Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1989 Apr;9(4):1498–1506. doi: 10.1128/mcb.9.4.1498

Forskolin inducibility and tissue-specific expression of the fibronectin promoter.

D C Dean 1, M S Blakeley 1, R F Newby 1, P Ghazal 1, L Hennighausen 1, S Bourgeois 1
PMCID: PMC362566  PMID: 2542772

Abstract

The mechanism of cyclic AMP (cAMP) induction of fibronectin (FN) in HT-1080 and JEG-3 cells differs (D. C. Dean, R. F. Newby, and S. Bourgeois, J. Cell Biol. 106:2159-2170, 1988). In the fibrosarcoma cell line HT-1080, induction requires both protein synthesis and a lag period of 12 to 24 h. In the choriocarcinoma cell line JEG-3, protein synthesis is not required and induction peaks before 24 h, declining thereafter. We show that the FN promoter is transcribed in vitro and that the transcripts initiate at the proper site. Based on transfection experiments with these cells and FN promoter constructions, a cAMP-responsive element (CRE) was identified between -157 and -188 base pairs upstream of the human FN gene. This sequence also conferred cAMP inducibility in both cell lines on the herpesvirus thymidine kinase promoter when it was placed upstream of a thymidine kinase-chloramphenicol acetyltransferase fusion gene. DNase I protection analysis and gel retardation experiments revealed that the CRE was bound by a protein(s) that was present in both HT-1080 and JEG-3 cells as well as in NIH 3T3 cells. Multiple protein-CRE complexes were resolved by gel retardation with extracts of both cell lines. Forskolin treatment of these cells did not alter qualitatively or quantitatively the pattern of CRE-binding proteins that was observed. The FN promoter was at least 10 times more active in HT-1080 than in JEG-3 cells, even though in JEG-3 cells both the rate of FN biosynthesis and the level of accumulated FN mRNA were greater than those in HT-1080 cells. The difference in promoter activity in HT-1080 and JEG-3 cell was mediated by sequences that were located between positions -510 and -56. Deletion of the FN promoter from positions -510 to -56 resulted in an ~30-fold decrease in promoter activity when this construction was transfected into HT-1080 cells, and similar results were observed in NIH 3T3 cells; however, less than a 2-fold effect was observed in JEG-3 cells. Results of these studies suggest that there is some degree of tissue specificity of FN gene expression and reveal that cAMP induction is mediated, in part, by the same element (CRE) in both HT-1080 and JEG-3 cells.

