Defining and targeting mechanisms of smoke-mediated fibrosis progression

一些环境暴露，特别是吸烟，与以下风险增加有关： 特发性肺纤维化（IPF）是一种进行性且无法治愈的间质性肺病。这种疾病有一个 中位生存时间为3至5年，比许多癌症更差。41%-83%的IPF患者目前或 吸烟在IPF的发生和进展中起着至关重要的作用。可惜 吸烟促进纤维化的机制知之甚少。令人担忧的是，没有可靠的 可用于吸烟相关IPF的生物标志物和/或特异性治疗。发现IPF的调节因子 成纤维细胞活化，我们比较并整合了两种不同来源的IPF成纤维细胞的基因谱， 微阵列平台。在所鉴定的基因中，豆蔻酰化富含丙氨酸的C激酶底物（MARCKS）是一个重要的基因。 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 并且在IPF成纤维细胞中显著升高。我们先前鉴定了膜相关蛋白MARCKS 作为与肺癌进展相关的烟雾反应分子。我们的初步数据显示 证明磷酸化MARCKS的上调伴随着蛋白酪氨酸的增加， 在烟雾暴露的细胞磷酸化水平。使用磷酸激酶抗体阵列筛选，我们发现AXL 受体酪氨酸激酶（RTK）作为响应于烟雾的顶级RTK活性。此外，我们的LC-MS/MS数据 已经揭示了AXL是磷酸化MARCKS的假定结合伴侣。磷酸-MARCKS和 在暴露于主流和侧流香烟烟雾的肺组织中观察到磷酸-AXL。 令人惊讶的是，磷酸化MARCKS的抑制导致AXL自身磷酸化的下调， 下行信号。鉴于这些观察结果，我们假设香烟烟雾通过MARCKS-AXL 信号传导刺激成纤维细胞活化，从而促进肺纤维化进展。为了验证这个假设， 提出了三个具体目标：1）表征香烟烟雾激活的MARCKS-AXL轴在 体外成纤维细胞增殖、迁移和/或分化; 2）测定MARCKS抑制 有效地抑制体内烟雾介导的肺纤维化; 3）评估磷酸化的临床相关性， 吸烟相关IPF组织中的MARCKS和磷酸化AXL。实现这些具体目标将是 吸烟介导的纤维化进展的新分子机制，从而提供新的 例如磷酸-MARCKS和磷酸-AXL的治疗靶点，用于对抗肺纤维化。

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.