DNA Methylation Determine Hepatic Stellate Cell Activation And Fibrogenesis In The Rat Liver And Its Molecular Mechanism Of Action

Fibrosis of the liver occurs as a pathological response to hepatitis viral infectionand hepatotoxins such as ethanol, and is characterized by increased and altereddeposition of extracellular matrix (ECM) components. Activation of hepatic stellatecells (HSCs) is now known as a critical step involved in the development andmaintenance of liver fibrosis. Therefore, inhibition of HSC activation and its relatedsubsequent events, such as increased production of ECM components and enhancedproliferation, are crucial goals for intervention in the hepatic fibrogenesis cascade. Toexplore the cellular and molecular mechanisms of liver fibrosis, we choose HSC as atarget for the pharmacological, molecular, and other novel therapeutics for hepaticfibrosis. One focus of this review is the inhibition of DNA methylation. Transforminggrowth factor-β1, which are important in hepatic fibrogenesis. The main contents aredivided into four sections, as follows:1. PTEN methylation status and loss of PTEN expression during in the fibrotic liversfrom CCl4-treated ratsLiver fibrosis was generated by12-weeks treatment of adult male Sprague-Dawley(200-220g) rats with CCL4(CCl4/olive oil,1:1(vol/vol) per kg body weight byintraperitoneal injection twice weekly) as previously described. After12-Weeks, thepathological and histological Masson collage dyeing were also detected. The expressionof PTEN in rat liver tissues was measured by Western blotting, RT-PCR respectively. PTEN expression was decreased during hepatic fibrosis.Methylation-specific PCRanalysis was performed. Incubation of HSC-T6cells with TGF-β1significantly inducedhypermethylation of the PTEN gene. These results suggest that TGF-β1treatmentinduces an aberrant methylation of PTEN gene promoter, which likely contributes to theloss of PTEN expression in activated HSC-T6cells.2. PTEN down-regulation due to the hypermethylation of its promoter region is restoredafter treatment of HSC-T6cells with the DNMT inhibitor5-azadCTreatment with5-azadC markedly diminished TGF-β1-induced aberranthypermethylation of the PTEN gene, loss of PTEN mRNA and protein expression inHSC-T6cells. Exposure with5-azadC also abolished TGF-β-mediated upregulation ofCol1a1and α-SMA mRNA levels. A similar decrease in α-SMA protein was alsoobserved after5-azadC treatment. These results suggest that the decreased expression ofPTEN during HSC activation was related to reversible epigenetic mechanisms,including DNA promoter region methylation.3. DNMT1is involved in PTEN hypermethylation during HSC activationImmunohistochemical analyses showed that DNMT1expression was significantlyincreased in the liver tissues from CCl4-treated rats compared to those fromvehicle-treated rats. Moreover, expression of DNMT1mRNA and protein was alsomarkedly increased in the liver tissues from the rats treated with CCl4compared withvehicle-treated groups. TGF-β1treatment markedly induced nuclear translocation ofDNMT1and upregulated expression of DNMT1in HSC-T6cells. Transfection withDNMT1RNAi but not scrambled RNAi reduced the cell viability of activated HSC-T6cells. In addition, α-SMA and Col1a1mRNA expression levels were reduced inDNMT1knockdown HSC-T6cells as compared to control or scrambled RNAi-treatedcells. Similarly, DNMT1knockdown with RNAi in HSC-T6cells downregulatedα-SMA protein level, ameliorated PTEN methylation, and restored the expression ofPTEN mRNA and protein. These results suggest that PTEN hypermethylation duringHSC activation was dependent on DNMT1.4. PTEN down-regulation due to hypermethylation of its promoter region contributes tothe activation of ERK and AKT pathway in HSC-T6cells and the fibrotic liver fromCCl4-treated rats Expression of p-AKT and p-ERK was markedly elevated in the fibrotic livers fromCCl4-treated rats compared with the liver tissues from vehicle-treated rats, whereas theexpression of total AKT and ERK proteins remained unchanged. Similarly, the proteinlevels of both p-AKT and p-ERK were increased in activated HSC-T6cells afterTGF-β1stimulation. Furthermore,treatment with5-azadC diminished TGF-β1-mediatedupregulation of both p-AKT and p-ERK proteins in HSC-T6cells. Of note, the increasein the levels of both p-AKT and p-ERK was inversely correlated with the expression ofPTEN. Finally, the effects of DNMT1silencing on the activation of p-AKT and p-EKRwere examined in HSC-T6cells. Knockdown of DNMT1significantly preventedactivation of both ERK and AKT compared to control or scrambled RNAi-treated cells,indicating a role of PTEN downregulation in p-AKT and p-ERK activation associatedwith HSC activation. These results suggest that an aberrant methylation occurred inPTEN gene promoter contributes to the increase in PI3K/AKT and ERK activity duringHSC activation.