Abstract
BackgroundVascular 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 TGFβ has been implicated to play a role in the pathogenesis of atherosclerosis through numerous downstream signaling pathways. We showed previously that TGFβ upregulates CRP2 expression; however, the detailed signaling mechanisms remain unclear.ResultsTGFβ treatment of VSMCs activated both Smad2/3 and ATF2 phosphorylation. Individually knocking down Smad2/3 or ATF2 pathways with siRNA impaired the TGFβ induction of CRP2, indicating that both contribute to CRP2 expression. Inhibiting TβRI kinase activity by SB431542 or TβRI knockdown abolished Smad2/3 phosphorylation but did not alter ATF2 phosphorylation, indicating while Smad2/3 phosphorylation was TβRI-dependent ATF2 phosphorylation was independent of TβRI. Inhibiting Src kinase activity by SU6656 suppressed TGFβ-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 TGFβ-induced ATF2 (but not Smad2) phosphorylation and CRP2 protein expression while ROCK inhibition blocked JNK activation. These results indicate that downstream of TβRII, Src family kinase-RhoA-ROCK-JNK signaling pathway mediates TβRI-independent ATF2 activation. Promoter analysis revealed that the TGFβ induction of CRP2 was mediated through the CRE and SBE promoter elements that were located in close proximity.ConclusionsOur results demonstrate that two signaling pathways downstream of TGFβ 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 TGFβ.
Highlights
Vascular smooth muscle cells (VSMCs) of the arterial wall play a critical role in the development of occlusive vascular diseases
We identified in a previous study that TGFβ induces Cysteine-rich protein 2 (CRP2) expression in VSMCs and TGFβ treatment reduces wild type but not Mouse CRP2 gene symbol (Csrp2)-deficient VSMC migration, demonstrating the functional importance of CRP2 induction by TGFβ in regulating VSMC migration [13]
Wortmannin or LY294002 did not affect ATF2 or Smad2/3 phosphorylation (Figure 1B). These results suggest that in VSMCs, TβRI mediates TGFβ activation of Smad2/3 whereas neither TβRI kinase activity nor PI3K signaling is involved in TGFβ dependent stimulation of ATF2
Summary
Vascular smooth muscle cells (VSMCs) of the arterial wall play a critical role in the development of occlusive vascular diseases. Vascular smooth muscle cells (VSMCs) in the tunica media of the arteries play important roles in regulating blood pressure and vascular tone. In normal vessels, these VSMCs exhibit a quiescent and differentiated phenotype and express proteins involved in the contractile functions such as Cysteine-rich protein (CRP) 2, a LIM-only CRP family member [2], is highly expressed in VSMCs [3,4]. Our recent study determined that CRP2 sequesters the scaffold protein p130Cas at focal adhesions, controls lamellipodia formation and reduces cell motility These CRP2-p130Cas complexes function to blunt VSMC migration [5]. Maintaining or upregulating CRP2 expression during vascular injury might serve as a protective mechanism against intimal thickening
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