Certain lung injuries induce large increases in connective tissue content, particularly collagen, resulting in pulmonary fibrosis. During injury, inflammation, and repair, cells are exposed to molecules such as interferon-gamma (IFN-gamma) and transforming growth factor-beta (TGF-beta) that are released by inflammatory cells and regulate production of collagen. Collagen type I transcription is activated after inflammatory response to injury in order to repair damage. This process is followed by repression of transcription. Without transcriptional repression, progressive fibrosis results in the lung. We hypothesize that changes in transcription require multiple proteins interacting cooperatively to alter transcription and chromatin structure in response to cytokine signals. During the last funding period, we focused on the mechanism of IFN-gamma induced repression of collagen transcription. IFN-gamma increases expression of regulatory factor for X-box 5 (RFX5) complex proteins which localizes in the nucleus, interacts with the collagen gene transcriptional start site and represses collagen synthesis. Class II transactivator (CIITA) dramatically increases early during IFN-gamma treatment and interacts with RFX5 complex. IFN-gamma-induced CIITA protein is responsible for both activation of major histocompatibility complex (MHC) and repression of collagen gene expression. Clinical trials for interstitial pulmonary fibrosis with IFN-gamma have been unsuccessful due to increased inflammatory response. We hypothesize that RFX5/CIITA proteins may be responsible for activating inflammatory responses while repressing collagen through separate CIITA transactivation and repression domains. We have compelling evidence that many proteins including co-repressors bind to the collagen start site during repression. Activation by agents such as TGF-beta may recruit different proteins to the collagen gene.
The specific aims are to; 1) Determine the proteins interacting with the collagen start site during IFN-gamma treatment. 2) Examine the functional interactions of activation proteins binding upstream in the promoter with RFX family of proteins with and without TGF-beta. 3) Examine bleomycin induced fibrosis in animals with CIITA mutations and/or deficiencies with and without collagen-promoter-CAT constructs to investigate inflammation and collagen transcriptional regulation during fibrosis.
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