Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1997 Dec 1;328(Pt 2):707–715. doi: 10.1042/bj3280707

Induction of the E-selectin promoter by interleukin 1 and tumour necrosis factor alpha, and inhibition by glucocorticoids.

K P Ray 1, S Farrow 1, M Daly 1, F Talabot 1, N Searle 1
PMCID: PMC1218975  PMID: 9371735

Abstract

Cytokine-induced expression of the endothelial cell surface adhesion molecule E-selectin is inhibited by glucocorticoids (GCs). To investigate possible mechanisms for steroid inhibition, a reporter gene (ESAP) was constructed, comprising the cytokine responsive region of the E-selectin gene (nt -383 to +81) coupled to alkaline phosphatase (AP). In A549 cells stably transfected with the ESAP gene, AP production was highly responsive to the cytokines interleukin 1beta (IL-1beta) and tumour necrosis factor alpha, with ED50 values of 3 pM and 1000 pM respectively. Furthermore the cytokine-induced AP responses were inhibited by GCs, indicating that both transcriptional activation and GC suppression of the E-selectin gene were mediated via regulatory elements within the same region of the promoter. The relative potencies of GC drugs as inhibitors of IL-1beta (10 pM)-stimulated ESAP-gene activation were fluticasone> beclomethasone>dexamethasone, with IC50 values of 0.13, 1.1 and 2.7 nM respectively. Inhibition by fluticasone was blocked by the GC receptor (GR) antagonist drug mifepristone (Ru486), which is consistent with the suppressive effects of GCs being mediated via the GR. However, because the E-selectin promoter lacks a consensus glucocorticoid responsive element, mechanisms for inhibition independent of GR-DNA binding were investigated. Evidence that GCs also inhibited cytokine activation of a synthetic nuclear factor kappaB (NFkappaB)-driven reporter gene transiently transfected into A549 cells suggested that interference with the activation and/or function of this transcription factor was important for GC inhibition of ESAP. However, in A549-ESAP cells, fluticasone (100 nM) did not affect IL-1beta (10 pM)-induced IkBalpha degradation, NFkappaB-p65 nuclear translocation or the DNA-binding capacity of nuclear NFkappaB complexes, over a period during which cytokine-induced ESAP-gene activation was inhibited. Finally, there was no evidence to suggest that GC enhancement of IkBalpha gene expression contributed to the suppression of the cytokine response. We conclude that interference by GR with the transcriptional activation potential of DNA-bound NFkappaB complexes might contribute to mechanisms underlying the anti-inflammatory effects of GCs.

Full Text

The Full Text of this article is available as a PDF (518.0 KB).

