Previous studies have shown that hepatocyte nuclear factor-4␣ (HNF4␣) is a central regulator of differentiated hepatocyte phenotype and forced expression of HNF4␣ could promote reversion of tumors toward a less invasive phenotype. However, the effect of HNF4␣ on cancer stem cells (CSCs) and the treatment of hepatocellular carcinoma (HCC) with HNF4␣ have not been reported. In this study, an adenovirus-mediated gene delivery system, which could efficiently transfer and express HNF4␣, was generated to determine its effect on hepatoma cells (Hep3B and H epatocellular carcinoma (HCC) is one of the most common cancers worldwide, and in the United States its incidence has increased by more than 90% in the past three decades. 1 Despite great advances in detection and treatment of the disease, the mortality rate remains high-especially in the advanced stage, when the disease is usually diagnosed. Even if anticancer therapies could shrink primary and metastatic tumors, such effects are usually transient, and most metastatic cancers relapse frequently.Recent evidence has demonstrated that tumors are organized in a hierarchy of heterogeneous cell populations with different biologic properties and that the populations consist of cancer stem cells (CSCs), proliferating Abbreviations: CSC, cancer stem cell; GFP, green fluorescent protein; HCC, hepatocellular carcinoma; HNF4␣, mRNA, messenger RNA; PBS, PCR, polymerase chain reaction; From the

Extracellular signal-regulated kinase 1 (ERK1) is a critical part of the mitogen-activated protein kinase signal transduction pathway, which is involved in hepatic fibrosis. However, the effect of down-regulation of ERK1 on hepatic fibrosis has not been reported. Here, we induced hepatic fibrosis in rats with dimethylnitrosamine administration or bile duct ligation. An adenovirus carrying small interfering RNA targeting ERK1 (AdshERK1) was constructed to determine its effect on hepatic fibrosis, as evaluated by histological and immunohistochemical examination. Our results demonstrated that AdshERK1 significantly reduced the expression of ERK1 and suppressed proliferation and levels of fibrosis-related genes in hepatic stellate cells in vitro. More importantly, selective inhibition of ERK1 remarkably attenuated the deposition of the extracellular matrix in fibrotic liver in both fibrosis models. In addition, both hepatocytes and biliary epithelial cells were proven to exert the ability to generate the myofibroblasts depending on the insults of the liver, which were remarkably reduced by AdshERK1. Furthermore, up-regulation of ERK1 paralleled the increased expression of transforming growth factor ␤1 (TGF-␤1), vimentin, snail, platelet-derived growth factor-BB (PDGF-BB), bone morphogenetic protein 4 (BMP4), and small mothers against decapentaplegic-1 (p-Smad1), and was in reverse correlation with E-cadherin in the fibrotic liver. Nevertheless, inhibition of ERK1 resulted in the increased level of E-cadherin in parallel with suppression of TGF-␤1, vimentin, snail, PDGF-BB, BMP4, and p-Smad1. Interestingly, AdshERK1 treatment promoted hepatocellular proliferation. Conclusion: Our study provides the first evidence for AdshERK1 suppression of hepatic fibrosis through the reversal of epithelial-mesenchymal transition of both hepatocytes and biliary epithelial cells without interference of hepatocellular proliferation. This suggests that ERK1 is implicated in hepatic fibrogenesis and selective inhibition of ERK1 by small interfering RNA may present a novel option for hepatic fibrosis treatment. (HEPATOLOGY 2009;50:1524-1536 H epatic fibrosis is the response of the liver to different chronic insults, and is characterized by the excess production and deposition of extracellular matrix (ECM) components, leading to tissue scarring and the destruction of normal hepatic parenchyma. 1 A very large number of studies have identified the hepatic stellate cells (HSCs) as the predominant source of myofibroblasts. [2][3][4] Nevertheless, recent studies suggest that

Differentiation of stem cells is tightly regulated by the microenvironment which is mainly composed of nonparenchymal cells. Herein, we investigated effect of hepatic stellate cells (HSCs) in different states on mesenchymal stem cells (MSCs) differentiation. Rat HSCs were isolated and stayed quiescent within 5 days. Primary HSCs were activated by being in vitro cultured for 7 days or cocultured with Kupffer cells for 5 days. MSCs were cocultured with HSCs of different states. Expression of hepatic lineage markers was analyzed by RT-PCR and immunofluorescence. Glycogen deposition was detected by periodic acid-schiff staining. MSCs cocultured with HSC-T6 or Kupffer cell activated HSCs were morphologically transformed into hepatocyte-like cells. Hepatic-specific marker albumin was expressed in 78.3% of the differentiated MSCs 2 weeks after initiation of coculture. In addition, the differentiated MSCs also expressed alpha-fetoprotein, cytokeratin-18, glutamine synthetase and phosphoenolpyruvate carboxykinase. Glycogen deposition was detectable in 55.4% of the differentiated MSCs 6 weeks after initiation of coculture. However, the quiescent HSCs or culture activated HSCs did not exert the ability to modulate the differentiation of MSCs. Moreover, Kupffer cell activated HSCs rather than culture activated HSCs expressed hepatocyte growth factor mRNA. We draw the conclusion that fully activated HSCs could modulate MSCs differentiation into hepatocyte-like cells.

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