Interaction of hydrogen sulfide and estrogen on the proliferation of vascular smooth muscle cells

Abstract

Hydrogen sulfide (H(2)S) can be endogenously generated from cystathionine gamma-lyase (CSE) in cardiovascular system, offering a cardiovascular protection. It is also known that the lower risk of cardiovascular diseases in female is partially attributed to the protective effect of estrogen. The current study explores the interaction of H(2)S and estrogen on smooth muscle cell (SMC) growth. In the present study, we found that the proliferation of cultured vascular SMCs isolated from wild-type mice (WT-SMCs) was inhibited, but that from CSE gene knockout mice (CSE-KO-SMCs) increased, by estrogen treatments. The expression of estrogen receptor α (ERα), but not ERβ, was significantly decreased in CSE-KO-SMCs compared with that in WT-SMCs. Exogenously applied H(2)S markedly increased ERα but not ERβ expression. In addition, the inhibition of ER activation and knockdown of ERα expression in WT-SMCs or the overexpression of ERα in CSE-KO-SMCs reversed the respective effects of estrogen on cell proliferation. The expression of cyclin D1 was reduced in WT-SMCs but increased in CSE-KO-SMCs after estrogen treatments, which was reversed by knockdown of ERα in WT-SMCs or overexpression of ERα in CSE-KO-SMCs, respectively. The overexpression of cyclin D1 in WT-SMCs or knockdown of cyclin D1 expression in CSE-KO-SMCs reversed the effects of estrogen on cell proliferation. These results suggest that H(2)S mediates estrogen-inhibited proliferation of SMCs via selective activation of ERα/cyclin D1 pathways.

Figure 1. Inhibition of WT-SMC growth but promotion of CSE-KO-SMC growth by estrogen.

WT-SMCs and CSE-KO-SMCs were treated with estrogen at the indicated concentrations for 72 h, and then cell viability and cell proliferation were measured. Cell viability of WT-SMCs (A) and CSE-KO-SMCs (B) were measured by MTT assay and cell proliferation of WT-SMCs (C) and CSE-KO-SMCs (D) were analyzed by BrdU incorporation assay. The control cells without estrogen treatment were considered as 100% viable. Data were from five independent experiments. * p<0.05 vs. control group.

Figure 2. The effect of H₂S on ERα and ERβ expression. A

, NaHS up-regulated the protein expressions of ERα in both WT-SMCs and CSE-KO-SMCs. The cells were treated with NaHS at 100 µM for 24 h. * p<0.05 vs. WT-SMCs. B, NaHS up-regulated the expression of ERα mRNA in both WT-SMCs and CSE-KO-SMCs. The cells were treated with NaHS at 100 µM for 24 h. * p<0.05 vs. WT-SMCs. C, NaHS had no effect on the protein expressions of ERβ in both WT-SMCs and CSE-KO-SMCs. D, NaHS potentiated the inhibitory effect of estrogen on the growth of WT-SMCs. After WT-SMCs were treated with 100 nM estrogen for 72 h in the presence or absence of 100 µM NaHS, cell viability was measured. * p<0.05 vs. control group; ^# p<0.05 vs. estrogen group. All data were from three independent experiments.

Figure 3. The role of ERα 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 µM 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 ERα by ERα-specific siRNA (siRNA ERα) in WT-SMCs. The cells were transfected with 50 nM siRNA ERα or negative control siRNA (siRNA C) for 48 h. * p<0.05. C, Overexpression of ERα in CSE-KO-SMCs. The cells were transfected with the plasmid pEGFP-C1-ERα or control plasmid pEGFP-C1 for 24 h. * p<0.05. D, Knockdown of ERα reversed estrogen-decreased cell viability in WT-SMCs. The cells were transfected with siRNA ERα 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 ERα reversed estrogen-increased cell viability in CSE-KO-SMCs. The cells were transfected with pEGFP-C1-ERα 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.

Figure 4. Estrogen altered the expression of cyclin D1.

Estrogen reduced the expression of cyclin D1 in WT-SMCs (A) but up-regulated it in CSE-KO-SMCs (B). The cells were treated with 100 nM estrogen for 72 h. * p<0.05. C, Knockdown of ERα reversed estrogen-decreased expression of cyclin D1 in WT-SMCs. After transfection with siRNA ERα or siRNA C at 50 nM for 12 h, the cells were incubated with 100 nM estrogen for another 36 h. * p<0.05 vs. siRNA C group; ^# p<0.05 vs. siRNA C or siRNA C+estrogen group. D, Overexpression of ERα reversed estrogen-increased expression of cyclin D1 in CSE-KO-SMCs. After transfection with pEGFP-C1-ERα plasmid or control plasmid for 24 h, the cells were incubated with 100 nM estrogen for another 48 h. * p<0.05 vs. control vector group; ^# p<0.05 vs. control vector or control vector+estrogen group. All data were from three independent experiments.

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.

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 µM U0126 (a MEK/ERK inhibitor), or 10 µM SB 203580 (a p38 MAPK inhibitor), or 10 µM 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.