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，特别是吸烟，已经与增加的风险有关。 特发性肺纤维化(IPF)是一种进行性和不可治愈的间质性肺疾病。这种病有一种 中位生存期为3至5年，比许多癌症都要差。41%-83%的IPF患者是现症或 作为吸烟者，吸烟在IPF的发生发展中起着至关重要的作用。不幸的是， 人们对吸烟促进纤维化的机制知之甚少。值得关注的是，没有可靠的 可用于吸烟相关IPF的生物标志物和/或特定治疗。发现IPF的监管者 成纤维细胞激活，我们比较和整合了来自不同来源的IPF成纤维细胞的基因图谱 微阵列平台。在已鉴定的基因中，肉豆蔻酰化富含丙氨酸的C-激酶底物(MARCKS)是 发现与肌成纤维细胞标志物α-平滑肌肌动蛋白(α-SMA)的表达高度相关 在IPF成纤维细胞中显著升高。我们之前发现了膜相关蛋白Marcks 作为一种与肺癌进展相关的烟雾反应分子。我们的初步数据显示 证明了磷酸化Marcks的上调伴随着蛋白质酪氨酸的增加 烟雾暴露细胞中的磷酸化水平。使用磷酸化激酶抗体阵列筛选，我们发现了Axl 受体酪氨酸激酶(RTK)是吸烟反应中最活跃的一种RTK。此外，我们的LC-MS/MS数据 揭示了Ax1是一个可能的磷酸化Marcks结合伙伴。磷酸化Marcks和磷酸化Marcks的共表达 在暴露于主流和侧流香烟烟雾的肺组织中观察到磷酸化Axl。 令人惊讶的是，抑制磷酸化Marcks导致了Axl自动磷酸化和它的 下行信令。考虑到这些观察，我们假设香烟烟雾通过Marcks-Axl 信号刺激成纤维细胞激活，从而促进肺纤维化的进展。为了检验这一假设， 提出了三个具体的目标：1)表征香烟烟雾激活的Marcks-Axl轴在 成纤维细胞体外增殖、迁移和/或分化；2)确定Marcks的抑制作用 有效抑制体内烟雾介导的肺纤维化；3)评价磷酸-β-天冬氨酸的临床意义。 吸烟相关IPF组织中MARCKs和磷酸化Axl的表达。这些具体目标的实现将成为 烟雾介导的纤维化进展的新的分子机制，从而提供了新的 抗肺纤维化的治疗靶点，如磷酸化MARCKS和磷酸化Axl。