Interaction of Hydrogen Sulfide and Estrogen on the Proliferation of Vascular Smooth Muscle Cells

Hydrogen sulfide (H2S) 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 H2S 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 H2S 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 H2S mediates estrogen-inhibited proliferation of SMCs via selective activation of ERα/cyclin D1 pathways.


Introduction
Acquired proliferative phenotype of vascular smooth muscle cells (SMCs) is associated with development and progression of numerous vascular proliferative conditions, such as primary atherosclerosis and post-angioplasty restenosis [1]. Women generally experience initial manifestations of coronary artery disease 10 years later than men, suggesting that estrogen may offer a cardiovascular protection [2]. It is well known that estrogen inhibits the proliferation of SMCs [3]. Estrogen can bind to estrogen receptors (ERs), including ERa and ERb, which are expressed in all vascular cell types and appear to mediate the inhibitive effects of estrogen on SMC proliferation [3,4].
Estrogen activates many intracellular signaling responses [5]. Estrogen-stimulated mitogen-activated protein kinase (MAPK) cascade plays a key role in the cellular signal transduction pathway in response to vascular stimuli [6]. MAPK family in mammalian cells consists of three major members including ERK (an extracelluar signal-regulated kinase), JNK (c-Jun Nterminal kinase) and p38 MAPK [7]. Each of these MAPK plays a unique role in the regulation of gene expression and intracellular metabolism related to growth and development, apoptosis, and cellular responses to external stresses [7]. Cyclin D1 and its associated cyclin-dependent kinases (CDK4 and 6) are key regulatory proteins in controlling the reentry of quiescent cells from G0 into G1 [8]. Cyclin D1 was also reported to mediate the inhibitory effect of estrogen on cell proliferation [9].
Hydrogen sulfide (H 2 S) was traditionally viewed as a toxic gas detected in the contaminated environmental atmosphere [10]. Over the last decade, physiological importance of endogenously produced H 2 S has been realized [11]. Two key enzymes in the transsulfuration pathway, cystathionine beta-synthase (CBS) and cystathioine gamma-lyase (CSE), produce H 2 S, pyruvate and ammonium using homocysteine and/or L-cysteine as substrates [12,13]. In some tissues, both CBS and CSE function to catalyze H 2 S production. CSE is the major H 2 S-producing enzyme in vascular tissues [14]. As an important gasotransmitter in the cardiovascular system, H 2 S has important physiological functions, such as anti-atherosclerosis, anti-inflammatory, vasodilatation, protection of ischemia injury, and antioxidant effects [12,[14][15][16].
Although both estrogen and H 2 S can inhibit SMC proliferation [3,4,7,8,13], the interaction of H 2 S and estrogen on SMC growth has been unknown. In the present study, we compared the effects of estrogen on the proliferation of SMCs from CSE knockout mice (CSE-KO-SMCs) and those from wild-type mice (WT-SMCs). The underlying cellular signaling mechanisms, including the activation of ERa, cyclin D1 and MAPK pathways, were further explored.

Materials
Estrogen (17b-estradiol) was from Sigma (St. Louis, MO). The anti-CSE antibody was purchased from Novus Biologicals (Littleton, CO). The anti-MAPK antibodies and different MAPK inhibitors were obtained from New England Biolabs (Camarillo, CA). Anti-cyclin D1 antibody was from Lab Vision Corporation (Fremont, CA). Anti-ERa, ERb antibodies and siRNA for ERa as well as control siRNA were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Cyclin D1 siRNA and control siRNA were purchased from Ambion (Austin, TX). The plasmids pCMV-Cyclin D1 and pEGFP-C1-ERa were from Addgene (Cambridge, MA). Horseradish peroxidase-conjugated goat antirabbit IgG antibody and goat anti-mouse IgG antibody were from Sigma. All other chemicals were from Sigma or New England Biolabs.

