Interaction of H2S and Estrogen on Proliferation Figure 6. MAPK were not involved in estrogen-altered cell growth in WT-SMCs and CSE-KO-SMCs. WT-SMCs (A) and CSE-KO-SMCs (B) were pretreated with 10 mM U0126 (a MEK/ERK inhibitor), or 10 mM SB 203580 (a p38 MAPK inhibitor), or 10 mM SP 600125 (a JNK inhibitor) for 30 min followed by 100 nM estrogen for additional 72 h. The cell viability was evaluated by MTT assay. The data were from four independent experiments. * p,0.05 vs. control group; # p,0.05 vs. estrogen group. doi:10.1371/journal.pone.0041614.g006 of ERa overturned the pro-growth effect of estrogen in CSE-KO- SMCs (Figure 3E). These discoveries indicate the interaction of H2S and ERa plays a key role in the effects of estrogen on SMC growth. ERs belong to ligand-activated transcription factors [27,29]. Cyclin D1 is a downstream gene of ERs [9]. The present study found that estrogen at 100 nM significantly inhibited cyclin D1 expression in WT-SMCs, but increased cyclin D1 expression in CSE-KO-SMCs (Figure 4, A and B). Cell cycle progression is determined by the formation of protein complexes among cyclins and cyclin-dependent kinases (cdks), and the association of cyclin D1 with cdk2, cdk4, and cdk6 determines early G1 progression [8]. We have shown before that cyclin D1 is inhibited by exogenously applied H2S or CSE overexpression but induced by CSE deficiency, all of which contribute to the regulatory role of H2S on SMC growth [8,13]. Here, we further found that cyclin D1 mediated the anti-growth effect in WT-SMCs and the pro- growth effect in CSE-KO-SMCs by estrogen treatments (Figure 4 and 5), pointing to the critical role of endogenous H2S in ERa- mediated cyclin D1 expression following altered cell growth. In summary, our study for the first time demonstrates that H2S mediates estrogen-inhibited proliferation of SMCs via selective activation of ERa/cyclin D1 pathways. The elucidation of the interaction between H2S and estrogen on the regulation of SMC growth would provide new insight for prevention and treatment of proliferative cardiovascular disease such as atherosclerosis. The PLoS ONE | www.plosone.org 9 August 2012 | Volume 7 | Issue 8 | e41614