TRAF6 knockdown did not affect TGFB induced ATF2 or Smad2 phosphorylation. Moreover, knocking down TRAF6 did selleck kinase inhibitor not Inhibitors,Modulators,Libraries abrogate TGFB induction of CRP2 protein expression after 24 h. TBRI kinase independent ATF2 activation by TGFB Upon ligand binding, TBRII recruits TBRI into Inhibitors,Modulators,Libraries an active heterotetrameric signaling complex and transphosphory lates TBRI to activate its kinase function. TBRI can then phosphorylate Inhibitors,Modulators,Libraries Smad23 for TGFB signaling. Inter estingly, we found that inhibiting TBRI kinase activity blocked TGFB induced Smad23 but not ATF2 activation. This ATF2 phosphorylation is not mediated through TRAF6 either. To confirm further that ATF2 phosphorylation is independent of TBRI, we knocked down TBRI expression using siRNA.

Silencing of TBRI ab rogated TGFB induced Smad2 phosphorylation but not ATF2 phosphorylation, suggesting indeed TBRI is not required for TGFB induced ATF2 activation. Type III TGFB receptor has been demon strated to bind Inhibitors,Modulators,Libraries and present TGFB to type II TGFB recep tor, thereby increases type II receptor binding affinity and cell responsiveness to TGFB. To deter mine whether TBRIII participates in the ATF2 activation, we knocked down TBRIII levels in VSMCs by TBRIII siRNA, cells were then stimulated with or without TGFB. Compared with control siRNA, knocking down TBRIII levels did not affect the activation of ATF2 or Smad2 at the 10 min early time point or af fecting CRP2 protein expression 24 h later. These results suggest that TBRIII did not affect TGFB signaling in the CRP2 induction. We then knocked down TBRII expression by siRNA and exam ined phosphorylation Inhibitors,Modulators,Libraries of these signaling molecules.

Silen cing TBRII not only reduced ATF2 activation but also that of Smad2, indicating the critical import ance of TBRII in mediating TGFB signaling. To further assess the role of TBRII, we transfected VSMCs with a dominant negative type II receptor HA TBRII, which has an HA tag at the N terminus but lacks selleck chemical expected but not the activation of ATF2. In addition, TBRII attenuated TGFB induced CRP2 expression 24 h later. Src family kinase mediates TBRII dependent TGFB activation of RhoA ROCK and ATF2 in VSMCs We next investigated how TBRII mediates ATF2 activation independent of TBRI in VSMCs. In JEG3 choriocarcinoma cells, Src kinase is involved in TGFB induced early signaling. In addition, c Src has been reported to mediate TGFB induced SMC gene expression. We therefore examined whether in VSMCs Src activation was required to transmit TGFB signaling for ATF2 activation. The selective Src fam ily kinase inhibitor SU6656 dose dependently abrogated TGFB induced ATF2 activation. In contrast, SU6656 did not alter Smad2 phosphorylation by TGFB even at high doses.