Caveolin-1-deficient aortic smooth muscle cells show cell autonomous abnormalities in proliferation, migration, and endothelin-based signal transduction

Abstract

We previously showed that ablation of caveolin-1 (Cav-1) gene expression in mice promotes neointimal hyperplasia in vivo, a phenomenon normally characterized by smooth muscle cell (SMC) migration and proliferation. Whether these defects are cell autonomous, i.e., due to loss of Cav-1 within SMCs or loss of Cav-1 expression in other adjacent cell types in vivo, remains unknown. Cav-1 has been shown to associate with receptors for many vasoactive factors on the SMC surface. Therefore, Cav-1 might be an important regulator of SMC proliferation, migration, and signal transduction. To mechanistically dissect the role of Cav-1 in SMC signaling, we isolated SMCs from the aortas (AoSMCs) of Cav-1-deficient (Cav-1−/−) mice and characterized these cells with respect to their proliferation, migration, and Ca2+response to an important vasoactive factor, endothelin-1 (ET-1). 5-Bromo-2′-deoxyuridine incorporation and a wound-healing assay showed an increase in proliferation and migration rates in Cav-1−/−compared with wild-type (Cav-1+/+) AoSMCs. Cav-1−/−AoSMCs demonstrated upregulation of phosphorylated ERK1/2, cyclin D1, and proliferating cell nuclear antigen and reduced expression of the cyclin-dependent kinase inhibitor p27Kip1. The Ca2+response was examined in the presence of ET-1 and assessed by confocal microscopy with the Ca2+-sensitive fluorescent probe fluo 3. When treated with ET-1, Cav-1−/−AoSMCs exhibited a faster and larger increase in free intracellular Ca2+than Cav-1+/+cells. The ET-1-induced response in Cav-1−/−cells was mediated by the ETBreceptor, as shown using the ETBreceptor antagonist BQ-788 and the ETAreceptor antagonist BQ-123. In Cav-1−/−cells, ETAreceptor expression was reduced and ETBreceptor expression was upregulated. Therefore, Cav-1 ablation increased the ET-1-induced Ca2+response in SMCs by altering the type and expression level of the ET receptor (i.e., receptor isoform switching). These data suggest a novel regulatory role for Cav-1 in SMCs with respect to their proliferation, migration, and Ca2+-mediated signaling.