Hepatic stellate cells (HSCs) undergo transdifferentiation into hepatic myofibroblastic cells (HMs)to participate in liver fibrogenesis. We demonstrated that this transdifferentiation is caused by loss of transcriptional regulation essential to adipogenesis. Treatment with the adipocyte differentiation cocktail or expression of a key adipogenic transcription factor such as PPARy, reverses a culture-induced HM phenotype to quiescent HSCs. These results identify PPARy as a therapeutic target and also highlight an analogy between preadipocyte-adipocyte differentiation and HSC transdifferentiation. Emerging evidence points to the importance of epigenetic regulation in adipocyte differentiation. Preadipocytic fibroblasts have increased expression of histone deacetylases (HDACs) and their recruitment to a promoter of ppary, de- acetylated histones, and consequent repression of ppary. As adipocyte differentiation ensues, this negative regulation is lifted. We hypothesize that repression of ppary in HSC-HM transdifferentiation is caused by a similar epigenetic mechanism. In support, we show that the DMA methyltransferase inhibitor 5- azadeoxycytidine, that reduces methyl-CpG and HDAC recruitment, restores PPARy expression and HSC quiescence. Further, HSC-HM transdifferentiation causes increased expression of MeCP2, the methyl-CpG binding protein (MBP) that mediates HDAC recruitment. To test and extend our hypothesis to alcoholic liver fibrosis (ALF), we wish to determine: 1) whether in vitro HSC-HM transdifferentiation is caused by epigenetic repression of ppary; 2) whether HMs from experimental ALF exhibit similar negative epigenetic changes; and 3) whether knockdown of MeCP2 prevents HSC-HM transdifferentiation and ALF. To pursue the aims, we will collaborate with the laboratory of Derek Mann who brings in expertise in epigenetic regulation. DNA methylation will be analyzed for ppary gene. Expression of epigenetic regulators (HDACs, MBPs, HMTs) and their recruitment to the ppary promoter, will be assessed by immunoblot and chromatin immunoprecipitation (ChIP) assays. ChIP will also determine modifications of histones bound to the promoter. HCSs/HMs will be isolated from rats with intragastric ethanol infusion plus enteral LPS administration that produce accentuated ALF.Epigenetic analysis of these cells will be compared to that of HMs captured by laser micro-dissection of frozen sections. To assess the role of MeCP2, three complementary approaches will be used: HSCs isolated from MeCP2 deficient mice; siRNA knockdown of MeCP2 in LX2 human HSC line; and a Cre-lox adenoviral vector system to express MeCP2 shRNA in a HM-specific manner in vivo by Cre recombinase expressed under the control of a2(l) collagen enhancer in collaboration with Dr. Kazuo Ikeda, a developer of this system. This exploratory study will be first to disclose epigenetic regulation of HSC transdifferentiation in ALF.

Agency
National Institute of Health (NIH)
Institute
National Institute on Alcohol Abuse and Alcoholism (NIAAA)
Type
Exploratory/Developmental Grants (R21)
Project #
3R21AA016682-01S1
Application #
7408834
Study Section
Special Emphasis Panel (ZAA1-DD (72))
Program Officer
Radaeva, Svetlana
Project Start
2006-09-30
Project End
2008-08-31
Budget Start
2007-04-20
Budget End
2007-08-31
Support Year
1
Fiscal Year
2007
Total Cost
$16,300
Indirect Cost
Name
University of Southern California
Department
Pathology
Type
Schools of Medicine
DUNS #
072933393
City
Los Angeles
State
CA
Country
United States
Zip Code
90089
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Mann, Jelena; Chu, David C K; Maxwell, Aidan et al. (2010) MeCP2 controls an epigenetic pathway that promotes myofibroblast transdifferentiation and fibrosis. Gastroenterology 138:705-14, 714.e1-4
Zhu, Nian-Ling; Wang, Jiaohong; Tsukamoto, Hidekazu (2010) The Necdin-Wnt pathway causes epigenetic peroxisome proliferator-activated receptor gamma repression in hepatic stellate cells. J Biol Chem 285:30463-71
Tsukamoto, Hidekazu; Machida, Keigo; Dynnyk, Alla et al. (2009) ""Second hit"" models of alcoholic liver disease. Semin Liver Dis 29:178-87