Defining and targeting mechanisms of smoke-mediated fibrosis progression

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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.