Full text

PDF
1503

Images in this article

1499Image
on p.1499
1501Image
on p.1501
1503Image
on p.1503
1503Image
on p.1503

Selected References

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

  1. Akiyama S. K., Yamada K. M. Fibronectin. Adv Enzymol Relat Areas Mol Biol. 1987;59:1–57. doi: 10.1002/9780470123058.ch1. [DOI] [PubMed] [Google Scholar]
  2. Angel P., Allegretto E. A., Okino S. T., Hattori K., Boyle W. J., Hunter T., Karin M. Oncogene jun encodes a sequence-specific trans-activator similar to AP-1. Nature. 1988 Mar 10;332(6160):166–171. doi: 10.1038/332166a0. [DOI] [PubMed] [Google Scholar]
  3. Angel P., Imagawa M., Chiu R., Stein B., Imbra R. J., Rahmsdorf H. J., Jonat C., Herrlich P., Karin M. Phorbol ester-inducible genes contain a common cis element recognized by a TPA-modulated trans-acting factor. Cell. 1987 Jun 19;49(6):729–739. doi: 10.1016/0092-8674(87)90611-8. [DOI] [PubMed] [Google Scholar]
  4. Bohmann D., Bos T. J., Admon A., Nishimura T., Vogt P. K., Tjian R. Human proto-oncogene c-jun encodes a DNA binding protein with structural and functional properties of transcription factor AP-1. Science. 1987 Dec 4;238(4832):1386–1392. doi: 10.1126/science.2825349. [DOI] [PubMed] [Google Scholar]
  5. Brown R., Marshall C. J., Pennie S. G., Hall A. Mechanism of activation of an N-ras gene in the human fibrosarcoma cell line HT1080. EMBO J. 1984 Jun;3(6):1321–1326. doi: 10.1002/j.1460-2075.1984.tb01970.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Comb M., Birnberg N. C., Seasholtz A., Herbert E., Goodman H. M. A cyclic AMP- and phorbol ester-inducible DNA element. 1986 Sep 25-Oct 1Nature. 323(6086):353–356. doi: 10.1038/323353a0. [DOI] [PubMed] [Google Scholar]
  7. Dean D. C., Bowlus C. L., Bourgeois S. Cloning and analysis of the promotor region of the human fibronectin gene. Proc Natl Acad Sci U S A. 1987 Apr;84(7):1876–1880. doi: 10.1073/pnas.84.7.1876. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dean D. C., Newby R. F., Bourgeois S. Regulation of fibronectin biosynthesis by dexamethasone, transforming growth factor beta, and cAMP in human cell lines. J Cell Biol. 1988 Jun;106(6):2159–2170. doi: 10.1083/jcb.106.6.2159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Delegeane A. M., Ferland L. H., Mellon P. L. Tissue-specific enhancer of the human glycoprotein hormone alpha-subunit gene: dependence on cyclic AMP-inducible elements. Mol Cell Biol. 1987 Nov;7(11):3994–4002. doi: 10.1128/mcb.7.11.3994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dignam J. D., Lebovitz R. M., Roeder R. G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983 Mar 11;11(5):1475–1489. doi: 10.1093/nar/11.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Edlund T., Walker M. D., Barr P. J., Rutter W. J. Cell-specific expression of the rat insulin gene: evidence for role of two distinct 5' flanking elements. Science. 1985 Nov 22;230(4728):912–916. doi: 10.1126/science.3904002. [DOI] [PubMed] [Google Scholar]
  12. Ghazal P., Lubon H., Fleckenstein B., Hennighausen L. Binding of transcription factors and creation of a large nucleoprotein complex on the human cytomegalovirus enhancer. Proc Natl Acad Sci U S A. 1987 Jun;84(11):3658–3662. doi: 10.1073/pnas.84.11.3658. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hynes R. Molecular biology of fibronectin. Annu Rev Cell Biol. 1985;1:67–90. doi: 10.1146/annurev.cb.01.110185.000435. [DOI] [PubMed] [Google Scholar]
  14. Imagawa M., Chiu R., Karin M. Transcription factor AP-2 mediates induction by two different signal-transduction pathways: protein kinase C and cAMP. Cell. 1987 Oct 23;51(2):251–260. doi: 10.1016/0092-8674(87)90152-8. [DOI] [PubMed] [Google Scholar]
  15. Imler J. L., Schatz C., Wasylyk C., Chatton B., Wasylyk B. A Harvey-ras responsive transcription element is also responsive to a tumour-promoter and to serum. Nature. 1988 Mar 17;332(6161):275–278. doi: 10.1038/332275a0. [DOI] [PubMed] [Google Scholar]
  16. Jameson J. L., Deutsch P. J., Gallagher G. D., Jaffe R. C., Habener J. F. trans-acting factors interact with a cyclic AMP response element to modulate expression of the human gonadotropin alpha gene. Mol Cell Biol. 1987 Sep;7(9):3032–3040. doi: 10.1128/mcb.7.9.3032. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jones N. C., Rigby P. W., Ziff E. B. Trans-acting protein factors and the regulation of eukaryotic transcription: lessons from studies on DNA tumor viruses. Genes Dev. 1988 Mar;2(3):267–281. doi: 10.1101/gad.2.3.267. [DOI] [PubMed] [Google Scholar]
