Abstract
Vascular smooth muscle cells (VSMCs) of the arterial wall play a critical role in the development of occlusive vascular lesions. Cysteine-rich protein (CRP) 2, a member of the LIM-only CRP family that contains two LIM domains, plays an important role in vascular remodeling. CRP2 is expressed in VSMCs and functions to reduce vascular lesion formation by inhibiting cellular migration. The goals of this study are (i) to investigate the molecular mechanisms that control CRP2 expression in VSMCs, and (ii) to define the molecular mechanisms by which CRP2 regulates VSMC migration. We previously demonstrated that the 5’-flanking Csrp2 (gene symbol of the mouse CRP2 gene) promoter is sufficient for gene expression in the developing vessels but not sufficient for adult vasculature. In the present study, the first goal was to elucidate the molecular mechanisms that control CRP2 expression in the adult vasculature. By generating and analyzing a series of transgenic mice harboring potential Csrp2 regulatory regions with a lacZ reporter, we determined that the 12-kb first intron was necessary for transgene activity in adult but not in developing vasculature. Within the intron we identified a 6.3-kb region that contains 2 CArG boxes (CC(A/T)6GG). Serum response factor (SRF) preferentially bound to CArG2 box in gel mobility shift and chromatin immunoprecipitation assays; additionally, SRF coactivator myocardin and the related factors activated CRP2 expression via the CArG2 box. Mutational analysis revealed that CArG2 box was important in directing lacZ expression in VSMCs of adult vessels. Although CRP2 expression during development is independent of CArG box regulatory sites, CRP2 expression in adult VSMCs requires CArG2 element within the first intron. Our results suggest that distinct mechanisms regulate CRP2 expression in VSMCs that are controlled by separate embryonic and adult regulatory modules. Given that an absence of CRP2 enhances VSMC migration and increases neointima formation following arterial injury, the second specific goal of this study was to define the molecular mechanisms by which CRP2 regulates VSMC migration. Transfection of VSMCs with CRP2-EGFP constructs revealed that CRP2 is associated with the actin cytoskeleton, suggesting a cytoskeletal function of CRP2. Lack of CRP2 did not affect cell’s ability to adhere to or spread on extracellular matrix. In response to chemoattractant stimulation, Csrp2-deficient (Csrp2–/–) VSMCs exhibited increased lamellipodia formation. Re-introduction of CRP2 abrogated the enhanced lamellipodia formation and migration of Csrp2–/– VSMCs following chemoattractant stimulation. Mammalian two-hybrid and co-immunoprecipitation assays demonstrated that CRP2 interacts with p130Cas, a scaffold protein important for lamellipodia formation and cell motility. Intriguingly, vascular injury modulated p130Cas phosphorylation levels. Furthermore, suppression of p130Cas expression or its phosphorylation attenuated neointima formation following arterial injury, suggesting an important role of p130Cas and its phosphorylation in vascular remodeling. Immunofluorescence staining showed that CRP2 colocalized with phospho-p130Cas at focal adhesions (FAs)/terminal ends of stress fibers in non-migrating cells. Interestingly, in migrating cells phospho-p130Cas localized to the leading edge of lamellipodia and FAs, whereas CRP2 was restricted to FAs and stress fibers. Taken together, our results indicate that CRP2 sequesters p130Cas at FAs, thereby reducing lamellipodia formation and blunting VSMC migration.