The present study examined the roles of peroxisome proliferator-activated receptors (PPAR) in activation of hepatic stellate cells (HSC), a pivotal event in liver fibrogenesis. RNase protection assay detected mRNA for PPAR␥1 but not that for the adipocyte-specific ␥2 isoform in HSC isolated from sham-operated rats, whereas the transcripts for neither isoforms were detectable in HSC from cholestatic liver fibrosis induced by bile duct ligation (BDL). Semi-quantitative reverse transcriptasepolymerase chain reaction confirmed a 70% reduction in PPAR␥ mRNA level in HSC from BDL. Nuclear extracts from BDL cells showed an expected diminution of binding to PPAR-responsive element, whereas NF-B and AP-1 binding were increased. Treatment of culturedactivated HSC with ligands for PPAR␥ (10 M 15-deoxy-⌬ 12,14 -PGJ 2 (15dPGJ 2 ); 0.1ϳ10 M BRL49653) inhibited DNA and collagen synthesis without affecting the cell viability. Suppression of HSC collagen by 15dPGJ 2 was abrogated 70% by the concomitant treatment with a PPAR␥ antagonist (GW9662). HSC DNA and collagen synthesis were inhibited by WY14643 at the concentrations known to activate both PPAR␣ and ␥ (>100 M) but not at those that only activate PPAR␣ (<10 M) or by a synthetic PPAR␣-selective agonist (GW9578). 15dPGJ 2 reduced ␣1(I) procollagen, smooth muscle ␣-actin, and monocyte chemotactic protein-1 mRNA levels while inducing matrix metalloproteinase-3 and CD36. 15dPGJ 2 and BRL49653 inhibited ␣1(I) procollagen promoter activity. Tumor necrosis factor ␣ (10 ng/ml) reduced PPAR␥ mRNA, and this effect was prevented by the treatment with 15dPGJ 2 . These results demonstrate that HSC activation is associated with the reductions in PPAR␥ expression and PPAR-responsive element binding in vivo and is reversed by the treatment with PPAR␥ ligands in vitro. These findings implicate diminished PPAR␥ signaling in molecular mechanisms underlying activation of HSC in liver fibrogenesis and the potential therapeutic value of PPAR␥ ligands for liver fibrosis.

Agonistic antibodies against the Fas receptor, when administered to mice in vivo, cause significant apoptosis in the liver. In this study we show that anti-Fas antibody not only causes apoptosis of liver cells but also provokes hepatic inflammation. Two hours after injection of antiFas, when mice displayed evidence of caspase-3 activation and apoptosis, we found significant hepatic induction of the CXC chemokines macrophage inflammatory protein-2 and KC. Coincident with the chemokine induction was infiltration of the hepatic parenchyma by neutrophils. Neutralization experiments identified that chemokines were the cause of Fas-induced hepatic inflammation, with KC having the predominant effect. Chemokine induction in the livers of anti-Fas-treated mice was not associated with activation of NF-B. Instead, it coincided with nuclear translocation of activator protein-1 (AP-1). AP-1 activation in liver was detected 1-2 h after anti-Fas treatment, suggesting a connection to the onset of apoptosis. When apoptosis was prevented by pretreating mice with a caspase-3 inhibitor, AP-1 activation and hepatic chemokine production were both significantly reduced. Hepatic inflammation was also reduced by 70%. Taken together, these findings indicate that Fas ligation can induce inflammation in the liver in vivo. Inflammation does not arise from Fas-mediated signaling through NF-B; rather, it represents an indirect effect, requiring activation of caspase-3 and nuclear translocation of AP-1.

Hepatic stellate cell (HSC) activation is a pivotal event in initiation and progression of hepatic fibrosis and a major contributor to collagen deposition driven by transforming growth factor beta (TGFβ). microRNAs (miRs), small non-coding RNAs modulating mRNA and protein expression, have emerged as key regulatory molecules in chronic liver disease. We investigated differentially expressed miRs in quiescent and activated HSCs to identify novel regulators of profibrotic TGFβ signaling. miR microarray analysis was performed on quiescent and activated rat HSCs. Members of the miR-17-92 cluster (19a, 19b, 92a) were significantly down-regulated in activated HSCs. Since miR 19b showed the highest fold-change of the cluster members, activated HSCs were transfected with miR 19b mimic or negative control and TGFβ signaling and HSC activation assessed. miR 19b expression was determined in fibrotic rat and human liver specimens. miR 19b mimic negatively regulated TGFβ signaling components demonstrated by decreased TGFβ receptor II (TGFβRII) and SMAD3 expression. Computational prediction of miR 19b binding to the 3’UTR of TGFβRII was validated by luciferase reporter assay. Inhibition of TGFβ signaling by miR 19b was confirmed by decreased expression of type I collagen and by blocking TGFβ-induced expression of α1(I) and α2(I) procollagen mRNAs. miR 19b blunted the activated HSC phenotype by morphological assessment and decreased αSMA expression. Additionally, miR 19b expression was markedly diminished in fibrotic rat liver compared to normal liver; similarly, miR 19b expression was markedly down-regulated in fibrotic compared to normal human livers. CONCLUSIONS miR 19b is a novel regulator of TGFβ signaling in HSCs suggesting a potential therapeutic approach for hepatic fibrosis.