  18. Kohler P. O., Bridson W. E. Isolation of hormone-producing clonal lines of human choriocarcinoma. J Clin Endocrinol Metab. 1971 May;32(5):683–687. doi: 10.1210/jcem-32-5-683. [DOI] [PubMed] [Google Scholar]
  19. Lee W., Haslinger A., Karin M., Tjian R. Activation of transcription by two factors that bind promoter and enhancer sequences of the human metallothionein gene and SV40. Nature. 1987 Jan 22;325(6102):368–372. doi: 10.1038/325368a0. [DOI] [PubMed] [Google Scholar]
  20. Lee W., Mitchell P., Tjian R. Purified transcription factor AP-1 interacts with TPA-inducible enhancer elements. Cell. 1987 Jun 19;49(6):741–752. doi: 10.1016/0092-8674(87)90612-x. [DOI] [PubMed] [Google Scholar]
  21. Montminy M. R., Bilezikjian L. M. Binding of a nuclear protein to the cyclic-AMP response element of the somatostatin gene. Nature. 1987 Jul 9;328(6126):175–178. doi: 10.1038/328175a0. [DOI] [PubMed] [Google Scholar]
  22. Montminy M. R., Sevarino K. A., Wagner J. A., Mandel G., Goodman R. H. Identification of a cyclic-AMP-responsive element within the rat somatostatin gene. Proc Natl Acad Sci U S A. 1986 Sep;83(18):6682–6686. doi: 10.1073/pnas.83.18.6682. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Ohlsson H., Edlund T. Sequence-specific interactions of nuclear factors with the insulin gene enhancer. Cell. 1986 Apr 11;45(1):35–44. doi: 10.1016/0092-8674(86)90535-0. [DOI] [PubMed] [Google Scholar]
  24. Oliver N., Newby R. F., Furcht L. T., Bourgeois S. Regulation of fibronectin biosynthesis by glucocorticoids in human fibrosarcoma cells and normal fibroblasts. Cell. 1983 May;33(1):287–296. doi: 10.1016/0092-8674(83)90357-4. [DOI] [PubMed] [Google Scholar]
  25. Patel R. S., Odermatt E., Schwarzbauer J. E., Hynes R. O. Organization of the fibronectin gene provides evidence for exon shuffling during evolution. EMBO J. 1987 Sep;6(9):2565–2572. doi: 10.1002/j.1460-2075.1987.tb02545.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Rasheed S., Nelson-Rees W. A., Toth E. M., Arnstein P., Gardner M. B. Characterization of a newly derived human sarcoma cell line (HT-1080). Cancer. 1974 Apr;33(4):1027–1033. doi: 10.1002/1097-0142(197404)33:4<1027::aid-cncr2820330419>3.0.co;2-z. [DOI] [PubMed] [Google Scholar]
  27. Rauscher F. J., 3rd, Sambucetti L. C., Curran T., Distel R. J., Spiegelman B. M. Common DNA binding site for Fos protein complexes and transcription factor AP-1. Cell. 1988 Feb 12;52(3):471–480. doi: 10.1016/s0092-8674(88)80039-4. [DOI] [PubMed] [Google Scholar]
  28. Rawlins D. R., Rosenfeld P. J., Wides R. J., Challberg M. D., Kelly T. J., Jr Structure and function of the adenovirus origin of replication. Cell. 1984 May;37(1):309–319. doi: 10.1016/0092-8674(84)90327-1. [DOI] [PubMed] [Google Scholar]
  29. Ryder K., Lau L. F., Nathans D. A gene activated by growth factors is related to the oncogene v-jun. Proc Natl Acad Sci U S A. 1988 Mar;85(5):1487–1491. doi: 10.1073/pnas.85.5.1487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Short J. M., Wynshaw-Boris A., Short H. P., Hanson R. W. Characterization of the phosphoenolpyruvate carboxykinase (GTP) promoter-regulatory region. II. Identification of cAMP and glucocorticoid regulatory domains. J Biol Chem. 1986 Jul 25;261(21):9721–9726. [PubMed] [Google Scholar]
  31. Sistonen L., Keski-Oja J., Ulmanen I., Hölttä E., Wikgren B. J., Alitalo K. Dose effects of transfected c-Ha-rasVal 12 oncogene in transformed cell clones. Exp Cell Res. 1987 Feb;168(2):518–530. doi: 10.1016/0014-4827(87)90024-3. [DOI] [PubMed] [Google Scholar]
  32. Tarpley W. G., Hopkins N. K., Gorman R. R. Reduced hormone-stimulated adenylate cyclase activity in NIH-3T3 cells expressing the EJ human bladder ras oncogene. Proc Natl Acad Sci U S A. 1986 Jun;83(11):3703–3707. doi: 10.1073/pnas.83.11.3703. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Treisman R. Identification of a protein-binding site that mediates transcriptional response of the c-fos gene to serum factors. Cell. 1986 Aug 15;46(4):567–574. doi: 10.1016/0092-8674(86)90882-2. [DOI] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES