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 α3β1 integrin, a high-affinity adhesion receptor for laminin isoforms. Transforming growth factor (TGF)-β1 stimulates non-invasive HCC cells to acquire invasive phenotypes in association with the enhanced expression of α3 integrin. In this study, we investigated the molecular mechanism underlying the upregulation of α3β1 integrin by TGF-β1 in non-invasive HepG2 HCC cells. The treatment of HepG2 cells with TGF-β1 induced the expression of α3 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 α3 integrin gene, and the results showed that the −260/−119 region (relative to the major transcription start site) contained elements responsive to TGF-β1 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-β1 stimulation. The nuclear proteins extracted from TGF-β1-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-β1 stimulates HepG2 cells to express a higher level of α3 integrin by transcriptional upregulation via Ets transcription factors and to exhibit a more invasive phenotype.