Interaction of H2S and Estrogen on Proliferation Figure 3. The role of ERa in the actions of estrogen on the growth of WT-SMCs and CSE-KO-SMCs. A, Blockage of ERs activity reversed estrogen-inhibited cell viability in WT-SMCs. The cells were treated with 10 mM ICI 182780 for 30 min following another 72 h treatment with 100 nM estrogen. * p,0.05 vs. control group; # p,0.05 vs. estrogen group. B, Knockdown of ERa by ERa-specific siRNA (siRNA ERa) in WT-SMCs. The cells were transfected with 50 nM siRNA ERa or negative control siRNA (siRNA C) for 48 h. * p,0.05. C, Overexpression of ERa in CSE-KO-SMCs. The cells were transfected with the plasmid pEGFP-C1-ERa or control plasmid pEGFP-C1 for 24 h. * p,0.05. D, Knockdown of ERa reversed estrogen-decreased cell viability in WT-SMCs. The cells were transfected with siRNA ERa or siRNA C at 50 nM for 12 h following another 36 h treatment with estrogen (100 nM). * p,0.05 vs. siRNA C group; # p,0.05 vs. siRNA C+estrogen group. e, Overexpression of ERa reversed estrogen-increased cell viability in CSE-KO-SMCs. The cells were transfected with pEGFP-C1-ERa plasmid or control plasmid for 24 h following another 48 h treatment with estrogen (100 nM). * p,0.05 vs. control vector group; # p,0.05 vs. control vector+estrogen group. All data were from four independent experiments. doi:10.1371/journal.pone.0041614.g003 potentiated the inhibitory effect of estrogen on cell viability of WT- SMCs (p,0.05) (Figure 2D). On the other hand, neither CSE expression nor H2S production rate was changed by estrogen treatments of WT-SMCs (p.0.05) (Figure S1A and S1B). The Role of ERa in Estrogen-altered Cell Growth of WT- SMCs and CSE-KO-SMCs To explore whether estrogen receptor mediates the differential effects of estrogen on the growth of WT-SMCs and CSE-KO- SMCs, pretreatment of WT-SMCs with ICI 182780 (an ERs inhibitor) significantly reversed estrogen-inhibited cell viability (p,0.05), and ICI 182780 alone had no effect on cell viability (Figure 3A). Next we manipulated endogenous expression levels of ERa in both WT-SMCs and CSE-KO-SMCs. Compared with siRNA control group, siRNA ERa at 50 nM significantly inhibited ERa protein expression in WT-SMCs (p,0.05) (Figure 3B). The transfection of pEGFP-C1-ERa significantly overexpressed ERa protein in CSE-KO-SMCs when compared with pEGFP control vector (p,0.05) (Figure 3C). We further found that knockdown of ERa in WT-SMCs attenuated estrogen-inhibited cell viability in WT-SMCs, but overexpression of ERa in CSE-KO-SMCs reversed estrogen-enhanced cell viability (p,0.05) (Figure 3D and 3E). Estrogen-induced Down-regulation of Cyclin D1 in WT- SMCs but up-regulation in CSE-KO-SMCs Cyclin D1 plays a critical role in the transition and progress of the cell cycle [8,13]. Incubation of WT-SMCs with 100 nM estrogen for 72 h significantly decreased cyclin D1 expression (p,0.05) (Figure 4A). Quite differently, estrogen significantly increased the expression of cyclin D1 in CSE-KO-SMCs (Figure 4B). Knockdown of ERa in WT-SMCs or overexpression of ERa in CSE-KO-SMCs markedly reversed estrogen-altered cyclin D1 expression in respective cells (p,0.05) (Figure 4C and 4D). Furthermore, overexpression of cyclin D1 in WT-SMCs (Figure 5A) or knockdown of cyclin D1 in CSE-KO-SMCs (Figure 5B) reversed the effect of estrogen on cell viability (Figure 5C and 5D). Estrogen Activated MAPK in WT-SMCs and CSE-KO-SMCs To determine whether estrogen differentially activates MAPK signaling pathways between WT-SMCs and CSE-KO-SMCs, we treated both cells with 100 nM estrogen for different durations. Estrogen treatment resulted in strong activation of ERK, p38 MAPK and JNK in both WT-SMCs