Canonical and noncanonical TGF-β signaling regulate fibrous tissue differentiation in the axial skeleton

Previously, we showed that embryonic deletion of TGF-{beta} type 2 receptor in mouse sclerotome resulted in defects in fibrous connective tissues in the spine. Here we investigated how TGF-{beta} regulates expression of fibrous markers: Scleraxis, Fibromodulin and Adamtsl2. We showed that TGF-{beta} stimulated expression of Scleraxis mRNA by two hours and Fibromodulin and Adamtsl2 mRNAs by eight hours of treatment. Regulation of Scleraxis by TGF-{beta} did not require new protein synthesis; however, protein synthesis was required for expression of Fibromodulin and Adamtsl2 indicating the necessity of an intermediate. We subsequently showed Scleraxis was a potential intermediate for TGF-{beta}-regulated expression of Fibromodulin and Adamtsl2. The canonical effector Smad3 was not necessary for TGF-{beta}-mediated regulation of Scleraxis. Smad3 was necessary for regulation of Fibromodulin and Adamtsl2, but not sufficient to super-induce expression with TGF-{beta} treatment. Next, the role of several noncanonical TGF-{beta} pathways were tested. We found that ERK1/2 was activated by TGF-{beta} and required to regulate expression of Scleraxis, Fibromodulin, and Adamtsl2. Based on these results, we propose a model in which TGF-{beta} regulates Scleraxis via ERK1/2 and then Scleraxis and Smad3 cooperate to regulate Fibromodulin and Adamtsl2. These results define a novel signaling mechanism for TGF{beta}-mediated fibrous differentiation in sclerotome.