No abstract

The hepatic stellate cell (HSC) is the predominant cell type responsible for excess collagen deposition during liver fibrosis. Both transforming growth factor-␤ (TGF-␤), the most potent fibrogenic cytokine for HSCs, which classically activates Smad signaling, and p38 MAPK signaling have been shown to influence collagen gene expression; however, the relative contribution and mechanisms that these two signaling pathways have in regulating collagen gene expression have not been investigated. The aim of this study was to investigate the relative roles and mechanisms of both Smad and p38 MAPK signaling in ␣1(I) collagen gene expression in HSCs. Inhibiting either p38 MAPK or Smad signaling reduced ␣1(I) collagen mRNA expression in untreated or TGF-␤-treated HSCs, and when both signaling pathways were simultaneously inhibited, ␣1(I) collagen gene expression was essentially blocked. Both signaling pathways were found to independently and additively increase ␣1(I) collagen gene expression by transcriptional mechanisms. TGF-␤ treatment increased ␣1(I) collagen mRNA half-life, mediated by increased stability of ␣1(I) collagen mRNA through p38 MAPK signaling but not through Smad signaling. In conclusion, both p38 MAPK and Smad signaling independently and additively regulate ␣1(I) collagen gene expression by transcriptional activation, whereas p38 MAPK and not Smad signaling increased ␣1(I) collagen mRNA stability.

During fibrosis the hepatic stellate cell (HSC) undergoes a complex activation process characterized by increased proliferation and extracellular matrix deposition. The 70-kDa ribosomal S6 kinase (p70S6K) is activated by mitogens, growth factors, and hormones in a phosphatidylinositol 3-kinase-dependent manner. p70S6K regulates protein synthesis, proliferation, and cell cycle control. Because these processes are involved in HSC activation, we investigated the role of p70S6K in HSC proliferation, cell cycle control, and type I collagen expression. Platelet-derived growth factor (PDGF) stimulated p70S6K phosphorylation, which was blocked by LY294002, an inhibitor of phosphatidylinositol 3-kinase. Rapamycin blocked phosphorylation of p70S6K but had no affect on PDGF-induced Akt phosphorylation, positioning p70S6K downstream of Akt. Transforming growth factor-beta, which inhibits HSC proliferation, did not affect PDGF-induced p70S6K phosphorylation. Rapamycin treatment did not affect alpha1(I) collagen mRNA but reduced type I collagen protein secretion. Expression of smooth muscle alpha-actin was not affected by rapamycin treatment, indicating that HSC activation was not altered. Rapamycin inhibited serum-induced DNA synthesis approximately 2-fold. Moreover, rapamycin decreased expression of cyclins D1, D3, and E but not cyclin D2, Rb-Ser780, and Rb-Ser795. Together, p70S6K plays a crucial role in HSC proliferation, collagen expression, and cell cycle control, thus representing a potential therapeutic target for liver fibrosis.

Transforming growth factor-␤ (TGF-␤) is a potent inhibitor of cell proliferation. This study investigated whether overexpression of Smad7, which blocks TGF-␤-induced activation of Smad2/3, could prevent the suppression of regeneration of small-for-size liver grafts. Rats were intravenously given adenoviruses (2 ϫ 10 9 pfu/rat) carrying the LacZ gene or the Smad7 gene (Ad-Smad7) 3 days prior to liver harvesting. Half-size livers were implanted into recipients of the same weight or twice the donor weight, and this resulted in half-size or quarter-size liver grafts. Cell proliferation, detected by 5-bromo-2Ј-deoxyuridine (BrdU) incorporation, increased to 23% in half-size grafts at 38 hours after implantation but was only 4% in quarter-size grafts. Graft weight did not increase after 38 hours in full-size and quarter-size grafts but increased 28% in half-size grafts. Ad-Smad7 restored BrdU labeling to 32%, and the graft weight increased to 43% in quarter-size grafts. Serum total bilirubin increased approximately 30-fold after the implantation of quarter-size grafts. Ad-Smad7 blunted hyperbilirubinemia by 80%. The basal hepatic TGF-␤ 1 level was 7 ng/g of liver wet weight, and this increased to 30 ng/g at 1.5 hours after the transplantation of full-size grafts but decreased rapidly afterwards. After the transplantation of quarter-size grafts, however, TGF-␤ 1 progressively increased to 159 ng/g in 38 hours. Nuclear phosphorylated Smad2/3 was barely detectable, and p21Cip1 expression was negligible in full-size grafts but increased markedly in quarter-size grafts. Ad-Smad7 blocked Smad2/3 activation and expression of p21Cip1. Together, these data show that TGF-␤ is responsible, at least in part, for the defective liver regeneration in small-for-size grafts by activating the Smad signaling pathway. Liver Transpl 16:181-190, 2010.