Divergent signaling pathways cooperatively regulate TGFbeta induction of cysteine-rich protein 2 in vascular smooth muscle cells

[[abstract]]BACKGROUND:Vascular smooth muscle cells (VSMCs) of the arterial wall play a critical role in the development of occlusive vascular diseases. Cysteine-rich protein 2 (CRP2) is a VSMC-expressed LIM-only protein, which functionally limits VSMC migration and protects against pathological vascular remodeling. The multifunctional cytokine TGFbeta has been implicated to play a role in the pathogenesis of atherosclerosis through numerous downstream signaling pathways. We showed previously that TGFbeta upregulates CRP2 expression; however, the detailed signaling mechanisms remain unclear.RESULTS:TGFbeta treatment of VSMCs activated both Smad2/3 and ATF2 phosphorylation. Individually knocking down Smad2/3 or ATF2 pathways with siRNA impaired the TGFbeta induction of CRP2, indicating that both contribute to CRP2 expression. Inhibiting TbetaRI kinase activity by SB431542 or TbetaRI knockdown abolished Smad2/3 phosphorylation but did not alter ATF2 phosphorylation, indicating while Smad2/3 phosphorylation was TbetaRI-dependent ATF2 phosphorylation was independent of TbetaRI. Inhibiting Src kinase activity by SU6656 suppressed TGFbeta-induced RhoA and ATF2 activation but not Smad2 phosphorylation. Blocking ROCK activity, the major downstream target of RhoA, abolished ATF2 phosphorylation and CRP2 induction but not Smad2 phosphorylation. Furthermore, JNK inhibition with SP600125 reduced TGFbeta-induced ATF2 (but not Smad2) phosphorylation and CRP2 protein expression while ROCK inhibition blocked JNK activation. These results indicate that downstream of TbetaRII, Src family kinase-RhoA-ROCK-JNK signaling pathway mediates TbetaRI-independent ATF2 activation. Promoter analysis revealed that the TGFbeta induction of CRP2 was mediated through the CRE and SBE promoter elements that were located in close proximity.CONCLUSIONS:Our results demonstrate that two signaling pathways downstream of TGFbeta converge on the CRE and SBE sites of the Csrp2 promoter to cooperatively control CRP2 induction in VSMCs, which represents a previously unrecognized mechanism of VSMC gene induction by TGFbeta