: Hepatic fibrosis is the result of chronic liver injury and can lead to end-stage liver disease and death, affecting millions of patients worldwide. The overall goal of this project is to elucidate the role of KLF6, a transcription factor, in hepatic stellate cell activation and liver fibrosis. The initial discovery that KLF6, a growth suppressive gene, is rapidly induced when stellate cells undergo a proliferative burst, presented a paradox that has now been resolved with the discovery that KLF6 can undergo alternative splicing in rodents and humans to shorter, dominant negative isoforms (""""""""KLF6sv1"""""""" and """"""""KLF6sv2"""""""") that lack all or part of the DMA binding domain. Indeed, alternative splicing of KLF6 increases during stellate cell activation in vivo and in Vitro, and KLF6 splice proteins antagonize full length KLF6 function. The hypothesis is that during liver injury increased KLF6 splicing is stimulated by TGFbeta1 and oxidant stress, and mediated by Ras, which then stimulates expression of profibrotic genes. This hypothesis will be tested through the following Specific Aims: 1. To assess, using specific siRNAs, how the main KLF6 alternative splice form (KLF6sv1) regulates human stellate cell expression of collagen alpha 1(1), MMP-2, TIMP-1&2,TGFbeta1, and beta-PDGF-R, and the stellate cell's transcriptional profile as determined by cDNA microarray. 2) To explore the roles of Ras, oxidant stress and TGFbeta 1 in regulating KLF6 splicing in human stellate cells; 3) To determine the impact of KLF6 full-length, or KLF6sv1 over-expression in stellate cells in transgenic mice by: a) assessing fibrogenic responses in these mice using two models of liver injury; b) comparing responses of normal vs. transgenic stellate cells in terms of growth, apoptosis and expression of fibrogenic genes. 4) To evaluate the response of KLF6 mice to hepatic injury from bile duct occlusion and thioacetamide. These innovative studies could lead to new insights into stellate cell activation that may offer novel approaches to treating hepatic fibrosis, for which there are currently no effective therapies.
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