI3K/Akt/mTOR signaling, but not canonical Smad signaling, downstream of TGFß, blocked TGFß-induced synthesis of vimentin, and inhibited ATF4-dependent Ocn transcription in osteoblasts. Thus, our study identifies that TGFß stimulates vimentin production via PI3K-Akt-mTOR signaling, which leads to suppression of ATF4-dependent Ocn transcription and osteoblast differentiation.