Fibroblast Biology and Pulmonary Fibrosis

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PROJECT SUMMARY Idiopathic pulmonary fibrosis (IPF) is a disease characterized by progressive scarring of the lungs resulting in deterioration in lung function with a median survival of 3.5 years. Pulmonary fibrosis is thought to be a disorder of abnormal wound healing, wherein the initial trigger to the fibrotic response is injury to the alveolar epithelium, followed by an exuberant, non-resolving wound-healing response. TGF-β is the key cytokine in the pathogenesis of pulmonary fibrosis. TGF-β-driven differentiation of fibroblasts into myofibroblasts is a key process in the pathogenesis of pulmonary fibrosis; however, the mechanisms of myofibroblast activation are not completely understood. In an unbiased approach using microarray studies, we identified Anoctamin-1 (ANO1), also known as TMEM16A, as one of highly upregulated genes in response to TGF-β contributing to the proliferation, migration and differentiation of lung fibroblasts to myofibroblasts. Our data also suggest that ANO1 is increased in the lungs of IPF patients and is localized to the fibrotic areas in the IPF lung. The role of ANO1 in myofibroblast activation and pulmonary fibrosis has been poorly investigated. Since ANO1 is a calcium-activated chloride channel, we evaluated the role of chloride-sensitive protein kinase, With-No-Lysine Kinase-1 (WNK1), which similarly to ANO1 contributed to myofibroblast activation. Based on our preliminary data, we hypothesize that ANO1 is a pro-fibrotic protein promoting myofibroblast activation and pulmonary fibrosis through WNK1 pathway. To test our hypothesis, we propose the following specific aims: Specific Aim #1. Determine how ANO1-mediated control of intracellular chloride levels activates human lung fibroblasts (HLFs). Specific Aim #2. Determine the signaling mechanisms of fibroblast activation through ANO1 and WNK1 in HLF. Specific Aim #3. Elucidate the profibrotic role of ANO1 in the lung in vivo by a conditional knockout of ANO1 in fibroblasts.