Specificity Protein-1 as a Critical Regulator of Human Cystathionine γ-Lyase in Smooth Muscle Cells

Cystathionine gamma-lyase (CSE) is the major enzyme in vascular smooth muscle cells (SMCs) that catalyzes the endogenous production of H2S. Phenotypic switching of SMCs is affected by endogenous H2S level and alterations of this switching may result in vascular disorders. To date, the mechanisms underlying the alteration of CSE expression and H2S production in vascular proliferative diseases have been unclear. In the present study, we found that serum deprivation induced SMC differentiation marker gene expressions and increased CSE expression and H2S production in cultured human aorta SMCs (HASMCs). Carotid artery ligation in mice resulted in enhanced neointima formation and down-regulation of CSE expression, suggesting an important role of CSE in SMC differentiation. Transient transfection of HASMCs with human CSE (hCSE) promoter/luciferase reporter revealed that the region between -226 to + 140 base pair contains the core promoter for the hCSE gene. Deletion and mutation analysis demonstrated that two specificity protein-1 (Sp1) consensus binding sites were present in the core promoter region of the hCSE gene. Incubation of HASMCs with Sp1 binding inhibitor mithramycin inhibited CSE mRNA expression in a dose-dependent manner. Overexpression of Sp1 alone was sufficient to increase the activity of the hCSE core promoter and CSE protein expression. Chromatin immunoprecipitation assay showed that the binding of Sp1 to the hCSE promoter was increased in differentiated HASMCs compared with that in proliferated HASMCs. Exogenously applied H2S at 100 mu M stimulated SMC differentiation, which was reversed by p38 MAPK inhibitor SB203580. These results suggest that transcript factor Sp1 is a critical regulator of the hCSE expression during SMC differentiation, and CSE/H2S system is essential for maintenance of SMC phenotype.