Centro de Regulación Celular y Patología, Centro de Regeneración y Envejecimiento, Departamento de Biología Celular y Molecular, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile.
Int J Biochem Cell Biol. 2012 Nov;44(11):1993-2002. doi: 10.1016/j.biocel.2012.07.028. Epub 2012 Aug 7.
Fibrotic disorders are typically characterised by excessive connective tissue and extracellular matrix (ECM) deposition that preclude the normal healing of different tissues. Several skeletal muscle dystrophies are characterised by extensive fibrosis. Among the factors involved in skeletal muscle fibrosis is angiotensin II (Ang-II), a key protein of the renin-angiotensin system (RAS). We previously demonstrated that myoblasts responded to Ang-II by increasing the ECM protein levels mediated by AT-1 receptors, implicating an Ang-II-induced reactive oxygen species (ROS) by a NAD(P)H oxidase-dependent mechanism. In this paper, we show that in myoblasts, Ang-II induced the increase of transforming growth factor beta 1 (TGF-β1) and connective tissue growth factor (CTGF) expression through its AT-1 receptor. This effect is dependent of the NAD(P)H oxidase (NOX)-induced ROS, as indicated by a decrease of the expression of both pro-fibrotic factors when the ROS production was inhibited via the NOX inhibitor apocynin. The increase in pro-fibrotic factors levels was paralleled by enhanced p38MAPK and ERK1/2 phosphorylation in response to Ang-II. However, only the p38MAPK activity was critical for the Ang-II-induced fibrotic effects, as indicated by the decrease in the Ang-II-induced TGF-β1 and CTGF expression and fibronectin levels by SB-203580, an inhibitor of the p38MAPK, but not by U0126, an inhibitor of ERK1/2 phosphorylation. Furthermore, we showed that the Ang-II-dependent p38MAPK activation, but not the ERK1/2 phosphorylation, was necessary for the NOX-derived ROS. In addition, we demonstrated that TGF-β1 expression was required for the Ang-II-induced pro-fibrotic effects evaluated by using SB-431542, an inhibitor of TGF-βRI kinase activity, and by knocking down TGF-β1 levels by shRNA technique. These results strongly suggest that the fibrotic response to Ang-II is mediated by the AT-1 receptor and requires the p38MAPK phosphorylation, NOX-induced ROS, and TGF-β1 expression increase mediated by Ang-II in skeletal muscle cells.
纤维化疾病的特征通常是过度的结缔组织和细胞外基质 (ECM) 沉积，这会阻碍不同组织的正常愈合。几种骨骼肌疾病的特征是广泛的纤维化。在骨骼肌纤维化涉及的因素中，血管紧张素 II (Ang-II) 是肾素-血管紧张素系统 (RAS) 的关键蛋白。我们之前证明，成肌细胞对 Ang-II 的反应是通过增加 ECM 蛋白水平来介导的，这涉及到通过 NAD(P)H 氧化酶依赖性机制诱导的 Ang-II 诱导的活性氧 (ROS)。在本文中，我们表明在成肌细胞中，Ang-II 通过其 AT-1 受体诱导转化生长因子β 1 (TGF-β1) 和结缔组织生长因子 (CTGF) 表达的增加。这种作用依赖于 NAD(P)H 氧化酶 (NOX) 诱导的 ROS，如通过 NOX 抑制剂 apocynin 抑制 ROS 产生时两种促纤维化因子的表达减少所表明的。促纤维化因子水平的增加伴随着 Ang-II 诱导的 p38MAPK 和 ERK1/2 磷酸化的增强。然而，只有 p38MAPK 活性对于 Ang-II 诱导的纤维化效应是关键的，如 SB-203580（p38MAPK 的抑制剂）降低 Ang-II 诱导的 TGF-β1 和 CTGF 表达和纤维连接蛋白水平以及 U0126（ERK1/2 磷酸化的抑制剂）所表明的。此外，我们表明，Ang-II 依赖性 p38MAPK 激活，而不是 ERK1/2 磷酸化，对于 NOX 衍生的 ROS 是必要的。此外，我们通过使用 TGF-βRI 激酶活性抑制剂 SB-431542 和通过 shRNA 技术降低 TGF-β1 水平来证明 TGF-β1 表达是评估 Ang-II 诱导的促纤维化效应所必需的。这些结果强烈表明，成肌细胞对 Ang-II 的纤维化反应是由 AT-1 受体介导的，并且需要 p38MAPK 磷酸化、NOX 诱导的 ROS 和 Ang-II 介导的 TGF-β1 表达增加。
