Transforming Growth Factor-beta1 Upregulates Transcription of Alpha3 Integrin Gene in Hepatocellular Carcinoma Cells Via Ets-transcription Factor-binding Motif in the Promoter Region

The invasive and metastatic potentials of hepatocellular carcinoma (HCC) are positively correlated with the expression level of alpha3beta1 integrin, a high-affinity adhesion receptor for laminin isoforms. Transforming growth factor (TGF)-beta1 stimulates non-invasive HCC cells to acquire invasive phenotypes in association with the enhanced expression of alpha3 integrin. In this study, we investigated the molecular mechanism underlying the upregulation of alpha3beta1 integrin by TGF-beta1 in non-invasive HepG2 HCC cells. The treatment of HepG2 cells with TGF-beta1 induced the expression of alpha3 integrin and potentiated these cells to adhere to laminin-5 and to migrate through laminin-5-coated membranes. The promoter activity was measured by luciferase assay with a series of deletion constructs of the 5'-flanking region of the mouse alpha3 integrin gene, and the results showed that the -260/-119 region (relative to the major transcription start site) contained elements responsive to TGF-beta1 stimulation. The introduction of mutations into the putative consensus binding sequence for the Ets-family of transcription factors located at -133 greatly decreased the promoter activity responding to TGF-beta1 stimulation. The nuclear proteins extracted from TGF-beta1-stimulated HepG2 cells yielded a larger amount of DNA-nuclear protein complexes than did those extracted from unstimulated cells, as determined by an electrophoretic mobility shift assay using an oligonucleotide containing the Ets-site as a probe. These results suggest that TGF-beta1 stimulates HepG2 cells to express a higher level of alpha3 integrin by transcriptional upregulation via Ets transcription factors and to exhibit a more invasive phenotype.