Inhibition of Clcn3 promoter activity by Myocyte Enhancer Factor‐2 (MEF‐2C) contributes to the control of vascular smooth muscle differentiation

Reduction of ClC‐3 anion channel expression inhibits vascular smooth muscle proliferation ( Circ. Res. 91:E28–32, 2002). We found that cultured mouse aortic (VSM) cells lacking ClC‐3 ( Clcn3 −/ − ) incorporate tritium‐labeled thymidine and proliferate more slowly than do wild‐type ( Clcn3 +/+ ) cells. They also express more smooth muscle alpha‐actin and myosin heavy chain, suggesting that they are more differentiated. We hypothesized that ClC‐3 expression plays a role in determining a proliferative vs. a differentiated phenotype. The human CLCN3 and murine Clcn3 genes consist of 15 exons, two are alternatively spliced (2 and 13) and one is derived from activation of an alternative promoter (exon 1B) encoding a novel, 26 aa N‐terminus. We analyzed both Clcn3 promoters and created deletion mutants driving a luciferase reporter. Both promoter/enhancer core regions revealed recurrent putative binding sites for MEF‐2, which has been shown to promote muscle cell differentiation. Biotin‐streptavidin pull‐down assays confirmed the binding of endogenous MEF‐2C to both promoters. Over‐expression of MEF‐2C inhibited the promoters in T293 and M1 epithelial cells and in Clcn3 +/+ VSM cells. In contrast, native MEF‐2C was more abundant in Clcn3 −/ − VSM cells and enhanced expression failed to further suppress the activity of the Clcn3 promoters. The ability of MEF‐2 to slow growth and promote differentiation of VSM may be at least in part related to suppression of the expression of ClC‐3, which is required for rapid cell proliferation. HL062483 and an EI from the AHA to FSL .