SUPPRESSION OF ALBUMIN ENHANCER ACTIVITY BY H-RAS AND AP-1 IN HEPATOCYTE CELL-LINES

We demonstrated, using a transient transfection assay, that the albumi n enhancer increased the expression of the albumin promoter in a highl y differentiated, simian virus 40 (SV40)-immortalized hepatocyte cell line, CWSV1, but was not functional in two ras-transformed cell lines (NR3 and NR4) derived from CWSV1 by stable transfection with the T24ra s oncogene. A transient cotransfection assay showed that T24ras and no rmal c-Ha-ras were each able to inhibit the activity of the albumin en hancer in an immortal hepatocyte cell line. DNase I footprinting and g el mobility shift assays demonstrated that the DNA binding activities specific to the albumin enhancer were not decreased in the ras-transfo rmed cells. ras also did not diminish the expression of HNF1 alpha, C/ EBP alpha, HNF3 alpha, HNF3 beta, or HNF3 gamma but did significantly increase AP-1 binding activity. Three AP-1 binding sites were identifi ed within the albumin enhancer, and DNA binding activities specific to these AP-1 sites were induced in the ras-transformed hepatocytes. Sub sequent functional assays showed that overexpression of c-jun and c-fo s inhibited the activity of the albumin enhancer. Site-directed mutage nesis of the AP-1 binding sites in the albumin enhancer partially abro gated the suppressing effect of ras and c-jun/c-fos on the enhancer. T hese functional Studies therefore supported the results of the structu ral studies with AP-1. We conclude that the activity of the albumin en hancer is subject to regulation by ras signaling pathways and that the effect of ras on the albumin enhancer activity may be mediated by AP- 1.