University of Texas Health Science Center Department of Medicine, San Antonio, Texas 78229-3900, USA.
Am J Physiol Cell Physiol. 2012 Jan 1;302(1):C122-30. doi: 10.1152/ajpcell.00141.2011. Epub 2011 Sep 21.
Extracellular matrix accumulation contributes to the progression of chronic kidney disease. Many growth factors including insulin-like growth factor-I (IGF-I) enhance matrix protein accumulation. Proximal tubular epithelial cells (PTCs) synthesize matrix proteins. NADPH oxidases are major sources of reactive oxygen species (ROS), important signaling molecules that mediate biological responses in a variety of cells and tissue. We investigated the mechanism by which IGF-I regulates fibronectin accumulation in PTCs and the role of a potential redox-dependent signaling pathway. IGF-I induces an increase in NADPH-dependent superoxide generation, enhances the release of hydrogen peroxide, and increases the expression of NADPH oxidase 4 (Nox4) in PTCs. IGF-I also stimulates phosphorylation of Akt, and inhibition of Akt or its upstream activator phosphatidylinositol 3-kinase attenuates IGF-I-induced fibronectin accumulation. Expression of dominant negative Akt also inhibits IGF-I-induced expression of fibronectin, indicating a role for this kinase in fibronectin accumulation. Expression of dominant negative adenovirus Nox4 inhibits IGF-I-induced NADPH oxidase activity, Akt phosphorylation, and fibronectin protein expression. Moreover, transfection of small interfering RNA targeting Nox4 decreases Nox4 protein expression and blocks IGF-I-induced Akt phosphorylation and the increase in fibronectin, placing Nox4 and ROS upstream of Akt signaling pathway. To confirm the role of Nox4, PTCs were infected with adenovirus construct expressing wild-type Nox4. Ad-Nox4, but not control Ad-green fluorescent protein, upregulated Nox4 expression and increased NADPH oxidase activity as well as fibronectin expression. Taken together, these results provide the first evidence for a role of Nox4 in IGF-I-induced Akt phosphorylation and fibronectin expression in tubular epithelial cells.
细胞外基质的积累是导致慢性肾脏病进展的原因之一。许多生长因子，包括胰岛素样生长因子-I（IGF-I），可增强基质蛋白的积累。近端肾小管上皮细胞（PTCs）合成基质蛋白。NADPH 氧化酶是活性氧（ROS）的主要来源，ROS 是一种重要的信号分子，可介导多种细胞和组织中的生物学反应。我们研究了 IGF-I 调节 PTCs 纤维连接蛋白积累的机制，以及潜在的氧化还原依赖信号通路的作用。IGF-I 诱导 NADPH 依赖性超氧阴离子生成增加，增强过氧化氢的释放，并增加 PTCs 中 NADPH 氧化酶 4（Nox4）的表达。IGF-I 还刺激 Akt 的磷酸化，抑制 Akt 或其上游激活物磷脂酰肌醇 3-激酶可减弱 IGF-I 诱导的纤维连接蛋白积累。表达显性失活 Akt 也可抑制 IGF-I 诱导的纤维连接蛋白表达，表明该激酶在纤维连接蛋白积累中起作用。表达显性失活腺病毒 Nox4 可抑制 IGF-I 诱导的 NADPH 氧化酶活性、Akt 磷酸化和纤维连接蛋白蛋白表达。此外，针对 Nox4 的小干扰 RNA 的转染可降低 Nox4 蛋白表达并阻断 IGF-I 诱导的 Akt 磷酸化和纤维连接蛋白的增加，将 Nox4 和 ROS 置于 Akt 信号通路的上游。为了确认 Nox4 的作用，将 PTC 用表达野生型 Nox4 的腺病毒构建体感染。Ad-Nox4，但不是对照 Ad-绿色荧光蛋白，可上调 Nox4 表达并增加 NADPH 氧化酶活性以及纤维连接蛋白表达。综上所述，这些结果首次提供了 Nox4 在 IGF-I 诱导的 Akt 磷酸化和管状上皮细胞中纤维连接蛋白表达中的作用的证据。
