Endothelin-1 driven proliferation of pulmonary arterial smooth muscle cells is c-fos dependent

Abstract

Pulmonary hypertension (PH) is characterized by enhanced pulmonary artery smooth muscle cell (PASMC) proliferation leading to vascular remodeling. Although, multiple factors have been associated with pathogenesis of PH the underlying mechanisms are not fully understood. Here, we hypothesize that already very short exposure to hypoxia may activate molecular cascades leading to vascular remodeling.

Microarray studies from lung homogenates of mice exposed to only 3 h of hypoxia revealed endothelin-1 (ET-1) and connective tissue growth factor (CTGF) as the most upregulated genes, and the mitogen-activated protein kinase (MAPK) pathway as the most differentially regulated pathway. Evaluation of these results in vitro showed that ET-1 but not CTGF stimulation of human PASMCs increased DNA synthesis and expression of proliferation markers such as Ki67 and cell cycle regulator, cyclin D1. Moreover, ET-1 treatment elevated extracellular signal-regulated kinase (Erk)-dependent c-fos expression and phosphorylation of c-fos and c-jun transcription factors. Silencing of c-fos with siRNA abrogated the ET-1-induced proliferation of PASMCs. Expression and immunohistochemical analyses revealed higher levels of total and phosphorylated c-fos and c-jun in the vessel wall of lung samples of human idiopathic pulmonary arterial hypertension patents, hypoxia-exposed mice and monocrotaline-treated rats as compared to control subjects.

These findings shed the light on the involvement of c-fos/c-jun in the proliferative response of PASMCs to ET-1 indicating that already very short hypoxia exposure leads to the regulation of mediators involved in vascular remodeling underlying PH.

Introduction

Increased intrapulmonary pressure leading to right heart hypertrophy, pressure overload and eventually to right heart failure are the hallmarks of pulmonary hypertension (PH) (Olschewski et al., 2001). The increase in pulmonary pressure is mainly due to a persistent vasoconstriction of the pulmonary arteries and thickening of the vessel wall, which contribute to the reduction of lumen, further increasing blood pressure (Schermuly et al., 2011). All three layers of the vessel wall (intima, media and adventitia) are involved in the remodeling processes with the most prominent contribution of smooth muscle cells (SMCs). Different animal models such as chronic hypoxia exposure in mice and monocrotaline-induced injury in rats have been established in order to study the development and molecular mechanisms underlying PH (Stenmark et al., 2009). Although, none of the mentioned animal models can fully reproduce the complexity of human disease (Bauer et al., 2007), they can recapitulate several aspects of PH such as the increased intrapulmonary pressure and the increased thickening of vessel wall due to the hyperproliferation of SMCs. The increased expansion of SMCs may be caused by a cumulative effect of several growth factors acting in an autocrine and/or paracrine manner (Toshner et al., 2010). Many of these factors are secreted by SMCs (platelet-derived growth factor (PDGF)-BB) (Perros et al., 2008), by fibroblasts (transforming growth factor (TGF-β) (Kelley et al., 1991) or endothelial cells (endothelin-1(ET-1)) (Vane and Botting, 1992).

ET-1 is a 21 amino acid peptide released from endothelial cells under hypoxic conditions (Kourembanas et al., 1991) that can bind to two receptors: endothelin receptor A (ET_A) expressed only by SMCs or endothelin receptor B (ET_B) expressed by both endothelial and SMCs (Hall et al., 2011). The binding of ET-1 to ET_A induces calcium influx, contraction and proliferation of SMCs (Wagner et al., 1992). Moreover, increase in calcium has been demonstrated to enhance expression of activator protein (AP)-1 family members which can, in a positive loop, be responsible for transcription of ET-1(Yamashita et al., 2001) and other growth factors such as TGF-β and connective tissue growth factor (CTGF) (Moritani et al., 2003, Gonzalez-Ramos et al., 2012).

AP-1 protein complex is a family of transcription factors, including jun (c-jun, junB and junD) and fos (c-fos, fosl2, fosl1 and fosB) components (Shaulian and Karin, 2001). These transcription factors are also called the immediate early genes, due to their capability to be activated transiently and rapidly in response to many different stimuli (Shaulian and Karin, 2001). C-fos and c-jun were first identified as oncogenic genes activated by the FBJ murine osteosarcoma virus (Silbermann et al., 1987) and avian sarcoma virus (Nishimura and Vogt, 1988), respectively. Consequently, they were shown to be highly relevant for cancer development and progression (Healy et al., 2013). While c-fos and c-jun are the most studied transcription factors in cancer field, their role and contribution to PH is still not fully addressed. Therefore, in the present study we focused on the possible role of c-fos and c-jun in proliferation of SMCs and development of PH in response to ET-1.