Survivin expression induced by endothelin-1 promotes myofibroblast resistance to apoptosis

Abstract

Fibrosis of the lungs and other organs is characterized by the accumulation of myofibroblasts, effectors of wound-repair that are responsible for the deposition and organization of new extracellular matrix (ECM) in response to tissue injury. During the resolution phase of normal wound repair, myofibroblast apoptosis limits the continued deposition of ECM. Mounting evidence suggests that myofibroblasts from fibrotic wounds acquire resistance to apoptosis, but the mechanisms regulating this resistance have not been fully elucidated. Endothelin-1 (ET-1), a soluble peptide strongly associated with fibrogenesis, decreases myofibroblast susceptibility to apoptosis through activation of phosphatidylinositol 3′-OH kinase (PI3K)/AKT. Focal adhesion kinase (FAK) also promotes myofibroblast resistance to apoptosis through PI3K/AKT-dependent and -independent mechanisms, although the role of FAK in ET-1 mediated resistance to apoptosis has not been explored. The goal of this study was to investigate whether FAK contributes to ET-1 mediated myofibroblast resistance to apoptosis and to examine potential mechanisms downstream of FAK and PI3K/AKT by which ET-1 regulates myofibroblast survival. Here, we show that ET-1 regulates myofibroblast survival by Rho/ROCK-dependent activation of FAK. The anti-apoptotic actions of FAK are, in turn, dependent on activation of PI3K/AKT and the subsequent increased expression of Survivin, a member of the inhibitor of apoptosis protein (IAP) family. Collectively, these studies define a novel mechanism by which ET-1 promotes myofibroblast resistance to apoptosis through upregulation of Survivin.

Introduction

Fibroblasts are mesenchymal cells which, in response to tissue injury, differentiate into alpha-smooth muscle actin expressing myofibroblasts. These myofibroblasts function as the effector cells in wound healing through their synthesis, secretion, organization and contraction of new extracellular matrix (ECM) (Hinz and Gabbiani, 2010). Ultimately, the normal resolution of the wound repair response following injury requires the elimination of myofibroblasts by apoptosis. To date, the mechanistic triggers for myofibroblast apoptosis upon the conclusion of successful repair remain undefined (Desmouliere et al., 1995, Hernandez-Gea and Friedman, 2011, Thannickal and Horowitz, 2006). In contrast to normal wound healing, tissue fibrosis is characterized by the accumulation of myofibroblasts and the excessive deposition of ECM (Tomasek et al., 2002). The organization and remodeling of this excessive ECM can result in the progressive destruction of tissue architecture and lead to impaired organ function. Indeed, fibrosis of various organs is estimated to contribute to 45% of deaths in the developed world (Wynn, 2008).

Idiopathic pulmonary fibrosis (IPF) is a fibrotic disease of the lung parenchyma with no effective therapy and a mortality of approximately 50% within 2–3 years of the diagnosis (Horowitz and Thannickal, 2006). Mortality in IPF correlates with the profusion of fibroblastic foci, the active sites of fibrosis in IPF which are composed of accumulated myofibroblasts in close proximity to an abnormal alveolar epithelium (King et al., 2001). These foci represent an apoptosis paradox with evidence of robust apoptosis in the epithelial compartment and a distinct absence of apoptosis in the adjacent myofibroblasts (Korfei et al., 2008, Lepparanta et al., 2010, Maher et al., 2010, Thannickal and Horowitz, 2006). Importantly, the absence of apoptosis in fibroblastic foci is associated with an increased resistance to apoptosis in mesenchymal cells isolated from fibrotic lungs (Buhling et al., 2005, Chang et al., 2010, Hinz and Gabbiani, 2010, Huang et al., 2009, Moodley et al., 2004).

The mechanisms regulating resistance to apoptosis in fibrotic lung mesenchymal cells are poorly understood. Studies have shown that normal lung fibroblasts have basal resistance to Fas-mediated apoptosis that is overcome in the presence of cycloheximide (CHX), an inhibitor of protein translation, or by exposure to the inflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (Buhling et al., 2005, Frankel et al., 2006, Huang et al., 2009, Kulasekaran et al., 2009). Endothelin-1 (ET-1) and transforming growth factor beta-1 (TGF-β1) are soluble mediators implicated in the pathogenesis of fibrosis, and each of these has been shown to promote myofibroblast differentiation, cell contraction and collagen synthesis. We have shown that ET-1 and TGF-β1 independently increase myofibroblast resistance to apoptosis induced by the combination of Fas-activation and CHX (Fas/CHX) through activation of phosphatidylinositol 3′-OH kinase/protein kinase B (PI3K/AKT) (Kulasekaran et al., 2009). Additional studies show that focal adhesion kinase (FAK), a non-receptor tyrosine kinase that mediates myofibroblast differentiation by TGF-β1, also promotes myofibroblast resistance to apoptosis through PI3K/AKT-dependent and -independent mechanisms (Ding et al., 2008, Horowitz et al., 2007, Thannickal et al., 2003, Xia et al., 2004). Both FAK and AKT are activated in murine lungs following a fibrotic injury and pharmacologic inhibition of these kinases attenuates lung fibrosis in vivo (Vittal et al., 2005). The downstream mechanisms by which FAK and AKT mediate myofibroblast susceptibility to apoptosis have not been shown.

In this study we investigated the role of FAK and the relationship between FAK and PI3K/AKT in the regulation of myofibroblast resistance to apoptosis by ET-1. Additionally, we examined the downstream mechanism by which these pro-survival kinases regulate susceptibility to apoptosis in myofibroblasts. Herein, we demonstrate that FAK is rapidly activated by ET-1 through a mechanism that is dependent on Rho-associated coiled-coil forming protein kinase (ROCK), that FAK phosphorylation is required for PI3K/AKT activation by ET-1, and that this ET-1/ROCK/FAK/PI3K/AKT signaling cascade regulates myofibroblast susceptibility to apoptosis. Additionally, we show that the expression of Survivin, a member of the inhibitor of apoptosis protein (IAP) family, is increased by ET-1 and that inhibition of Survivin reverses the anti-apoptotic effects of ET-1. Collectively, these studies elucidate a novel mechanism by which ET-1 decreases myofibroblast susceptibility to apoptosis.