ONE OF 3 CCARGG BOX SERUM RESPONSE ELEMENTS OF THE BETA-ACTIN GENE ISAN INSULIN-RESPONSIVE ELEMENT

The cytoskeletal actins are abundant proteins in mammalian nonmuscle c ells. We have previously reported that physiological concentrations of insulin induced beta-actin transcription in rat H4 hepatoma cells. To define whether one or more of the three CCArGG box elements or other elements within the beta-actin gene promoter is an insulin response el ement, we transfected H4 cells with regions of the human beta-actin ge ne promoter fused to the chloramphenicol acetyltransferase gene. A 350 -basepair DNA fragment was isolated that mediates both insulin and ser um effects. This fragment contains at least two up-stream elements, a CCAAT box and a CCArGG box, and accounts for more than 70% of the basa l activity of the beta-actin promoter in H4 cells. There was a small, but significant, stimulatory effect of insulin over maximal serum indu ction, suggesting a difference in their mechanisms of action. Mutation of the CCAAT box drastically reduced basal expression, with no effect on insulin induction. In contrast, a mutation of the CCArGG element r educed basal expression and completely abolished insulin inducibility. Electrophoretic mobility shift assays suggested that insulin regulate d the activity, but not the binding, of a factor(s) that associates wi th the CCArGG box. These data demonstrate that in H4 cells, insulin in duction of beta-actin gene expression was mediated at least in part th rough one of the three beta-actin CCArGG elements.