Z Gastroenterol 2006; 44 - A1_10
DOI: 10.1055/s-2006-931632

Ski/SnoN as negative regulators of certain TGF-β signaling pathways in liver cells

L Ciuclan 1, K Breitkopf 2, P Godoy 1, E Wiercinska 1, MV Singer 1, S Dooley 1
  • 1Abt. Mol. Alkoholforschung Gateroenterology, Medizin II, Universitätskrankenhaus Mannheim, Universität Heidelberg, Mannheim
  • 2Dept. of Medicine II, Mol. Alcohol Research in Gastroenterology, University Hospital Mannheim, University of Heidelberg, Germany, Mannheim

TGF-β1 is a critical factor promoting stellate cell transdifferentiation into myofibroblasts, with subsequent shift of balance in the extracellular matrix degradation toward synthesis. Since hepatocytes are the predominant liver cell type and are responsive to TGF-β, they may also actively contribute to fibrogenesis.

Aims: To study the TGF-β1 pathway in isolated hepatocytes and hepatocyte-derived cell lines, with special impact on the regulation of cell proliferation and apoptosis. Further, to investigate the Ski/ SnoN interference in specific TGF-β signaling pathways.

Methods: Primary cultured hepatocytes were characterized using RT-PCR to show expression of typical cell markers. RNA and protein expression were quantified using RT-PCR, Northern and Western blot analysis. Implication of Ski/SnoN as negative regulators for TGF-β signaling was investigated by proliferation and apoptosis assays. Finally, TGF-β target gene regulation was investigated by Real-Time PCR, transient transfections and reporter gene experiments.

Results: Mouse hepatocytes at early times of culture express little TGF-β, Smad7, Ski and SnoN. By reporter gene assays and Western blot, we found that addition of TGF-β to primary cultured mouse hepatocytes and Hepatoma1–6 cells activates both classical cascades, Smad1/5 and Smad2/3. SnoN expression is rapidly but transiently induced by TGF-β treatment in Hepatoma1–6 cells and hepatocytes.

Overexpression of Ski/SnoN does not interfere with TGF-β mediated phosphorylation of Smad2 and Smad3. Overexpression of Ski and/or SnoN in Hepatoma1–6 cells and primary hepatocytes prevents activation of an artificial Smad3/4 reporter construct, (CAGA)9-MLP-Luc, PAI–1 and Smad7 promoters, a Smad2 reporter construct, 2XARE-Luc and a Smad1/5 reporter construct, BRE-Luc.

High levels of Ski/SnoN interdict TGF-β antiproliferative effects, but not its induction of apoptosis. Smad7 expression is rapidly induced by TGF-β and can inhibit TGF-β induced apoptosis in this cell type. In this case, parallel overexpression of Ski and SnoN restores the TGF-β apoptotic effect.

Conclusions: These results suggest that Ski and SnoN interfere, directly or indirectly, with apoptotic, proliferative and profibrogenic actions of TGF-β in hepatocytes and Hepatoma1–6 cells. Our data define new details of TGF-β -induced cellular responses, which may help to define more specific TGF-β neutralizing strategies to prevent or effectively treat liver damage in the future.

Key words

Ski/SnoN - TGF-ß - fibrogenesis - liver cells