Introduction: In early stages of hepatocellular carcinoma (HCC) TGF-β is proposed to have a tumour suppressive function whereas in advanced HCC it acts as a tumor promoter [1]. However, the relevance of a potential TGF-β1 target gene, i.e. Id1, is discussed controversial during HCC development and progression [2 – 4]. In HCC cell lines expressing various types of TGF-β receptors and being growth inhibited by TGF-β1, Id1 is suppressed by this ligand [5]. In order to refine the response pattern to TGF-β1 and identify putative regulatory circuits in HCC cells, we analysed the gene activation pattern in the TGF-β responsive HepG2 cell line. Methods and results: The time dependent mRNA expression in response to TGF-β1 was analysed by microarray analysis. 166 candidate genes were found to be differentially regulated during the time course. These were grouped in five clusters according to their expression kinetics. Candidate genes were validated by qRT-PCR and the corresponding protein expression and activation of signalling intermediates was confirmed by Western blot analysis. Most genes were localized in the immediate early cluster including pathway inhibitors, i.e. Smad6, Smad7, Skil, and Id1. The latter showed a biphasic expression, being immediate early induced (2 hrs) and thereafter repressed (4 hrs), an expression pattern that was also found in hepatic stellate cells. The function of Id1 was elucidated by transient overexpression in HepG2 and found to influence Connective Tissue Growth Factor protein expression and Smad activation. Induction and repression were direct responses since cycloheximide failed to abrogate the observed effects on Id1 expression. In contrast the ALK5 inhibitor SB-431542 partially blocked induction and repression of Id1. Conclusion: Id1 is an important target gene of TGF-β1-signalling in hepatoma cells which are sensitive to TGF-β1 and express ALK5 since this receptor is crucial for the complex regulation of this target gene.
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