Isolation and Culture of SMCs
Twelve-week-old male CSE-KO offspring and age matched male WT littermates on C57BL/6J6129SvEv background were used to isolate primary SMCs from mesenteric arteries [17]. All animals were maintained on standard rodent chow and had free access to food and water. All animal experiments were conducted in compliance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and approved by the Animal Care Committees of Lakehead University, Canada. In brief, 12week male WT and CSE-KO mice were generally anaesthetized by intraperitoneal injection with a mixture of 10 mg/kg xylazine (Bimeda-MTC Animal Health, CA) and 100 mg/kg ketamine hydrochloride (BIONICHE Animal Health, CA). When mice have lost muscle tone and blink reflexes, pupils become fixed and respiration is regular, the operation was carried out. Mice were killed by decapitation. Small mesenteric arteries below the second branch of the main mesenteric artery were dissected out and kept in ice-cold physiological salt solution (PSS) containing (in mM) NaCl 137, KCl 5.6, NaH 2 PO 4 0.4, Na 2 HPO 4 0.4, NaHCO 3 4.2, MgCl 2 1, CaCl 2 2.6, Hepes 10, and glucose 5 with pH adjusted to 7.4 with NaOH. The small arteries were cut into 5 mm-long pieces, and incubated at 37uC in low-Ca 2+ PSS (0.1 mM CaCl 2 ) containing 1 mg/ml albumin, 0.5 mg/ml papain, and 1 mg/ml dithioerythritol for 30 min, and for another 20 min in the Ca 2+free PSS containing 1 mg/ml albumin, 0.8 mg/ml collagenase, and 0.8 mg/ml hyaluronidase. Single cells were released by gentle trituration through a Pasteur pipette, stored in the nominally Ca 2+ -free PSS at 4uC, and used the same day. These primary SMCs were grown in phenol red-free Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 mg/ml streptomycin. The experiments were performed when the cells reached 70-80% confluence between passages 6 and 10. In all studies, cells were first incubated in the serum-free medium for 24 h and then 1% serum added together with different treatments, the media were changed every 2 days.

Cell Viability Assay
Cell viability was determined by the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay as described previously [18]. In brief, equal numbers of cells were seeded into each well of 96-well plates for 24 h. After different treatments, MTT (final concentration, 5 mg/ml) was added to each well, and then the cells were incubated for 4 h at 37uC. The cell culture medium was removed, and dimethyl sulfoxide (200 ml/well) was added. The absorbance of formazan products at 570 nm was measured in a FLUOstar OPTIMA microplate spectrophotometer (BMG LABTEch, Offenburg, Germany). The cells incubated with control medium were considered 100% viable.

Cell Proliferation Assay
Cell proliferation was assessed by bromodeoxyuridine (BrdU) incorporation as described previously [8]. SMCs were plated at equal numbers of cells (2.0610 3 per well) in a 96-well plate for 24 h in the presence of 10% FBS. BrdU label was added into the culture media for another 24 h. Incorporated BrdU was detected following the procedures provided by the manufacturer (Calbiochem, Gibbstown, NJ, USA). For the experiments with estrogen treatments the cells were first seeded in a 96-well plate for 24 h in media containing 10% FBS, and then changed to a serum-free medium for another 24 h. Afterwards, 1% FBS was added back together with estrogen (0.1-1000 nM) for 48 h following BrdU label for additional 24 h.

Measurement of H 2 S Production
H 2 S production rate was measured as described previously [19]. In brief, after different treatments, the cells were collected and homogenized in 50 mM ice-cold potassium phosphate buffer (pH 6.8). The flasks containing the reaction mixture (100 mM potassium phosphate buffer, 10 mM l-cysteine, 2 mM pyridoxal 5phosphate, and 10% cell homogenates) and center wells containing 0.5 ml 1% zinc acetate and a piece of filter paper (262.5 cm) were flushed with N 2 gas and incubated at 37uC for 90 min. The reaction was stopped by adding 0.5 ml of 50% trichloroacetic acid, and the flasks were incubated at 37uC for another 60 min. The contents of the center wells were transferred to test tubes, each containing 3.5 ml of water. Then 0.5 ml of 20 mM N, Ndimethyl-p-phenylenediamine sulfate in 7.2 M HCl and 0.5 ml 30 mM FeCl 3 in 1.2 M HCl was added. The absorbance of the resulting solution at 670 nm was measured 20 min later with a FLUOstar OPTIMA microplate spectrophotometer.

