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

• Published in: The Journal of biological chemistry Vol. 277; no. 31; pp. 27935 - 27944
• Main Authors: Ayala, Julio E., Streeper, Ryan S., Svitek, Christina A., Goldman, Joshua K., Oeser, James K., O'Brien, Richard M.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 02.08.2002; American Society for Biochemistry and Molecular Biology
• Subjects: Base Sequence; beta-Galactosidase - genetics; Cell Line; Chloramphenicol O-Acetyltransferase - genetics; Collagenases - genetics; DNA Primers; Gene Expression Regulation, Enzymologic - drug effects; HeLa Cells; Humans; Insulin - physiology; Malate Dehydrogenase - genetics; Mutagenesis; Oligodeoxyribonucleotides; Phorbol Esters - pharmacology; Polymerase Chain Reaction; Promoter Regions, Genetic; Recombinant Proteins - metabolism; Transcription Factor AP-1 - metabolism; Transcription Factors - metabolism; Transfection
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M203682200

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.