Ding Qiang, Subramanian Indhu, Luckhardt Tracy R, Che Pulin, Waghray Meghna, Zhao Xue-Ke, Bone Nathaniel, Kurundkar Ashish R, Hecker Louise, Hu Meng, Zhou Yong, Horowitz Jeffrey C, Vittal Ragini, Thannickal Victor J
Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, University of Alabama Birmingham, Birmingham, Alabama;
Division of Pulmonary, Allergy, and Critical Care Medicine, Alameda Health System, Oakland, California.
Am J Physiol Lung Cell Mol Physiol. 2017 Jun 1;312(6):L926-L935. doi: 10.1152/ajplung.00121.2016. Epub 2017 Mar 30.
Alveolar epithelial cell (AEC) injury and apoptosis are prominent pathological features of idiopathic pulmonary fibrosis (IPF). There is evidence of AEC plasticity in lung injury repair response and in IPF. In this report, we explore the role of focal adhesion kinase (FAK) signaling in determining the fate of lung epithelial cells in response to transforming growth factor-β1 (TGF-β1). Rat type II alveolar epithelial cells (RLE-6TN) were treated with or without TGF-β1, and the expressions of mesenchymal markers, phenotype, and function were analyzed. Pharmacological protein kinase inhibitors were utilized to screen for SMAD-dependent and -independent pathways. SMAD and FAK signaling was analyzed using siRNA knockdown, inhibitors, and expression of a mutant construct of FAK. Apoptosis was measured using cleaved caspase-3 and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. TGF-β1 induced the acquisition of mesenchymal markers, including α-smooth muscle actin, in RLE-6TN cells and enhanced the contraction of three-dimensional collagen gels. This phenotypical transition or plasticity, epithelial-myofibroblast plasticity (EMP), is dependent on SMAD3 and FAK signaling. FAK activation was found to be dependent on ALK5/SMAD3 signaling. We observed that TGF-β1 induces both EMP and apoptosis in the same cell culture system but not in the same cell. While blockade of SMAD signaling inhibited EMP, it had a minimal effect on apoptosis; in contrast, inhibition of FAK signaling markedly shifted to an apoptotic fate. The data support that FAK activation determines whether AECs undergo EMP vs. apoptosis in response to TGF-β1 stimulation. TGF-β1-induced EMP is FAK- dependent, whereas TGF-β1-induced apoptosis is favored when FAK signaling is inhibited.
肺泡上皮细胞（AEC）损伤和凋亡是特发性肺纤维化（IPF）的突出病理特征。有证据表明AEC在肺损伤修复反应和IPF中具有可塑性。在本报告中，我们探讨了粘着斑激酶（FAK）信号在决定肺上皮细胞对转化生长因子-β1（TGF-β1）反应的命运中的作用。用或不用TGF-β1处理大鼠II型肺泡上皮细胞（RLE-6TN），并分析间充质标志物的表达、表型和功能。使用药理学蛋白激酶抑制剂筛选SMAD依赖性和非依赖性途径。使用siRNA敲低、抑制剂和FAK突变构建体的表达分析SMAD和FAK信号。使用裂解的半胱天冬酶-3和末端脱氧核苷酸转移酶dUTP缺口末端标记（TUNEL）染色测量凋亡。TGF-β1诱导RLE-6TN细胞中包括α-平滑肌肌动蛋白在内的间充质标志物的获得，并增强三维胶原凝胶的收缩。这种表型转变或可塑性，即上皮-肌成纤维细胞可塑性（EMP），依赖于SMAD3和FAK信号。发现FAK激活依赖于ALK5/SMAD3信号。我们观察到TGF-β1在同一细胞培养系统中诱导EMP和凋亡，但不是在同一细胞中。虽然阻断SMAD信号抑制EMP，但对凋亡影响最小；相反，抑制FAK信号明显转向凋亡命运。数据支持FAK激活决定AEC在对TGF-β1刺激的反应中是经历EMP还是凋亡。TGF-β1诱导的EMP是FAK依赖性的，而当FAK信号被抑制时，TGF-β1诱导的凋亡更易发生。
