Characterization of the TGF-β/Smad signalling pathways and their negative feedback regulators Ski and SnoN in hepatocytes

Introduction: TGF-β is a profibrotic cytokine that plays a central role in the onset and progression of chronic liver diseases. TGF-β activity is tightly controlled by multiple mechanisms, in which antagonising Smads activities by different co-repressors is an important regulatory component. Aims: In this study, we focus on identifying the role of Ski and SnoN as negative regulators of signal transduction by TGF-β in mouse hepatocytes and Hepa1–6 cell line, with special impact on the regulation of cell proliferation, fibrogenesis and apoptosis. Methods: To obtain a comprehensive view on TGF-β effects in hepatocytes, Affymetrix microarray analysis was performed. Results were confirmed by qRT-PCR, Northern blot and Western blot analysis. Implication of Ski/SnoN as negative regulators of TGF-β signaling was investigated by proliferation, apoptosis and reporter gene assays. Results: During culture of mouse hepatocytes expression of antagonists Ski and Snon is up-regulated whereas parenchymal cell markers are unchanged. Snon/Ski transcription is transiently increased by TGF-β treatment thus providing a negative feedback loop control. TGF-β stimulation activates both Smad1/5 and Smad2/3 cascades. Overexpression of the mentioned antagonists does not interfere with TGF-β mediated phosphorylation of Smad2/3. Ski/SnoN expression prevents activation of specific Smad 2/3/4 reporter constructs, (CAGA)9-MLP-Luc, PAI-1 Smad7 or 2XARE-Luc promoter and a Smad1/5 reporter construct, BRE-Luc. High levels of Ski/SnoN interdict TGF-β antiproliferative effects, but not its induction of apoptosis. Furthermore, at the protein level SnoN expression abrogated TGF-β induction of tested fibrogenesis related genes, for example, PAI-1 and CTGF. Conclusions: Our data provide evidence for pro-fibrogenic activity of TGF-β in hepatocytes and Hepatoma1–6 cells and suggest that Ski and SnoN interfere, directly or indirectly with apoptotic, proliferative and profibrogenic actions of TGF-β.