L. Ciuclan

Hepatocyte-Specific Smad7 Expression Attenuates TGF-β–Mediated Fibrogenesis and Protects Against Liver Damage

BACKGROUND & AIMS The profibrogenic role of transforming growth factor (TGF)-beta in liver has mostly been attributed to hepatic stellate cell activation and excess matrix synthesis. Hepatocytes are believed to contribute to increased rates of apoptosis. METHODS Primary hepatocyte outgrowths and AML12 cells were used as an in vitro model to detect TGF-beta effects on the cellular phenotype and expression profile. Furthermore, a transgenic mouse model was used to determine the outcome of hepatocyte-specific Smad7 expression on fibrogenesis following CCl(4)-dependent damage. Samples from patients with chronic liver diseases were assessed for (partial) epithelial-to-mesenchymal transition (EMT) in hepatocytes. RESULTS In primary cell cultures and in vivo, the majority of hepatocytes survive despite activated TGF-beta signaling. These cells display phenotypic changes and express proteins characteristic for (partial) EMT and fibrogenesis. Experimental expression of Smad7 in hepatocytes of mice attenuated TGF-beta signaling and EMT, resulted in less accumulation of interstitial collagens, and improved CCl(4)-provoked liver damage and fibrosis scores compared with controls. CONCLUSIONS The data indicate that hepatocytes undergo TGF-beta-dependent EMT-like phenotypic changes and actively participate in fibrogenesis. Furthermore, ablation of TGF-beta signaling specifically in this cell type is sufficient to blunt the fibrogenic response.

Profibrogenic transforming growth factor‐β/activin receptor–like kinase 5 signaling via connective tissue growth factor expression in hepatocytes

Connective tissue growth factor (CTGF) is important for transforming growth factor‐β (TGF‐β)–induced liver fibrogenesis. Hepatic stellate cells have been recognized as its major cellular source in the liver. Here we demonstrate the induction of CTGF expression in hepatocytes of damaged livers and identify a molecular mechanism responsible for it. CTGF expression was found by immunohistochemistry in bile duct epithelial cells, hepatic stellate cells, and hepatocytes in fibrotic liver tissue from patients with chronic hepatitis B infection. Similarly, CTGF expression was induced in hepatocytes of carbon tetrachloride–treated mice. CTGF expression and secretion were detected spontaneously in a medium of hepatocytes after 3 days of culture, which was enhanced by stimulation with TGF‐β. TGF‐β–induced CTGF expression was mediated through the activin receptor–like kinase 5 (ALK5)/Smad3 pathway, whereas activin receptor–like kinase 1 activation antagonized this effect. CTGF expression in the liver tissue of TGF‐β transgenic mice correlated with serum TGF‐β levels. Smad7 overexpression in cultured hepatocytes abrogated TGF‐β–dependent and intrinsic CTGF expression, indicating that TGF‐β signaling was required. In line with these data, hepatocyte‐specific transgenic Smad7 reduced CTGF expression in carbon tetrachloride–treated animals, whereas in Smad7 knockout mice, it was enhanced. Furthermore, an interferon gamma treatment of patients with chronic hepatitis B virus infection induced Smad7 expression in hepatocytes, leading to decreased CTGF expression and fibrogenesis. Conclusion: Our data provide evidence for the profibrogenic activity of TGF‐β directed to hepatocytes and mediated via the up‐regulation of CTGF. We identify ALK5‐dependent Smad3 signaling as the responsible pathway inducing CTGF expression, which can be hindered by an activated activin receptor–like kinase 1 pathway and completely inhibited by TGF‐β antagonist Smad7. (HEPATOLOGY 2007.)

Disruption of the Smad7 gene enhances CCI4-dependent liver damage and fibrogenesis in mice