Short Interfering RNA (siRNA) and Plasmid Transfection
Transfection of SMCs by siRNA or plasmids (pCMV-Cyclin D1 and pEGFP-C1-ERa as well as corresponding control plasmid) was achieved by using the Lipofectamine TM 2000 transfection agent from Invitrogen (Burlington, ON). In brief, SMCs were seeded at equal number of cells (2.0610 5 per plate) in 60 mm 2 plates with the medium containing 10% FBS. The cells were plated to form 60-70% confluent monolayers for siRNA transfection, and 80-90% confluence for plasmid transfection. siRNA or plasmid and the transfection reagent complex were added to the serum-free medium for 4 h, and the transfection continued for another 48 h (for siRNA transfection) or 24 h (for plasmid transfection) in serum-containing regular medium. After that, the cells were collected for detection of protein expressions with western blotting analysis.

Western Blotting Analysis
Cells were harvested and lysed. Equal amounts of proteins were boiled and separated with SDS-PAGE and electrophoretically transferred to a nitrocellulose membrane, as described previously [20]. In each lane of a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, equal amounts of proteins were applied, electrophoresed and transferred to a polyvinylidene fluoride membrane. Membranes were blocked with Tris-buffered saline containing 5% non-fat milk at room temperature for 1 h, then incubated overnight at 4uC with primary antibody. The primary antibody dilutions were 1:1,000 for CSE, ERa, ERb and Cyclin D1, phosphorylated or total ERK, p38 MAPK, or JNK, and 1:10,000 for b-actin. The membrane was then washed three times with 16Tris-buffer saline-Tween 20 (TBST) buffer and incubated in TBST solution with horseradish peroxidase-conjugated secondary antibody (diluted 1:5,000) for 1 h at room temperature on a shaker. Finally, the membrane was washed with TBST solution for 3 times. The immunoreactions were visualized with ECL and exposed to x-ray film (Kodak Scientific Imaging film, Kodak, Rochester, NY).

Real-time PCR Analysis
Total RNA was isolated using an RNeasy Mini Kit (Qiagen, Germantown, MD) and converted to cDNA with an iScriptTM cDNA Synthesis Kit (Bio-Rad, Hercules, CA). Real-time PCR was performed in an iCycler iQ5 apparatus (Bio-Rad) associated with the iCycler optical system software (version 3.1) using SYBR Green PCR Master Mix, as described previously [8]. The primers of ERa were 59-ATTCTGACAATCGACGCCAG-39 (forward) and 59-GTGCATTGGTTTGTAGCTGG-39 (reverse). The primers for b-actin were 59-ATGGTGGGAATGGGTCAGAA-39 (forward) and 59-CTTTTCACGGTTGGCCTTAG-39 (reverse). The PCR conditions were as follows: denaturation at 95uC for 60 s, followed by 40 cycles of denaturation at 95uC for 15 s, annealing at 55uC for 15s, and extension at 72uC for 45 s. Relative mRNA quantification was calculated by using the arithmetic formula ''2-DDCT'', where DCT is the difference between the threshold cycle of a given target cDNA and an endogenous reference b-actin cDNA.

Statistical Analysis
All data were expressed as the mean 6 SE and represented at least three independent experiments. Statistical comparisons were made using student's t-test or one-way ANOVA followed by a post hoc analysis (Tukey test) where applicable. Significance level was set at p,0.05.

