One of three CCArGG box/serum response elements of the beta-actin gene is an insulin-responsive element.
The cytoskeletal actins are abundant proteins in mammalian nonmuscle cells. 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 element, we transfected H4 cells with regions of the human beta-actin gene promoter fused to the chloramphenicol acetyltransferase gene. A 350-basepair DNA fragment was isolated that mediates both insulin and serum 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 basal activity of the beta-actin promoter in H4 cells. There was a small, but significant, stimulatory effect of insulin over maximal serum induction, 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 reduced basal expression and completely abolished insulin inducibility. Electrophoretic mobility shift assays suggested that insulin regulated the activity, but not the binding, of a factor(s) that associates with the CCArGG box. These data demonstrate that in H4 cells, insulin induction of beta-actin gene expression was mediated at least in part through one of the three beta-actin CCArGG elements.
Onyia
J E
JE
Department of Physiology, State University of New York Health Science Center, Syracuse 13210.
Halladay
D L
DL
Messina
J L
JL
D000199:Actins / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D001790:Blood Physiological Phenomena; D004247:DNA / Q000378:metabolism; D017353:Gene Deletion; D015870:Gene Expression / Q000187:drug effects; D005796:Genes; D007328:Insulin / Q000494:pharmacology; D015335:Molecular Probes / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic; D051381:Rats; D012045:Regulatory Sequences, Nucleic Acid; D014407:Tumor Cells, Cultured
D000199:Actins; D007328:Insulin; D015335:Molecular Probes; D004247:DNA