Cell-type specific DNA-protein interactions at the tissue-type plasminogen activator promoter in human endothelial and HeLa cells in vivo and in vitro.
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 GC-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.
Arts
J
J
Gaubius Laboratory, TNO-PG, PO Box 2215, 2301 CE Leiden, The Netherlands.
Herr
I
I
Lansink
M
M
Angel
P
P
Kooistra
T
T
D022762:CCAAT-Enhancer-Binding Proteins; D018983:DNA Footprinting; D017931:DNA Primers / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases; D004727:Endothelium / Q000378:metabolism; D005786:Gene Expression Regulation / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D051839:NFI Transcription Factors; D009687:Nuclear Proteins / Q000737:chemistry; D015513:Oncogene Proteins; D015514:Oncogene Proteins, Fusion / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011518:Proto-Oncogene Proteins; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D010959:Tissue Plasminogen Activator / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D043222:Ubiquitin Thiolesterase; D014470:Umbilical Cord
D022762:CCAAT-Enhancer-Binding Proteins; C077305:CTF-1 transcription factor; D017931:DNA Primers; D004268:DNA-Binding Proteins; D051839:NFI Transcription Factors; D009687:Nuclear Proteins; D015513:Oncogene Proteins; D015514:Oncogene Proteins, Fusion; D011518:Proto-Oncogene Proteins; D014157:Transcription Factors; D010450:Endopeptidases; C473330:USP6 protein, human; D043222:Ubiquitin Thiolesterase; D010959:Tissue Plasminogen Activator; D013755:Tetradecanoylphorbol Acetate