Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1997 Jan 15;25(2):311–317. doi: 10.1093/nar/25.2.311

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

J Arts 1, I Herr 1, M Lansink 1, P Angel 1, T Kooistra 1
PMCID: PMC146448  PMID: 9016559

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.

Full Text

The Full Text of this article is available as a PDF (187.2 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Angel P., Karin M. The role of Jun, Fos and the AP-1 complex in cell-proliferation and transformation. Biochim Biophys Acta. 1991 Dec 10;1072(2-3):129–157. doi: 10.1016/0304-419x(91)90011-9. [DOI] [PubMed] [Google Scholar]
  2. Auwerx J., Sassone-Corsi P. IP-1: a dominant inhibitor of Fos/Jun whose activity is modulated by phosphorylation. Cell. 1991 Mar 8;64(5):983–993. doi: 10.1016/0092-8674(91)90322-p. [DOI] [PubMed] [Google Scholar]
  3. Carmeliet P., Collen D. Gene targeting and gene transfer studies of the biological role of the plasminogen/plasmin system. Thromb Haemost. 1995 Jul;74(1):429–436. [PubMed] [Google Scholar]
  4. Collen D., Lijnen H. R. Basic and clinical aspects of fibrinolysis and thrombolysis. Blood. 1991 Dec 15;78(12):3114–3124. [PubMed] [Google Scholar]
  5. Darrow A. L., Rickles R. J., Pecorino L. T., Strickland S. Transcription factor Sp1 is important for retinoic acid-induced expression of the tissue plasminogen activator gene during F9 teratocarcinoma cell differentiation. Mol Cell Biol. 1990 Nov;10(11):5883–5893. doi: 10.1128/mcb.10.11.5883. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fisher R., Waller E. K., Grossi G., Thompson D., Tizard R., Schleuning W. D. Isolation and characterization of the human tissue-type plasminogen activator structural gene including its 5' flanking region. J Biol Chem. 1985 Sep 15;260(20):11223–11230. [PubMed] [Google Scholar]
  7. Grulich-Henn J., Müller-Berghaus G. Regulation of endothelial tissue plasminogen activator and plasminogen activator inhibitor type 1 synthesis by diacylglycerol, phorbol ester, and thrombin. Blut. 1990 Jul;61(1):38–44. doi: 10.1007/BF01739432. [DOI] [PubMed] [Google Scholar]
  8. Henderson B. R., Sleigh M. J. TATA box-independent transcription of the human tissue plasminogen activator gene initiates within a sequence conserved in related genes. FEBS Lett. 1992 Sep 7;309(2):130–134. doi: 10.1016/0014-5793(92)81080-6. [DOI] [PubMed] [Google Scholar]
  9. Herr I., van Dam H., Angel P. Binding of promoter-associated AP-1 is not altered during induction and subsequent repression of the c-jun promoter by TPA and UV irradiation. Carcinogenesis. 1994 Jun;15(6):1105–1113. doi: 10.1093/carcin/15.6.1105. [DOI] [PubMed] [Google Scholar]
  10. Holmberg M., Leonardsson G., Ny T. The species-specific differences in the cAMP regulation of the tissue-type plasminogen activator gene between rat, mouse and human is caused by a one-nucleotide substitution in the cAMP-responsive element of the promoters. Eur J Biochem. 1995 Jul 15;231(2):466–474. doi: 10.1111/j.1432-1033.1995.tb20720.x. [DOI] [PubMed] [Google Scholar]
  11. Jaffe E. A., Nachman R. L., Becker C. G., Minick C. R. Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest. 1973 Nov;52(11):2745–2756. doi: 10.1172/JCI107470. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kooistra T., Bosma P. J., Toet K., Cohen L. H., Griffioen M., van den Berg E., le Clercq L., van Hinsbergh V. W. Role of protein kinase C and cyclic adenosine monophosphate in the regulation of tissue-type plasminogen activator, plasminogen activator inhibitor-1, and platelet-derived growth factor mRNA levels in human endothelial cells. Possible involvement of proto-oncogenes c-jun and c-fos. Arterioscler Thromb. 1991 Jul-Aug;11(4):1042–1052. doi: 10.1161/01.atv.11.4.1042. [DOI] [PubMed] [Google Scholar]
  13. Kooistra T., Bosma P. J., Toet K., Cohen L. H., Griffioen M., van den Berg E., le Clercq L., van Hinsbergh V. W. Role of protein kinase C and cyclic adenosine monophosphate in the regulation of tissue-type plasminogen activator, plasminogen activator inhibitor-1, and platelet-derived growth factor mRNA levels in human endothelial cells. Possible involvement of proto-oncogenes c-jun and c-fos. Arterioscler Thromb. 1991 Jul-Aug;11(4):1042–1052. doi: 10.1161/01.atv.11.4.1042. [DOI] [PubMed] [Google Scholar]
