Department of Pharmacology, The University of Illinois at Chicago, Chicago, IL, USA; Department of Medicine, The University of Illinois at Chicago, Chicago, IL, USA.
Women's Hospital School of Medicine Zhejiang University, Hangzhou, Zhejiang, China.
Free Radic Biol Med. 2017 Nov;112:162-173. doi: 10.1016/j.freeradbiomed.2017.07.023. Epub 2017 Jul 24.
Lysocardiolipin acyltransferase (LYCAT), a cardiolipin remodeling enzyme, plays a key role in mitochondrial function and vascular development. We previously reported that reduced LYCAT mRNA levels in peripheral blood mononuclear cells correlated with poor pulmonary function outcomes and decreased survival in IPF patients. Further LYCAT overexpression reduced lung fibrosis, and LYCAT knockdown accentuated experimental pulmonary fibrosis. NADPH Oxidase 4 (NOX4) expression and oxidative stress are known to contribute to lung fibroblast differentiation and progression of fibrosis. In this study, we investigated the role of LYCAT in TGF-β mediated differentiation of human lung fibroblasts to myofibroblasts, and whether this occurred through mitochondrial superoxide and NOX4 mediated hydrogen peroxide (HO) generation. Our data indicated that LYCAT expression was up-regulated in primary lung fibroblasts isolated from IPF patients and bleomycin-challenged mice, compared to controls. In vitro, siRNA-mediated SMAD3 depletion inhibited TGF-β stimulated LYCAT expression in human lung fibroblasts. ChIP immunoprecipitation assay revealed TGF-β stimulated SMAD2/3 binding to the endogenous LYCAT promoter, and mutation of the SMAD2/3 binding sites (-179/-183 and -540/-544) reduced TGF-β-stimulated LYCAT promoter activity. Overexpression of LYCAT attenuated TGF-β-induced mitochondrial and intracellular oxidative stress, NOX4 expression and differentiation of human lung fibroblasts. Further, pretreatment with Mito-TEMPO, a mitochondrial superoxide scavenger, blocked TGF-β-induced mitochondrial superoxide NOX4 expression and differentiation of human lung fibroblasts. Treatment of human lung fibroblast with NOX1/NOX4 inhibitor, GKT137831, also attenuated TGF-β induced fibroblast differentiation and mitochondrial oxidative stress. Collectively, these results suggest that LYCAT is a negative regulator of TGF-β-induced lung fibroblast differentiation by modulation of mitochondrial superoxide and NOX4 dependent HO generation, and this may serve as a potential therapeutic target for human lung fibrosis.
溶血磷脂酰甘油酰基转移酶（LYCAT）是一种心磷脂重塑酶，在心磷脂代谢和线粒体功能中发挥重要作用。我们之前的研究发现，间质性肺纤维化（IPF）患者外周血单个核细胞中 LYCATmRNA 水平降低与肺功能不良结局和生存率降低相关。进一步的 LYCAT 过表达可减轻肺纤维化，而 LYCAT 敲低则加剧实验性肺纤维化。已知 NADPH 氧化酶 4（NOX4）表达和氧化应激可促进肺成纤维细胞分化和纤维化进展。在这项研究中，我们研究了 LYCAT 在 TGF-β 介导的人肺成纤维细胞向肌成纤维细胞分化中的作用，以及这种作用是否通过线粒体超氧化物和 NOX4 介导的过氧化氢（HO）生成来实现。我们的数据表明，与对照组相比，从 IPF 患者和博来霉素处理的小鼠分离的原代肺成纤维细胞中 LYCAT 表达上调。在体外，siRNA 介导的 SMAD3 耗竭抑制了 TGF-β 刺激的人肺成纤维细胞中 LYCAT 的表达。染色质免疫沉淀实验显示，TGF-β 刺激 SMAD2/3 结合到内源性 LYCAT 启动子上，并且 SMAD2/3 结合位点（-179/-183 和-540/-544）的突变降低了 TGF-β 刺激的 LYCAT 启动子活性。LYCAT 的过表达减轻了 TGF-β 诱导的线粒体和细胞内氧化应激、NOX4 表达和人肺成纤维细胞的分化。此外，线粒体超氧化物清除剂 Mito-TEMPO 的预处理阻断了 TGF-β 诱导的线粒体超氧化物、NOX4 表达和人肺成纤维细胞的分化。用 NOX1/NOX4 抑制剂 GKT137831 处理人肺成纤维细胞也可减轻 TGF-β 诱导的成纤维细胞分化和线粒体氧化应激。总之，这些结果表明，LYCAT 通过调节线粒体超氧化物和 NOX4 依赖性 HO 生成来负调控 TGF-β 诱导的肺成纤维细胞分化，这可能成为人类肺纤维化的潜在治疗靶点。
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