Interaction of Hydrogen Sulfide and Estrogen on the Proliferation of Vascular Smooth Muscle Cells Hongzhu Li1,2, Sarathi Mani1, Wei Cao3, Guangdong Yang4, Christopher Lai5, Lingyun Wu3, Rui Wang1,2* 1 Department of Biology, Lakehead University, Thunder Bay, Canada, 2 Department of Pathophysiology, Harbin Medical University, Harbin, China, 3 Department of Health Science, Lakehead University, Thunder Bay, Canada, 4 The School of Kinesiology, Lakehead University, Thunder Bay, Canada, 5 Northern Ontario School of Medicine, Lakehead University, Thunder Bay, Canada Abstract 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 a (ERa), but not ERb, was significantly decreased in CSE-KO-SMCs compared with that in WT-SMCs. Exogenously applied H2S markedly increased ERa but not ERb expression. In addition, the inhibition of ER activation and knockdown of ERa expression in WT-SMCs or the overexpression of ERa 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 ERa in WT-SMCs or overexpression of ERa 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 ERa/cyclin D1 pathways. Citation: Li H, Mani S, Cao W, Yang G, Lai C, et al. (2012) Interaction of Hydrogen Sulfide and Estrogen on the Proliferation of Vascular Smooth Muscle Cells. PLoS ONE 7(8): e41614. doi:10.1371/journal.pone.0041614 Editor: Tianqing Peng, University of Western Ontario, Canada Received April 21, 2012; Accepted June 22, 2012; Published August 3, 2012 Copyright: 2012 Li et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by a Discovery Grant from Natural Sciences and Engineering Research Council of Canada. SM was supported by a research fellowship award from Heart and Stroke Foundation of Canada (HSFC). GY was supported by a New Investigator Award from HSFC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: rwang@lakeheadu.ca 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 gener- ally 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 N- terminal 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 (H2S) was traditionally viewed as a toxic gas detected in the contaminated environmental atmosphere [10]. Over the last decade, physiological importance of endogenously produced H2S has been realized [11]. Two key enzymes in the transsulfuration pathway, cystathionine beta-synthase (CBS) and cystathioine gamma-lyase (CSE), produce H2S, pyruvate and ammonium using homocysteine and/or L-cysteine as substrates [12,13]. In some tissues, both CBS and CSE function to catalyze H2S production. CSE is the major H2S-producing enzyme in vascular tissues [14]. As an important gasotransmitter in the cardiovascular system, H2S has important physiological functions, such as anti-atherosclerosis, anti-inflammatory, vasodilatation, protection of ischemia injury, and antioxidant effects [12,14–16]. Although both estrogen and H2S can inhibit SMC proliferation [3,4,7,8,13], the interaction of H2S 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. PLoS ONE | www.plosone.org 1 August 2012 | Volume 7 | Issue 8 | e41614