Src Kinase Signaling Regulates Connective Tissue Growth Factor (CTGF/CCN2) Induction by Transforming Growth Factor-Beta 1 (TGF-b1) in Osteoblasts

Abstract

Connective tissue growth factor (CTGF/CCN2) is a cysteine rich, extracellular matrix protein that acts as an anabolic growth factor to regulate osteoblast differentiation and function. In osteoblasts, CTGF is induced by transforming growth factor beta 1 (TGF-β1) where it acts as a downstream mediator of TGF-β1 induced extracellular matrix production. The molecular mechanisms that control CTGF induction by TGF-β1 in osteoblasts are not understood. We have previously demonstrated the requirement of Src, Erk and Smad signaling for TGF-β1 induced CTGF promoter activity in primary osteoblasts, however the potential interaction among these signaling pathways in osteoblasts remains unknown. In this study, we demonstrate that CTGF is induced by TGF-β1 in rat osteosarcoma osteoblast like cells (ROS17/2.8). TGF-β1 activates Src and blocking of Src family kinases by PP2 abrogates TGF-β1 induced CTGF up-regulation. Western blot analysis revealed that primary osteoblasts and ROS 17/2.8 cells express not only Src, but also other Src family members, such as Fyn, Yes and Hck. In order to determine whether CTGF up-regulation is controlled by Src or other members, we used either kinase-dead dominant negative Src constructs in primary osteoblasts or Src siRNA in ROS17/2.8 cells to block Src function. Inactivation of Src by both kinase-dead and siRNA prevented TGF-β1 induced CTGF induction, demonstrating that TGF-β1 induced CTGF up-regulation is mediated only by Src not by other members. In addition, we also demonstrated that Erk is activated by TGF-β1 and that blocking of Erk activation using pharmacological inhibitors, PD98059 and U0126, prevents TGF-β1 induced CTGF induction, demonstrating the requirement of Erk for CTGF induction. These results prompted us to further explore the cross-talk between Src, Erk and Smads in ROS17/2.8 cells. Inhibition of Src using PP2 prevented Erk activation, demonstrating that Src is upstream of Erk. To investigate how Src and Erk regulate the canonical TGF-β1 signaling pathway, including Smad2/3 phosphorylation and nuclear translocation of activated Smads, we treated cells with TGF-β1 in the presence or absence of the Src inhibitor, PP2, or the Erk inhibitors, PD98059 or U0126. PP2 pre-treatment prevented the phosphorylation of Smad2/3 at both the SSXS motif and the linker region and consequently blocked their nuclear translocation, demonstrating that Src can regulate Smad signaling. In contrast, the Erk inhibitors did not have any effects on Smad phosphorylation and/or nuclear translocation. To examine whether Erk can modulate Smad signaling indirectly through the activation/ inactivation of required nuclear coactivators/ co-repressors that mediate Smad DNA binding, we used electro-mobility shift assays. These experiments showed that inhibition of Erk activation impaired transcriptional complex formation on the Smad binding element (SBE) and TGF- β responsive element (TRE) of the CTGF promoter, demonstrating that Erk activation is required for SBE and TRE transactivation. Taking together, these data demonstrate that Src is an essential upstream signaling transducer for Erk and Smad signaling in osteoblasts, and that while the Smad and Erk signaling cascades appear to function independent of each other, they are both essential for the formation of a transcriptionally active complex on the CTGF promoter.