and CSE-KO-SMCs (Figure S2A, S2B, S2C). The total amount of MAPK protein remained unchanged with estrogen stimulation. To determine whether MAPK are involved in estrogen-altered cell viability in WT-SMCs and CSE-KO-SMCs, U0126 (a selective inhibitor of the MEK/ ERK signaling pathway), SB203580 (a p38 MAPK inhibitor), SP600125 (a JNK inhibitor), and ICI 182780 were used in the following experiments. We first validated that treatment of WT- SMCs and CSE-KO-SMCs with MAPK inhibitors significantly suppressed estrogen-induced phosphorylation of ERK, p38 MAPK and JNK (Figure S3A, S3B, S3C). Blockage of ER activation by ICI 182780 significantly inhibited estrogen-induced phosphorylation of MAPK (Figure S3A, S3B, S3C). SB203580 and SP600125 did not affect estrogen-changed cell viability, but U0126 decreased the viability of both WT-SMCs and CSE-KO- SMCs (Figure 6A and 6B). We further found that inhibition of ERK phosphorylation did not affect estrogen-altered expression of cyclin D1 in both WT-SMCs and CSE-KO-SMCs (Figure S4A and S4B). Therefore, MAPK may not be involved in the effects of estrogen on the growth of both WT-SMCs and CSE-KO-SMCs. Discussion The incidence of proliferative cardiovascular diseases is higher among men than women, but it rises in aging women after the menopause, suggesting a protective role of estrogen [2,21]. H2S is a novel gasotransmitter and provides cardiovascular protection [7,8,12,15,16,22,23]. Both H2S and estrogen can inhibit cell proliferation [3,4,7,8,13]. However, the interaction between estrogen and H2S as well as its effects on cardiovascular function are not clear. Estrogen has been used in previous studies at concentrations from 1 pM to 1000 nM [1,24,25]. Our present study uses estrogen at the similar range and in most of our experiments 100 nM estrogen was used. In this study, we demonstrated that estrogen significantly reduced the growth of WT-SMCs which produce H2S normally, but increased that of CSE-KO-SMCs which lack the endogenous H2S production (Figure 1). High dose of estrogen (1000 nM) inhibited both cell proliferations, suggesting very higher concentration of estrogen displays a toxic effect and kills the cells via a nonspecific pathway. It is also well known that estrogen at 1000 nM is far beyond of pathophysiological range. We previously showed that over-produced H2S derived from CSE overexpression reduced cell growth and CSE deficiency led to a significant increase in the growth of cultured SMCs [7,8,26]. Estrogen may inhibit cell growth by stimulating CSE/H2S system. However, we found that estrogen has little effect on CSE expression and H2S production. Therefore, it is endogenous H2S level that decides the pro- or anti-growth effect of estrogen on SMCs. However, the mechanism by which H2S assumes this switch role is not clear. It is generally accepted that estrogen receptors (ERs) mediate the anti-growth effect of estrogen on SMCs [9,27]. Two types of estrogen receptor exist: ERs and estrogen G protein-coupled receptor (GPER) [27,28]. ERs include ERa and ERb, which are the members of the nuclear hormone family of intracellular receptors [27]. GPER belong to G protein-coupled receptor [28]. GPER is a new discovered ER at the moment and need to explored in the future. In present study, we only observed the change of ERa and ERb, both of which are DNA-binding transcription factors and expressed in SMCs [27]. Estrogen has been shown to regulate gene transcription by activating both ERa and ERb [9,27,29]. ERb differs from ERa in 2 important functional domains [29]. The N-terminus containing the activa- PLoS ONE | www.plosone.org 6 August 2012 | Volume 7 | Issue 8 | e41614