Defining and targeting mechanisms of smoke-mediated fibrosis progression

烟雾介导的纤维化进展的定义和靶向机制

• 批准号: 10372090
• 负责人: Ching-Hsien Chen
• 金额: $ 39.25万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10372090
• 关键词: Achievement; Adoptive Transfer; Affect; Antibodies; Binding; Biological Assay; Biological Markers; Biopsy; Bleomycin; Cell Proliferation; Cells; Chronic lung disease; Co-Immunoprecipitations; Complement; Complex; Data; Development; Disease; Down-Regulation; Environmental Exposure; Exposure to; FDA approved; Fibroblasts; Fibrosis; Future; Gene Expression Profiling; Genes; Human; Immunofluorescence Immunologic; In Vitro; Interstitial Lung Diseases; Intervention; Knock-out; Ligands; Lung; MARCKS gene; Mainstreaming; Malignant Neoplasms; Malignant neoplasm of lung; Mediating; Medical; Membrane Proteins; Modeling; Molecular; Myofibroblast; Pathogenesis; Pathogenicity; Patients; Peptides; Pharmaceutical Preparations; Pharmacology; Phenotype; Phosphorylation; Phosphotransferases; Play; Prognosis; Proteins; Pulmonary Fibrosis; Receptor Protein-Tyrosine Kinases; Reporting; Risk; Role; Series; Signal Transduction; Signaling Protein; Smoke; Smoking; Smoking History; Smoking Status; Smooth Muscle Actin Staining Method; Stains; Structure of parenchyma of lung; Testing; Therapeutic; Time; Tissues; Tobacco smoke; Tobacco smoking behavior; Tyrosine Phosphorylation; Up-Regulation; Western Blotting; axl receptor tyrosine kinase; cigarette smoke; cigarette smoking; clinically relevant; druggable target; epidemiology study; evidence base; exposed human population; exposure to cigarette smoke; fibrotic lung; former smoker; idiopathic pulmonary fibrosis; in vivo; inhibitor; knock-down; lung cancer cell; migration; new therapeutic target; nintedanib; novel; response; small hairpin RNA; therapeutic target; tumor progression

Several environmental exposures, especially tobacco smoking, have been associated with increased risk for idiopathic pulmonary fibrosis (IPF), a progressive and incurable interstitial lung disease. This disease has a median survival time of 3 to 5 years, worse than many cancers. With 41%-83% of IPF patients being current or former smokers, cigarette smoking plays a crucial role in IPF development and progression. Unfortunately, the mechanisms by which smoking promotes fibrosis are poorly understood. Of great concern, there are no reliable biomarkers and/or specific therapies available for smoking-associated IPF. To discover the regulators of IPF fibroblast activation, we compared and integrated the gene profiles of IPF fibroblasts from two different microarray platforms. Among the genes identified, myristoylated alanine-rich C-kinase substrate (MARCKS) was found to be highly correlated with the expression of the myofibroblast marker alpha smooth muscle actin (α-SMA) and was notably elevated in IPF fibroblasts. We previously identified the membrane-associated protein MARCKS as a smoke-responsive molecule associated with lung cancer progression. Our preliminary data have demonstrated that up-regulation of phospho-MARCKS concomitant with an increase of protein tyrosine phosphorylation level in smoke-exposed cells. Using a phospho-kinase antibody array screen, we found the AXL receptor tyrosine kinase (RTK) as a top-one RTK active in response to smoke. In addition, our LC-MS/MS data have revealed AXL is a putative binding partner of phospho-MARCKS. Co-expression of phospho-MARCKS and phospho-AXL were observed in lung tissues with exposure to both mainstream and sidestream cigarette smoke. Surprisingly, the inhibition of phospho-MARCKS resulted in downregulation of AXL autophosphorylation and its downstream signaling. Given these observations, we hypothesize that cigarette smoke through MARCKS-AXL signaling stimulates fibroblast activation, thereby contributing to lung fibrosis progression. To test this hypothesis, three specific aims are proposed: 1) characterize the role of cigarette smoke-activated MARCKS-AXL axis in fibroblast cell proliferation, migration, and/or differentiation in vitro; 2) to determine that MARCKS inhibition effectively suppresses smoke-mediated lung fibrosis in vivo; 3) to evaluate the clinical relevance of phospho- MARCKS and phospho-AXL in smoking-related IPF tissues. Achievement of these specific aims will characterize novel molecular mechanisms underlying smoke-mediated fibrosis progression, thereby providing novel therapeutic targets such as phospho-MARCKS and phospho-AXL for combating pulmonary fibrosis.

几种环境暴露，尤其是吸烟，与增加的风险有关 特发性肺纤维化（IPF），一种进行性且无法治愈的间质性肺疾病。这种疾病有 中位生存时间为3至5年，比许多癌症差。 IPF患者中有41％-83％是当前或 前吸烟者吸烟在IPF的开发和发展中起着至关重要的作用。不幸的是， 吸烟促进纤维化的机制知之甚少。非常关心的是，没有可靠的 可用于吸烟相关的IPF的生物标志物和/或特定疗法。发现IPF的调节器 成纤维细胞激活，我们比较并整合了来自两个不同的IPF成纤维细胞的基因谱 微阵列平台。在确定的基因中，肉豆蔻酰化富含丙氨酸的C-激酶底物（MARCKS）为 发现与肌纤维细胞标记平滑肌肌动蛋白（α-SMA）的表达高度相关 IPF成纤维细胞的升高。我们先前鉴定了与膜相关的蛋白质马克克斯 作为与肺癌进展相关的烟雾反应性分子。我们的初步数据 证明磷酸 - 马克克的上调随蛋白酪氨酸的增加 暴露于烟雾的细胞中的磷酸化水平。使用磷酸激酶抗体阵列筛网，我们找到了AXL 受体酪氨酸激酶（RTK）是响应烟雾的顶级RTK。此外，我们的LC-MS/MS数据 已经揭示了AXL是磷酸化 - 马克克的假定结合伙伴。磷酸 - 摩尔克的共表达和 在肺组织中观察到磷酸 - 轴，暴露于主流和侧源香烟烟雾中。 令人惊讶的是，磷酸摩尔克菌的抑制导致AXL自磷酸化及其下调 下游信号传导。鉴于这些观察，我们假设通过Marcks-axl烟雾 信号传导刺激成纤维细胞激活，从而导致肺纤维化进展。为了检验这一假设， 提出了三个具体目的：1）表征香烟烟雾激活的马克克斯轴的作用 体外成纤维细胞增殖，迁移和/或分化； 2）确定马克克斯抑制 有效地抑制体内烟雾介导的肺纤维化； 3）评估磷酸的临床相关性 与吸烟相关的IPF组织中的MARCK和磷酸轴。这些特定目标的实现将表征 烟雾介导的纤维化进展的新型分子机制，从而提供新颖 治疗靶标，例如磷酸化 - 马克斯和磷酸轴，用于对抗肺纤维化。