A transcriptional factor induced by HGF

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Growth factors induce cell proliferation in hepatocytes by stimulating progression through the G1 phase of the cell cycle. Induction of cyclin D1 expression is a critical feature of the mitogenic action of growth factors. Recently, we have reported that hepatocyte growth factor (HGF) and epidermal growth factor (EGF) additively stimulate a Ras/MAPK pathway, resulting in additive enhancement of cyclin D1 expression in primary cultured rat hepatocytes. In the present study, we isolated the rat cyclin D1 gene 5'-flanking sequence and identified two transcriptional regulatory regions by transient transfection studies using dRLh84 rat hepatoma cells. One (CD1CRE) was mapped to a potential cAMP-responsive element (CRE) at position -41 bp, while another (CD1E0.7) was located 753 bp upstream of the initiation site. Electrophoretic gel mobility shift assays (EMSAs) indicated that a protein binding to the CD1CRE was a CREB.In contrast, competition EMSA showed that CD1E0.7 did not interact with hepatocyte nuclear factor (HNF)-3β, nuclear factor of activated T cells (NF-AT), or the Ets family, although CD1E0.7 revealed high homology with a binding site for these nuclear factors through a computer search. Moreover, the specific interaction of CD1E0.7 with a protein extracted from primary cultured rat hepatocytes treated with HGF or EGF was detected. These results indicate that CREB binds to CD1CRE and mediates the activation of the cyclin D1 promoter in rat hepatoma cells, and suggest that CD1E0.7 is possibly occupied by a transcription factor induced by growth factors in rat hepatocytes.