Inhibitory Effects of CGRP on Vascular Smooth Muscle Cell Proliferation: Role of Caveolae/caveolin-1/ERK 1/2 Signal Pathway

Abstract

Caveolae and caveolin-1 participate in the transportation of cholesterol and cell signal transduction.Our previous studies showed that the inhibitory effect of calcitonin gene-related peptide(CGRP) on the vascular smooth muscle cells(VSMCs) was related to decreasing the activity of extracellular signal-regulated kinase(ERK) 1/2 and increasing the expression of caveolin-1.In the present study,we investigated the role of caveolae and caveolin-1 in proliferation of VSMCs and whether there are interaction between the caveolin-1 and ERK 1/2 in the inhibitory effect of CGRP signal pathway.VSMCs were prepared from thoracic aorta of male Sprague-Dawley rat by the classic explants method,the passage 3 ~ 10 VSMCs were used for the present study.10% fetal bovine serum(FBS) was employed as a stimulus for the proliferation of VSMCs.β-Cyclodextrin or filipin was used to deplete cholesterol in the caveolae.Proliferation of VSMCs was estimated by methylthiazoletrazolium(MTT) assay and Flow Cytometry.Western blotting and co-immunoprecipitation were used to determine interaction of p-ERK 1/2 or caveolin-1.Results showed that CGRP significantly inhibited VSMC proliferation and down-regulated phosphorylation of ERK 1/2.Incubation of VSMCs with β-cyclodextrin or filipin promoted cells proliferation,up-regulated phosphorylation of ERK 1/2,attenuated the inhibitory action of CGRP on VSMC proliferation and decreased caveolin-1 expression.Pretreatment with CGRP increased the direct binding of cavolin-1 with phosphorylated(p-) ERK 1/2 but not non-phosphorylated ERK 1/2 in the presence of 10%FBS.Our results revealed that caveolae and caveolin-1 may contribute to the inhibitory effect of CGRP on the VSMC proliferation,and the mechanism may be related to the deceased nuclei translocation of p-ERK 1/2 because of the increased binding of caveolin-1 with p-ERK 1/2.