Transforming growth factor-β1 upregulates transcription of α3 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 alpha 3 beta 1 integrin, a high-affinity adhesion receptor for laminin isoforms. Transforming growth factor (TGF)-beta 1 stimulates non-invasive HCC cells to acquire invasive phenotypes in association with the enhanced expression of alpha 3 integrin. In this study, we investigated the molecular mechanism underlying the upregulation of alpha 3 beta 1 integrin by TGF-beta 1 in non-invasive HepG2 HCC cells. The treatment of HepG2 cells with TGF-beta 1 induced the expression of alpha 3 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 alpha 3 integrin gene, and the results showed that the -260/-119 region (relative to the major transcription start site) contained elements responsive to TGF-beta 1 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-beta 1 stimulation. The nuclear proteins extracted from TGF-beta 1-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-beta 1 stimulates HepG2 cells to express a higher level of alpha 3 integrin by transcriptional upregulation via Ets transcription factors and to exhibit a more invasive phenotype.