Further supporting a protective role of TGFB against vascular lesion formation, targeted disruption of TGFB Smad3 signaling enhances neointimal hyperplasia after arterial injury. We identified in a previous study selleck chemicals that TGFB induces CRP2 expression in VSMCs and TGFB treatment reduces wild type but not Csrp2 defi cient VSMC Inhibitors,Modulators,Libraries migration, demonstrating the functional im portance of CRP2 induction by TGFB in regulating VSMC migration. TGFB upregulates CRP2 expression via a CRE promoter element and transcription factor ATF2 . however, the detailed signaling mechanisms underlying TGFB induction of CRP2 remain unclear. The goal of the present study was to delineate the signaling pathways by which TGFB upregulates CRP2 expression, which might provide an opportunity for developing targeted strategies to reduce intimal thickening.

Results TGFB induces CRP2 expression through Smad23 and ATF2 To investigate the signaling pathways that mediate Inhibitors,Modulators,Libraries CRP2 induction by TGFB, we first examined type I TGFB receptor downstream signaling. We pretreated VSMCs with vehicle or TBRI kinase inhibitor SB431542 for 30 min, followed by stimulation with or without TGFB for 24 h and then examined CRP2 expression levels. SB431542 significantly reduced TGFB induced CRP2 expression, indicating TBRI kinase ac tivity is required for TGFB induction of CRP2. It is well established that Smad23 transmits TGFB signaling, thus we examined Smad23 activation. Indeed, TGFB increased phosphorylation levels of Smad2 and Smad3 in VSMCs. In addition, as previously re ported, TGFB also increased ATF2 phosphorylation.

Interestingly, SB431542 blocked TGFB induced activation of Smad2 and Smad3 but did not block ATF2 phosphorylation. PI3K has also been implicated in TGFB signaling, thus we deter mined whether Inhibitors,Modulators,Libraries PI3K pathways participate in this regulation by treating cells with PI3K inhibitors. Wortmannin or LY294002 did not affect ATF2 or Smad23 Inhibitors,Modulators,Libraries phosphorylation. These results suggest that in VSMCs, TBRI mediates TGFB activation of Smad23 whereas neither TBRI kinase activity nor PI3K signaling is involved in TGFB dependent stimulation of ATF2. To define the role of Smad23 and ATF2 in CRP2 upregulation, we used siRNA to suppress their expression. In comparison with control siRNA, knockdown of Smad23 or ATF2 abrogated TGFB induced CRP2 expression, supporting the concept that both Smad23 and ATF2 contribute to CRP2 induction.

ATF2 activation by TGFB is independent of TAK1 and TRAF6 It has been shown in epithelial cells and fibroblasts that in dependent of TBRI kinase activity, TGFB activates TAK1 signaling through interaction of TBRI with TRAF6, whereas Inhibitors,Modulators,Libraries Smad2 activation those is not dependent on TRAF6. Thus, we examined whether TGFB activates ATF2 through TAK1 and TRAF6 in VSMCs. We transfected VSMCs with siRNA to TAK1 or TRAF6, or control siRNA and then stimulated cells with or without TGFB for 10 min.