Interaction of AP-1-, AP-2-, and Sp1-like proteins with two distinct sites in the upstream regulatory region of the plasminogen activator inhibitor-1 gene mediates the phorbol 12-myristate 13-acetate response

Phorbol 12-myristate 13-acetate induces a 3- and 10-fold induction of chloramphenicol acetyltransferase (CAT) activity in HT1080 and HeLa cells, respectively, following transient transfection of a 336-base pair plasminogen activator inhibitor-1 (PAI-1) promoter fragment linked to a CAT reporter gene...

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Bibliographic Details
Published in:	The Journal of biological chemistry Vol. 267; no. 21; pp. 15086 - 15091
Main Authors:	DESCHEEMAEKER, K. A, WYNS, S, NELLES, L, AUWERX, J, NY, T, COLLEN, D
Format:	Journal Article
Language:	English
Published:	Bethesda, MD American Society for Biochemistry and Molecular Biology 25-07-1992
Subjects:	Base Sequence Biological and medical sciences Chloramphenicol O-Acetyltransferase - biosynthesis Chloramphenicol O-Acetyltransferase - genetics DNA-Binding Proteins - metabolism Electrophoresis Enzyme Induction Fundamental and applied biological sciences. Psychology HeLa Cells Humans Immunoblotting Molecular and cellular biology Molecular genetics Molecular Sequence Data Mutagenesis, Site-Directed Plasmids Plasminogen Inactivators - metabolism Promoter Regions, Genetic Proto-Oncogene Proteins c-jun - metabolism Sp1 Transcription Factor - metabolism Tetradecanoylphorbol Acetate - pharmacology Transcription Factor AP-2 Transcription Factors - metabolism Transcription. Transcription factor. Splicing. Rna processing Transfection Tumor Cells, Cultured Regulation(control) Gene Transcription Enzyme Enzyme inhibitor Plasminogen activator Regulatory sequence Transcription factor
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Summary:	Phorbol 12-myristate 13-acetate induces a 3- and 10-fold induction of chloramphenicol acetyltransferase (CAT) activity in HT1080 and HeLa cells, respectively, following transient transfection of a 336-base pair plasminogen activator inhibitor-1 (PAI-1) promoter fragment linked to a CAT reporter gene. Substitution mutations in the regions encompassing nucleotides -78 to -69 (TGGGTGGGGC) or -61 to -54 (TGAGTTCA), but not in the regions -155 to -149 (TGCCTCA) or -84 to -76 (AGTGAGTGG) reduced this induction. Gel electrophoresis of double-stranded -65 to -50 oligonucleotides of the PAI-1 promoter region and nuclear extracts from Hela cells produced a gel shift pattern similar to that obtained with a AP-1 consensus oligomer, and excess unlabeled AP-1 oligomer reverted binding, suggesting that this region of the PAI-1 promoter is an AP-1-like binding site. Gel electrophoresis of double-stranded -82 to -65 oligonucleotides with HeLa nuclear extracts revealed a gel shift pattern of three bands; Sp1 consensus oligomer competed with the binding to two of these bands and AP-2 consensus sequence oligomer with the binding to the third band. The -82 to -65 oligomer also bound to purified AP-2 and Sp1 proteins. Southwestern blotting of HeLa nuclear extracts revealed that the labeled oligomer spanning region -82 to -65 bound to proteins with molecular masses of 52 and 72 kDa. Consensus AP-2 oligonucleotides competed for binding of the labeled -82 to -65 oligonucleotide to the 52-kDa protein, but consensus Sp-1 oligonucleotides did not compete for binding to the 72-kDa compound. The 72-kDa component binding to the -82 to -65 region may represent a new protein involved in transcriptional regulation.
Bibliography:	ObjectType-Article-2 SourceType-Scholarly Journals-1 ObjectType-Feature-1 content type line 23
ISSN:	0021-9258 1083-351X 1083-351X
DOI:	10.1016/S0021-9258(18)42149-7