Hepatocyte nuclear factor-1 alpha inhibits insulin promoter factor 1-dependent transactivation of the human insulin gene

To investigate the regulational interaction of hepatocyte nuclear factor-1 alpha (HNF-1 alpha) and insulin promoter factor 1 (IPF1) on insulin gene ex pression, either or both of the expression vectors carrying each transcript ion factor were transiently transfected into HeLa cells, RINm5F cells and M IN6 cells together with the luciferase reporter construct driven by a human preproinsulin gene promoter (-1998 to +237) designated as pINS-1998/luc. I PF1-transfection into HeLa cells strongly stimulated the luciferase activit y to 725 fold that of the basal level. In contrast, HNF-1 alpha -transfecti on resulted in only a 6.7 fold increase. In co-transfection experiments, in creasing the amount of HNF-1 alpha resulted in an 84.5% and 74.4% decrease in IPF1-stimulated luciferase activity in HeLa and RINm5F cells, respective ly Deletion constructs designated as pINS-248/luc, pINS-213/luc and pINS-18 5/luc were transfected into RINm5F cells to determine the role of the A3 el ement and its 5 ' flanking sequence in the inhibitory effect of HNF-1 alpha . The results showed that the inhibiting effects of HNF-1 alpha with pINS-2 13/luc and pINS-185/luc were significantly smaller than those with both pIN S-1998/luc and pINS-248/luc. Transfection into MIN6 cells with pINS-1998/lu c in the absence of IPF1 resulted in constitutional transactivation of the insulin gene, and this transactivation was abolished by the co-transfection with HNF-1 alpha. The present data indicate that IPF1 rather than HNF-1 al pha predominantly transactivates the insulin gene, and that HNF-1 alpha inh ibits IPF1-dependent insulin gene transactivation mediated through the 5 ' flanking sequence of the A3 element. It is suggested that HNF-1 alpha may b e involved in insulin gene expression as a negative regulator.