Transforming growth factor‐β (TGF‐β) signalling is induced in liver as a consequence of damage and contributes to wound healing with transient activation, whereas it mediates fibrogenesis with long‐term up‐regulation in chronic disease. Smad‐dependent TGF‐β effects are blunted by antagonistic Smad7, which is transcriptionally activated as an immediate early response upon initiation of TGF‐β signalling in most cell types, thereby providing negative feedback regulation. Smad7 can be induced by other cytokines, e.g. IFN‐γ, leading to a crosstalk of these signalling pathways. Here we report on a novel mouse strain, denoted S7ΔE1, with a deletion of exon I from the endogenous smad7 gene. The mice were viable and exhibited normal adult liver architecture. To obtain insight into Smad7‐depend‐ent protective effects, chronic liver damage was induced in mice by carbon tetrachloride (CCI4) administration. Subsequent treatment, elevated serum liver enzymes indicated enhanced liver damage in mice lacking functional Smad7. CCI4‐dependent Smad2 phosphoryla‐tion was pronounced in S7ΔE1 mice and accompanied by increased numbers of α‐smooth muscle actin positive ‘activated’ HSCs. There was evidence for matrix accumulation, with elevated collagen deposition as assessed morphometrically in Sirius red stained tissue and confirmed with higher levels of hydroxyproline in S7ΔE1 mice. In addition, the number of CD43 positive infiltrating lymphocytes as well as of apoptotic hepatocytes was increased. Studies with primary hepatocytes from S7ΔE1 and wild‐type mice indicate that in the absence of functional Smad7 protein, hepatocytes are more sensitive for TGF‐β effects resulting in enhanced cell death. Furthermore, S7ΔE1 hepatocytes display increased oxidative stress and cell damage in response to CCI4, as measured by reactive oxygen species production, glutathione depletion, lactate dehydrogenase release and lipid peroxidation. Using an ALK‐5 inhibitor all investigated CCI4 effects on hepatocytes were blunted, confirming participation of TGF‐β signalling. We conclude that Smad7 mediates a protective effect from adverse TGF‐β signalling in damaged liver, re‐iterating its negative regulatory loop on signalling.

TGF-β enhances alcohol dependent hepatocyte damage via down-regulation of alcohol dehydrogenase I

BACKGROUND & AIMS Adverse alcohol effects in the liver involve oxidative metabolism, fat deposition and release of fibrogenic mediators, including TGF-beta. The work presents an assessment of liver damaging cross-talk between ethanol and TGF-beta in hepatocytes. METHODS To investigate TGF-beta effects on hepatocytes, microarray analyses were performed and validated by qRT-PCR, Western blot analysis and immunohistochemistry. The cellular state was determined by assessing lactate dehydrogenase, cellular glutathione, reactive oxygen species, lipid peroxidation and neutral lipid deposition. RNA interference was used for gene silencing in vitro. RESULTS TGF-beta is induced in mouse livers after chronic ethanol insult, enhances ethanol induced oxidative stress and toxicity towards cultured hepatocytes plus induces lipid-, oxidative stress metabolism- and fibrogenesis-gene expression signatures. Interestingly, TGF-beta down-regulates alcohol metabolizing enzyme Adh1 mRNA in cultured hepatocytes and liver tissue from TGF-beta transgenic mice via the ALK5/Smad2/3 signalling branch, with Smad7 as a potent negative regulator. ADH1 deficiency is a determining factor for the increased lipid accumulation and Cyp2E1 dependent toxicity in liver cells upon alcohol challenge. Further, ADH1 expression was decreased during liver damage in an intragastric ethanol infusion mouse model. CONCLUSION In the presence of ethanol, TGF-beta displays pro-steatotic action in hepatocytes via decreasing ADH1 expression. Low ADH1 levels are correlated with enhanced hepatocyte damage upon chronic alcohol consumption by favoring secondary metabolic pathways.

105 NOTCH SIGNALING PLAYS A CRITICAL ROLE IN EXPERIMENTAL AND HUMAN LIVER FIBROGENSIS

105 NOTCH SIGNALING PLAYS A CRITICAL ROLE IN EXPERIMENTAL AND HUMAN LIVER FIBROGENSIS S. Dooley, Y. Liu, I. Ilkavets, H. Shen, V. Zimmer, R.M. Bohle, C. Zhu, C. Xu, C. Yu, C. Meyer, L. Ciuclan, C. Stump, A. Muller, T. Huang, Y. Li, F. Lammert, M.V. Singer, P.R. Mertens, H.-L. Weng. Molecular Hepatology – Alcohol Dependent Diseases, II. Medical Clinic Faculty of Medicine at Mannheim, University of Heidelberg, Mannheim, Department of Internal Medicine II, Institute of Pathology, University of the Saarland, Homburg, Department of Nephrology and Hypertention, Otto-von-Guericke-University, Magdeburg, Germany; Department of Gastroenterology, the First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, Department of Cardiac Vascular Medicine, Affiliated Hospital, Medical School, Ningbo University, Ningbo, China E-mail: honglei.weng@medma.uni-heidelberg.de