title: Accessory elements, flanking DNA sequence, and promoter context play key roles in determining the efficacy of insulin and phorbol ester signaling through the malic enzyme and collagenase-1 AP-1 motifs.
abstract: Insulin stimulates malic enzyme (ME)-chloramphenicol acetyltransferase (CAT) and collagenase-1-CAT fusion gene expression in H4IIE cells through identical activator protein-1 (AP-1) motifs. In contrast, insulin and phorbol esters only stimulate collagenase-1-CAT and not ME-CAT fusion gene expression in HeLa cells. The experiments in this article were designed to explore the molecular basis for this differential cell type- and gene-specific regulation. The results highlight the influence of three variables, namely promoter context, AP-1 flanking sequence, and accessory elements that modulate insulin and phorbol ester signaling through the AP-1 motif. Thus, fusion gene transfection and proteolytic clipping gel retardation assays suggest that the AP-1 flanking sequence affects the conformation of AP-1 binding to the collagenase-1 and ME AP-1 motifs such that it selectively binds the latter in a fully activated state. However, this influence of ME AP-1 flanking sequence is dependent on promoter context. Thus, the ME AP-1 motif will mediate both an insulin and phorbol ester response in HeLa cells when introduced into either the collagenase-1 promoter or a specific heterologous promoter. But even in the context of the collagenase-1 promoter, the effects of both insulin and phorbol esters, mediated through the ME AP-1 motif are dependent on accessory factors.
authors.affiliation: Department of Molecular Physiology and Biophysics, Vanderbilt University Medical School, Nashville, Tennessee 37232, USA.
authors.forename: Julio E
authors.initials: JE
authors.lastname: Ayala
authors.forename: Ryan S
authors.initials: RS
authors.lastname: Streeper
authors.forename: Christina A
authors.initials: CA
authors.lastname: Svitek
authors.forename: Joshua K
authors.initials: JK
authors.lastname: Goldman
authors.forename: James K
authors.initials: JK
authors.lastname: Oeser
authors.forename: Richard M
authors.initials: RM
authors.lastname: O'Brien
mesh_terms: D001483:Base Sequence; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D017364:Collagenases / Q000235:genetics*; D017931:DNA Primers; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D007328:Insulin / Q000502:physiology*; D008291:Malate Dehydrogenase / Q000235:genetics*; D016296:Mutagenesis; D009838:Oligodeoxyribonucleotides; D010703:Phorbol Esters / Q000494:pharmacology*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection; D001616:beta-Galactosidase / Q000235:genetics
chemical_list: D017931:DNA Primers; D007328:Insulin; D009838:Oligodeoxyribonucleotides; D010703:Phorbol Esters; D011994:Recombinant Proteins; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D008291:Malate Dehydrogenase; D015500:Chloramphenicol O-Acetyltransferase; D001616:beta-Galactosidase; D017364:Collagenases; C417249:collagenase 1