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Cell-type specific DNA-protein interactions at the tissue-type plasminogen activator promoter in human endothelial and HeLa cells in vivo and in vitro

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Author: Arts, J. · Herr, I. · Lansink, M. · Angel, P. · Kooistra, T.
Type:article
Date:1997
Institution: TNO Preventie en Gezondheid
Source:Nucleic Acids Research, 2, 25, 311-317
Identifier: 233997
doi: doi:25.2.311
Keywords: Health · Phorbol 13 acetate 12 myristate · Endothelium cell · Gel mobility shift assay · Gene control · Gene expression · Human cell · Oncogene c fos · Oncogene c jun · Promoter region · Protein dna interaction · Transcription regulation · CCAAT-Enhancer-Binding Proteins · DNA Footprinting · DNA Primers · DNA-Binding Proteins · Electrophoresis, Polyacrylamide Gel · Endopeptidases · Endothelium · Gene Expression Regulation · Hela Cells · Humans · NFI Transcription Factors · Nuclear Proteins · Oncogene Proteins · Oncogene Proteins, Fusion · Polymerase Chain Reaction · Promoter Regions (Genetics) · Tetradecanoylphorbol Acetate · Tissue Plasminogen Activator · Transcription Factors · Umbilical Cord · Murinae

Abstract

Tissue-type plasminogen activator (t-PA) gene expression in human endothelial cells and HeLa cells is stimulated by the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) at the level of transcription. To study the mechanism of transcriptional regulation, we have characterized a segment of the t-PA gene extending from -135 to +100 by in vivo footprinting analysis [dimethyl sulphate (DMS) method] and gel mobility shift assay. In vivo footprinting analysis revealed changes in cleavage pattern in five distinct promoter elements in both endothelial cells and HeLa cells, including a PMA-responsive element (TRE), a CTF/NF-1 binding site and three CC-boxes, and an altered cleavage pattern of the TRE and CTF/NF-1 element after PMA treatment of HeLa cells. Although endothelial cells and Hela cells differed in the exact G residues protected by nuclear proteins, in vitro bandshift analysis showed that nuclear protein binding to the t-PA promoter was qualitatively and quantitatively very similar in both cell types, except for the TRE. Protein binding to the TRE under non-stimulated conditions was much higher in human endothelial cells than in HeLa cells, and this TRE-bound protein showed a lower dissociation rate in the endothelial cells than in HeLa cells. In endothelial cells, the proteins bound to the TRE consisted mainly of the AP-1 family members JunD and Fra-2, while in HeLa cells predominantly JunD, FosB and Fra-2 were bound. The proteins bound to the other protected promoter elements were identified as SP-1 (GC-box II and III) and CTF/NF-1 (CTF/NF-1 binding site). After PMA treatment of the cells, AP-1 and SP-1 binding was increased two-fold in endothelial cell nuclear extracts and > 20-fold in HeLa nuclear extracts. In the endothelial cells, all Jun and Fos forms (c-Jun, JunB, JunD, c-Fos, FosB, Fra-1 and Fra-2) were part of the AP-1 complex after PMA induction. In HeLa cells, the complex consisted predominantly of c-Jun and the Fos family members FosB and Fra-2. In the light of previous studies involving mutational analysis of the human and murine t-PA promoter our results underline an important role of the five identified promoter regions in basal and PMA-stimulated t-PA gene expression in intact human endothelial cells and HeLa cells. The small differences in DMS protection pattern and differences in the individual AP-1 components bound in endothelial cells and HeLa cells point to subtle cell-type specific differences in t-PA gene regulation. Chemicals/CAS: CCAAT-Enhancer-Binding Proteins; CTF-1 transcription factor; DNA Primers; DNA-Binding Proteins; Endopeptidases, EC 3.4.-; NFI Transcription Factors; Nuclear Proteins; Oncogene Proteins; Oncogene Proteins, Fusion; Tetradecanoylphorbol Acetate, 16561-29-8; Tissue Plasminogen Activator, EC 3.4.21.68; Transcription Factors; USP6 protein, human, EC 3.4.99.-