Nucleic Acids Research, Vol 25, Issue 2 311-317, Copyright © 1997 by Oxford University Press
J Arts, I Herr, M Lansink, P Angel and T Kooistra
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.
ARTICLES
Cell-type specific DNA-protein interactions at the tissue-type plasminogen activator promoter in human endothelial and HeLa cells in vivo and in vitro
Gaubius Laboratory, TNO-PG, PO Box 2215, 2301 CE Leiden, The Netherlands.
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