Angiotensin II induces apoptosis in intestinal epithelial cells through the AT2 receptor, GATA-6 and the Bax pathway

Abstract

Angiotensin II (Ang II) has been shown to play an important role in cell apoptosis. However, the mechanisms of Ang-II-induced apoptosis in intestinal epithelial cells are not fully understood. GATA-6 is a zinc finger transcription factor expressed in the colorectal epithelium, which directs cell proliferation, differentiation and apoptosis. In the present study we investigated the underlying mechanism of which GATA-6 affects Ang-II induced apoptosis in intestinal epithelial cells. The in vitro intestinal epithelial cell apoptosis model was established by co-culturing Caco-2 cells with Ang II. Pretreatment with Angiotensin type 2 (AT2) receptor antagonist, PD123319, significantly reduced the expression of Bax and prevented the Caco-2 cells apoptosis induced by Ang II. In addition, Ang II up-regulated the expression of GATA-6. Interestingly, GATA-6 short hairpin RNA prevented Ang II-induced intestinal epithelial cells apoptosis and reduced the expression of Bax, but not Bcl-2. Taken together, the present study suggests that Angiotensin II promotes apoptosis in intestinal epithelial cells through GATA-6 and the Bax pathway in an AT2 receptor-dependent manner.

Introduction

Massive resection of small intestine leads to the development of short bowel syndrome (SBS), which is characterized as a state of inadequate digestion and impaired absorption of nutrients. SBS is capable of causing a series of adaptive response to increase absorptive surface area and establish nutritional homeostasis in residual intestine [1], [2]. This adaption is highlighted by an increase in villus length and crypt depth, and an increased number of microvillus. The adaptive processes include significant increases in epithelial cell (EC) proliferation and EC apoptosis rates [3], [4], [5]. To improve intestinal compensation after SBS, researchers usually focus on EC proliferation via an up-regulation in a number of growth factors including keratinocyte growth factor (KGF), glucagon like peptide 2 (GLP-2), epidermal growth factor (EGF) and transforming growth factor alpha (TGF-α) [2], [6], [7], [8], [9]. Also, a few studies showed that reducing intestinal epithelial cell apoptosis would significantly improve bowel adaptation [3]. For example, Wildhaber et al. reported that ACE inhibitors (ACE-I) significantly reduces EC apoptosis and enhances intestinal adaptation [10]. Furthermore, blockade of angiotensin II (Ang II) signaling by Ang II receptor antagonists reduced injury effects and apoptosis [2], [11]. These studies suggested that Ang II plays a key role in apoptosis of intestinal epithelial cells. However, the mechanisms by which Ang II regulates intestinal epithelial cells apoptosis are still elusive.

Ang II exerts its biological effects via two major isoforms of receptor known as angiotensin type 1 (AT1) and type 2 (AT2) receptor, both of which are seven-transmembrane-spanning G protein-coupled receptors [11], [12], [13]. The signaling of AT1 and AT2 is associated with cell proliferation and apoptosis. The delicate balance between proliferation and apoptosis plays an essential role in the pathophysiology of various diseases [14], [15]. Several studies demonstrated that Ang-II-induced apoptosis in coronary artery endothelial cells and transformed epithelial cells was mediated by the AT1 receptor [16], [17], [18], [19]. By contrast, some other researchers showed that AT2-receptor activation induced apoptosis in smooth muscle cells, HUVECs and PC12W cells whereas AT1 activation resulted in proliferative and anti-apoptotic cellular responses in these cells [20], [21], [22]. Conclusively, Ang-II-induced signaling has been suggested to be cell-type dependent, and the signaling specificity has been considered to determine the biological response.

GATA is a family of six transcriptional regulation proteins that plays important role in both developmental processed and tissue- or cell-specific gene transcription [23], [24]. Previous studies have indicated that GATA-1, GATA-2 and GATA-3 function mainly in hematopoietic cell lineages whereas GATA-4, GATA-5 and GATA-6 are found in tissues of the endodermal origin [25], [26], [27]. Among the GATA family members, GATA-6 is highly expressed in gastric, colonic, ovary, pulmonary and cardiovascular cell lines [25], [28], [29], [30], [31]. In the intestinal tract, GATA-6 expression is gradually increased along the crypt–villus axis [26], [27], [32]. Studies have suggested that GATA-6 is associated with proliferation, differentiation and apoptosis by regulating genes expression in gastrointestinal tissue [28], [32], [33], [34]. It has been shown that GATA-6 regulated the apoptosis-related proteins FasL in smooth muscle cells [21]. Moreover, GATA-6 has been shown to regulate Bax and Bcl-2 in human fetal testis [35]. But there is no report on the relationship between GATA-6 and apoptosis related genes in intestine so far. In this study, we explore the mechanism of Ang II induced apoptosis in intestinal epithelium cells involving GATA-6 transcriptional regulation of Bax and Bcl-2 expression.