  14. Kooistra T., Schrauwen Y., Arts J., Emeis J. J. Regulation of endothelial cell t-PA synthesis and release. Int J Hematol. 1994 Jun;59(4):233–255. [PubMed] [Google Scholar]
  15. Levin E. G., Marotti K. R., Santell L. Protein kinase C and the stimulation of tissue plasminogen activator release from human endothelial cells. Dependence on the elevation of messenger RNA. J Biol Chem. 1989 Sep 25;264(27):16030–16036. [PubMed] [Google Scholar]
  16. Levin E. G., Santell L. Stimulation and desensitization of tissue plasminogen activator release from human endothelial cells. J Biol Chem. 1988 Jul 5;263(19):9360–9365. [PubMed] [Google Scholar]
  17. Maciag T., Cerundolo J., Ilsley S., Kelley P. R., Forand R. An endothelial cell growth factor from bovine hypothalamus: identification and partial characterization. Proc Natl Acad Sci U S A. 1979 Nov;76(11):5674–5678. doi: 10.1073/pnas.76.11.5674. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  19. Medcalf R. L., Rüegg M., Schleuning W. D. A DNA motif related to the cAMP-responsive element and an exon-located activator protein-2 binding site in the human tissue-type plasminogen activator gene promoter cooperate in basal expression and convey activation by phorbol ester and cAMP. J Biol Chem. 1990 Aug 25;265(24):14618–14626. [PubMed] [Google Scholar]
  20. Mueller P. R., Wold B. In vivo footprinting of a muscle specific enhancer by ligation mediated PCR. Science. 1989 Nov 10;246(4931):780–786. doi: 10.1126/science.2814500. [DOI] [PubMed] [Google Scholar]
  21. Mul Y. M., Van der Vliet P. C. Nuclear factor I enhances adenovirus DNA replication by increasing the stability of a preinitiation complex. EMBO J. 1992 Feb;11(2):751–760. doi: 10.1002/j.1460-2075.1992.tb05108.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ohlsson M., Leonardsson G., Jia X. C., Feng P., Ny T. Transcriptional regulation of the rat tissue type plasminogen activator gene: localization of DNA elements and nuclear factors mediating constitutive and cyclic AMP-induced expression. Mol Cell Biol. 1993 Jan;13(1):266–275. doi: 10.1128/mcb.13.1.266. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Pecorino L. T., Darrow A. L., Strickland S. In vitro analysis of the tissue plasminogen activator promoter reveals a GC box-binding activity present in murine brain but undetectable in kidney and liver. Mol Cell Biol. 1991 Jun;11(6):3139–3147. doi: 10.1128/mcb.11.6.3139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Rickles R. J., Darrow A. L., Strickland S. Differentiation-responsive elements in the 5' region of the mouse tissue plasminogen activator gene confer two-stage regulation by retinoic acid and cyclic AMP in teratocarcinoma cells. Mol Cell Biol. 1989 Apr;9(4):1691–1704. doi: 10.1128/mcb.9.4.1691. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Santell L., Levin E. G. Cyclic AMP potentiates phorbol ester stimulation of tissue plasminogen activator release and inhibits secretion of plasminogen activator inhibitor-1 from human endothelial cells. J Biol Chem. 1988 Nov 15;263(32):16802–16808. [PubMed] [Google Scholar]
  26. Santell L., Marotti K., Bartfeld N. S., Baynham P., Levin E. G. Disruption of microtubules inhibits the stimulation of tissue plasminogen activator expression and promotes plasminogen activator inhibitor type 1 expression in human endothelial cells. Exp Cell Res. 1992 Aug;201(2):358–365. doi: 10.1016/0014-4827(92)90284-f. [DOI] [PubMed] [Google Scholar]
  27. Santoro C., Mermod N., Andrews P. C., Tjian R. A family of human CCAAT-box-binding proteins active in transcription and DNA replication: cloning and expression of multiple cDNAs. Nature. 1988 Jul 21;334(6179):218–224. doi: 10.1038/334218a0. [DOI] [PubMed] [Google Scholar]
  28. Stein B., Baldwin A. S., Jr, Ballard D. W., Greene W. C., Angel P., Herrlich P. Cross-coupling of the NF-kappa B p65 and Fos/Jun transcription factors produces potentiated biological function. EMBO J. 1993 Oct;12(10):3879–3891. doi: 10.1002/j.1460-2075.1993.tb06066.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. van Hinsbergh V. W., Binnema D., Scheffer M. A., Sprengers E. D., Kooistra T., Rijken D. C. Production of plasminogen activators and inhibitor by serially propagated endothelial cells from adult human blood vessels. Arteriosclerosis. 1987 Jul-Aug;7(4):389–400. doi: 10.1161/01.atv.7.4.389. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES