PI3K p110a Isoform-Dependent Rho GTPase Rac1 Activation Mediates H2S-Promoted Endothelial Cell Migration via Actin Cytoskeleton Reorganization Li-Jia Zhang, Bei-Bei Tao, Ming-Jie Wang, Hui-Ming Jin, Yi-Chun Zhu* Department of Physiology and Pathophysiology, Fudan University Shanghai Medical College, Shanghai, China Abstract Hydrogen sulfide (H2S) is now considered as the third gaseotransmitter, however, the signaling pathways that modulate the biomedical effect of H2S on endothelial cells are poorly defined. In the present study, we found in human endothelial cells that H2S increased cell migration rates and induced a marked reorganization of the actin cytoskeleton, which was prevented by depletion of Rac1. Pharmacologic inhibiting vascular endothelial growth factor receptor (VEGFR) and phosphoinositide 3- kinase (PI3K) both blunted the activation of Rac1 and the promotion of cell migration induced by H2S. Moreover, H2S- induced Rac1 activation was selectively dependent on the presence of the PI3K p110a isoform. Activated Rac1 by H2S thus in turn resulted in the phosphorylation of the F-actin polymerization modulator, cofilin. Additionally, inhibiting of extracellular signal-regulated kinase (ERK) decreased the augmented cell migration rate by H2S, but had no effect on Rac1 activation. These results indicate that Rac1 conveys the H2S signal to microfilaments inducing rearrangements of actin cytoskeleton that regulates cell migration. VEGFR-PI3K was found to be upstream pathway of Rac1, while cofilin acted as a downstream effector of Rac1. ERK was also shown to be involved in the action of H2S on endothelial cell migration, but independently of Rac1. Citation: Zhang L-J, Tao B-B, Wang M-J, Jin H-M, Zhu Y-C (2012) PI3K p110a Isoform-Dependent Rho GTPase Rac1 Activation Mediates H2S-Promoted Endothelial Cell Migration via Actin Cytoskeleton Reorganization. PLoS ONE 7(9): e44590. doi:10.1371/journal.pone.0044590 Editor: Chuen-Mao Yang, Chang Gung University, Taiwan Received May 4, 2012; Accepted August 3, 2012; Published September 7, 2012 Copyright: 2012 Zhang 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 work was supported by grants from the Ministry of Science and Technology (2010CB912601 and 2009ZX09303-006) of China and the National Natural Science Foundation of China (30825016, 30971064, 81000045). 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: yczhu@shmu.edu.cn Introduction Hydrogen sulfide (H2S), along with nitric oxide (NO) and carbon monoxide (CO), is now considered to be the third member of the gaseotransmitter family [1,2,3]. H2S is endogenously generated from L-cysteine in reactions that are catalyzed by cystathionine-b-synthase (CBS) [4], cystathionine-c-lyase (CSE) [4], and 3-mercaptopyruvate sulfurtransferase (3-MST) [5,6]. Recently, H2S has been recognized as an important signaling molecule for the nervous system, the cardiovascular system, and the inflammatory system [1,2,7]. As a signaling molecule, H2S modulates neuronal transmission by facilitating the induction of hippocampal long term potential (LTP) [8]. In the cardiovascular system, H2S has been shown to relax smooth muscles and to regulate blood pressure [9,10,11]. H2S can act as a cytoprotectant to protect cells from oxidative stress and as an anti-apoptosis agent by preserving mitochondrial function during ischemia-reperfusion [12,13]. H2S has also been found to regulate the release of insulin [14,15]. Both pro-inflammatory and anti-inflammatory effects of H2S have recently been discovered also [16,17]. While in our previous study, we have provided the first piece of evidence regarding the proangiogenic effect of exogenous H2S in a matrigel plug model [18]. And our subsequent study has also verified the proangiogenic effect of H2S in a rat model of hind limb ischemia [19]. Researches by Papapetropoulos et al also indicate that H2S is an endogenous stimulator of angiogenesis [20]. However, the concrete underlying molecular mechanisms of H2S on angiogen- esis are still poorly understood. Vascular endothelial cell migration has a pivotal role in angiogenesis. A series of cellular events including changes in the actin cytoskeleton, in cell-matrix adhesions, cell-cell interaction, and in the biosynthesis and degradation of extracellular matrix are involved in cell migration [21]. Cell migration is characterized with actin polymerization in the protruding front edges of the cells and retraction of the cell tail ends [22]. The migrating cells are driven with the mechanical force provided by dynamic remodeling of the actin cytoskeleton which is downstream of the intracellular signaling pathways involving the small GTPases of the Rho family, in particular Rac1, RhoA and Cdc42 [23]. However, it is not known if small Rho GTPases play a role in mediating the action of H2S in angiogenesis and migration of vascular endothelial cells. The aim of this study was to test the hypothesis that small Rho GTPases mediate the action of H2S on endothelial cells. Using multifaceted approaches, we provide the first evidence that H2S promotes the migration of human endothelial cells through Rho GTPase Rac1-mediated actin cytoskeleton reorganization. In addition, the upstream regulators and downstream effectors of Rac1 were also particularly monitored. An important physiolog- ical role of H2S as an endothelial cell migration promoting factor and the detailed signaling transduction pathway of H2S are thus elucidated. PLOS ONE | www.plosone.org 1 September 2012 | Volume 7 | Issue 9 | e44590