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

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 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.

Fra-1 Induces Morphological Transformation and Increases In Vitro Invasiveness and Motility of Epithelioid Adenocarcinoma Cells

“…The majority of genes selectively up-regulated in CSML100 cells, i.e., the tPA, TIMP-1, mts1, MMP-9, PAI-1, MMP-3, uPAR, and uPA genes, have been reported to be AP-1 dependent under appropriate conditions (5,18,32,33,43,45,54,59). Therefore, it was of interest to examine AP-1 in this cell system.…”

Conversion of HPV 18 positive non-tumorigenic HeLa-fibroblast hybrids to invasive growth involves loss of TNF-α mediated repression of viral transcription and modification of the AP-1 transcription complex

“…HPV 18 harbors two functional AP-1 binding sites within its URR (Garcia-Carranca et al, 1988), both equally contributing to the ®nal transcription level initiated at the P 105 promoter (Thierry et al, 1992). For EMSA analyses, we selected the distal AP-1 element at nucleotide position 7596 ± 7620 (5'-CGCACCTGGTATTAGT-CATTTTCC-3', Butz and Hoppe-Seyler, 1993), since it is located within the constitutive enhancer region, conferring not only tissue speci®city (Oord and Beard, 1990) but also inducibility to phorbol esters (Gius and Laimins, 1989), known to potentiate the eects of carcinogens and AP-1 activity (Arts et al, 1997). Control experiments using a AP-1 consensus sequence (5'-CGC TT GATGACTCAG CCG GA A -3', Lee et al, 1987) in comparison with the distal HPV 18-speci®c AP-1 binding site provided identical results, indicating that there are no dierences in DNA anity and selectivity of the binding reaction (Ryseck and Bravo, 1991).…”

The t-PA -7351C>T enhancer polymorphism decreases Sp1 and Sp3 protein binding affinity and transcriptional responsiveness to retinoic acid

“…26,27 HeLa cells were chosen as our EMSAs show that nuclear proteins derived from these cells produce the same binding pattern as those from HUVECs. In addition, Arts et al 9 have shown that t-PA gene expression is regulated in a similar fashion in HeLa cells as compared with HUVECs.…”