Interaction of H2S and Estrogen on Proliferation Figure 5. Cyclin D1 mediates estrogen-regulated cell proliferation in WT-SMCs and CSE-KO-SMCs. A, Overexpression of cyclin D1 in WT- SMCs. The cells were transfected with the vector pCMV-Cyclin D1 or control vector pShuttle-CMV for 24 h. * p,0.05. B, Knockdown of cyclin D1 in CSE-KO-SMCs. The cells were transfected with cyclin D1-specific siRNA (siRNA cyclin D1) or control siRNA (siRNA C) at 50 nM for 48 h. * p,0.05. C, Overexpression of cyclin D1 reversed estrogen-inhibited cell viability in WT-SMCs. The cells were transfected with pCMV-Cyclin D1 or control vector for 24 h following another 48 h treatment with 100 nM estrogen. * p,0.05 vs. control vector+control group, # p,0.05 vs. control vector+estrogen group. D, Knockdown of cyclin D1 reversed estrogen-induced cell viability in CSE-KO-SMCs. The cells were transfected with siRNA cyclin D1 or siRNA C at 50 nM for 12 h following another 36 h treatment with 100 nM estrogen. * p,0.05 vs. siRNA C control group, # p,0.05 vs. siRNA C+estrogen group. All experiments were repeated for four times. doi:10.1371/journal.pone.0041614.g005 tion function-1 (AF-1) domain of ERa and ERb has only 30% homology, and the hormone-binding (HBD) domains of ERa and ERb have only 53% homology [29]. Here, we demonstrated that the expression of ERa was significantly reduced in the absence of CSE expression but induced by exogenously applied H2S. However, CSE deficiency had no effect on the expression of ERb, suggesting that H2S may regulate ERa expression by targeting at the AF-1 or HBD domain. It is not clear at this moment how H2S regulates ERa expression. H2S can post- translationally modify the proteins by S-sulfhydration [30,31], and it is possible that H2S may S-sulfhydrate some transcript factors or proteins, which subsequently stimulate ERa expression. Altered ERa expression or activity contributes to different regulatory roles of estrogen in cell growth between WT-SMCs and CSE-KO-SMCs. First, blockage of ERs activity by ICI 182780 reversed the anti-growth effect of estrogen on WT-SMCs (Figure 3A). Second, supplement of WT-SMCs with exogenously applied H2S at 100 mM aggravated the anti-growth effect of estrogen (Figure 2D). Third, when ERa expression was knock- down by ERa-specific siRNA in WT-SMCs, estrogen had no effect on SMC growth (Figure 3, B and D). Fourth, overexpression PLoS ONE | www.plosone.org 8 August 2012 | Volume 7 | Issue 8 | e41614