The Opposite Effects of Estrogen on the Growth of WT-SMCs and CSE-KO-SMCs
Estrogen at 10 to 1000 nM significantly decreased cell viability of WT-SMCs in a dose-dependent manner. However, at 10 to 100 nM estrogen significantly increased cell viability of CSE-KO-SMCs ( Figure 1A and 1B). Cell viability data was further confirmed by cell proliferation analysis (Figure 1C and 1D). At the concentrations higher than 100 nM, the stimulatory effect of estrogen on proliferation of CSE-KO-SMCs could not be detected ( Figure 1D).

Effect of H 2 S on ERa Activation
The physiological functions of estrogen are mostly mediated by estrogen receptors [3,4]. Our results showed that both mRNA and protein expressions of ERa were significant higher in WT-SMCs than that in CSE-KO-SMCs, and treatment with 100 mM NaHS (a H 2 S donor) for 24 h up-regulated ERa mRNA and protein expressions in both WT-SMCs and CSE-KO-SMCs (p,0.05) (Figure 2A and 2B). We further found that ERb protein expression was not changed between WT-SMCs and CSE-KO-SMCs, and NaHS treatment had no effect on the expression of ERb in both cells (p.0.05) ( Figure 2C). In addition, NaHS at 100 mM 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 H 2 S 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]. H 2 S is a novel gasotransmitter and provides cardiovascular protection [7,8,12,15,16,22,23]. Both H 2 S and estrogen can inhibit cell proliferation [3,4,7,8,13]. However, the interaction between estrogen and H 2 S 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 H 2 S normally, but increased that of CSE-KO-SMCs which lack the endogenous H 2 S 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 H 2 S 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/H 2 S system. However, we found that estrogen has little effect on CSE expression and H 2 S production. Therefore, it is endogenous H 2 S level that decides the pro-or anti-growth effect of estrogen on SMCs. However, the mechanism by which H 2 S 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-  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 H 2 S. However, CSE deficiency had no effect on the expression of ERb, suggesting that H 2 S may regulate ERa expression by targeting at the AF-1 or HBD domain. It is not clear at this moment how H 2 S regulates ERa expression. H 2 S can posttranslationally modify the proteins by S-sulfhydration [30,31], and it is possible that H 2 S 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 H 2 S at 100 mM aggravated the anti-growth effect of estrogen ( Figure 2D). Third, when ERa expression was knockdown by ERa-specific siRNA in WT-SMCs, estrogen had no effect on SMC growth (Figure 3, B and D). Fourth, overexpression of ERa overturned the pro-growth effect of estrogen in CSE-KO-SMCs ( Figure 3E). These discoveries indicate the interaction of H 2 S 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 H 2 S or CSE overexpression but induced by CSE deficiency, all of which contribute to the regulatory role of H 2 S on SMC growth [8,13]. Here, we further found that cyclin D1 mediated the anti-growth effect in WT-SMCs and the progrowth effect in CSE-KO-SMCs by estrogen treatments (Figure 4 and 5), pointing to the critical role of endogenous H 2 S in ERamediated cyclin D1 expression following altered cell growth.
In summary, our study for the first time demonstrates that H 2 S mediates estrogen-inhibited proliferation of SMCs via selective activation of ERa/cyclin D1 pathways. The elucidation of the interaction between H 2 S and estrogen on the regulation of SMC growth would provide new insight for prevention and treatment of proliferative cardiovascular disease such as atherosclerosis. The physiological and pathological implications of the interactions between H 2 S and estrogen in proliferative vascular diseases merit further investigation. Figure S1 Estrogen had no effect on CSE expression and H 2 S production. A, The expression of CSE was not changed by estrogen treatments. After WT-SMCs were incubated with the indicated concentration of estrogen for 72 h, the cells were collected and subjected to western blotting with anti-CSE antibody. B, Estrogen did not affect H 2 S production. After WT-SMCs were treated with the indicated concentration of estrogen for 72 h, H 2 S production rate was measured. All the results were representative of five individual experiments.