Selected References

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

  1. Auphan N., DiDonato J. A., Rosette C., Helmberg A., Karin M. Immunosuppression by glucocorticoids: inhibition of NF-kappa B activity through induction of I kappa B synthesis. Science. 1995 Oct 13;270(5234):286–290. doi: 10.1126/science.270.5234.286. [DOI] [PubMed] [Google Scholar]
  2. Brostjan C., Anrather J., Csizmadia V., Natarajan G., Winkler H. Glucocorticoids inhibit E-selectin expression by targeting NF-kappaB and not ATF/c-Jun. J Immunol. 1997 Apr 15;158(8):3836–3844. [PubMed] [Google Scholar]
  3. Butcher E. C. Leukocyte-endothelial cell recognition: three (or more) steps to specificity and diversity. Cell. 1991 Dec 20;67(6):1033–1036. doi: 10.1016/0092-8674(91)90279-8. [DOI] [PubMed] [Google Scholar]
  4. Collins T., Williams A., Johnston G. I., Kim J., Eddy R., Shows T., Gimbrone M. A., Jr, Bevilacqua M. P. Structure and chromosomal location of the gene for endothelial-leukocyte adhesion molecule 1. J Biol Chem. 1991 Feb 5;266(4):2466–2473. [PubMed] [Google Scholar]
  5. Cronstein B. N., Kimmel S. C., Levin R. I., Martiniuk F., Weissmann G. A mechanism for the antiinflammatory effects of corticosteroids: the glucocorticoid receptor regulates leukocyte adhesion to endothelial cells and expression of endothelial-leukocyte adhesion molecule 1 and intercellular adhesion molecule 1. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):9991–9995. doi: 10.1073/pnas.89.21.9991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cupps T. R., Fauci A. S. Corticosteroid-mediated immunoregulation in man. Immunol Rev. 1982;65:133–155. doi: 10.1111/j.1600-065x.1982.tb00431.x. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Guesdon F., Ikebe T., Stylianou E., Warwick-Davies J., Haskill S., Saklatvala J. Interleukin 1-induced phosphorylation of MAD3, the major inhibitor of nuclear factor kappa B of HeLa cells. Interference in signalling by the proteinase inhibitors 3,4-dichloroisocoumarin and tosylphenylalanyl chloromethylketone. Biochem J. 1995 Apr 1;307(Pt 1):287–295. doi: 10.1042/bj3070287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Heck S., Kullmann M., Gast A., Ponta H., Rahmsdorf H. J., Herrlich P., Cato A. C. A distinct modulating domain in glucocorticoid receptor monomers in the repression of activity of the transcription factor AP-1. EMBO J. 1994 Sep 1;13(17):4087–4095. doi: 10.1002/j.1460-2075.1994.tb06726.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Jonat C., Rahmsdorf H. J., Park K. K., Cato A. C., Gebel S., Ponta H., Herrlich P. Antitumor promotion and antiinflammation: down-modulation of AP-1 (Fos/Jun) activity by glucocorticoid hormone. Cell. 1990 Sep 21;62(6):1189–1204. doi: 10.1016/0092-8674(90)90395-u. [DOI] [PubMed] [Google Scholar]
  11. Kamei Y., Xu L., Heinzel T., Torchia J., Kurokawa R., Gloss B., Lin S. C., Heyman R. A., Rose D. W., Glass C. K. A CBP integrator complex mediates transcriptional activation and AP-1 inhibition by nuclear receptors. Cell. 1996 May 3;85(3):403–414. doi: 10.1016/s0092-8674(00)81118-6. [DOI] [PubMed] [Google Scholar]
  12. Kaszubska W., Hooft van Huijsduijnen R., Ghersa P., DeRaemy-Schenk A. M., Chen B. P., Hai T., DeLamarter J. F., Whelan J. Cyclic AMP-independent ATF family members interact with NF-kappa B and function in the activation of the E-selectin promoter in response to cytokines. Mol Cell Biol. 1993 Nov;13(11):7180–7190. doi: 10.1128/mcb.13.11.7180. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kerppola T. K., Luk D., Curran T. Fos is a preferential target of glucocorticoid receptor inhibition of AP-1 activity in vitro. Mol Cell Biol. 1993 Jun;13(6):3782–3791. doi: 10.1128/mcb.13.6.3782. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kleinert H., Euchenhofer C., Ihrig-Biedert I., Förstermann U. Glucocorticoids inhibit the induction of nitric oxide synthase II by down-regulating cytokine-induced activity of transcription factor nuclear factor-kappa B. Mol Pharmacol. 1996 Jan;49(1):15–21. [PubMed] [Google Scholar]
  15. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  16. Lewis H., Kaszubska W., DeLamarter J. F., Whelan J. Cooperativity between two NF-kappa B complexes, mediated by high-mobility-group protein I(Y), is essential for cytokine-induced expression of the E-selectin promoter. Mol Cell Biol. 1994 Sep;14(9):5701–5709. doi: 10.1128/mcb.14.9.5701. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Mukaida N., Mahe Y., Matsushima K. Cooperative interaction of nuclear factor-kappa B- and cis-regulatory enhancer binding protein-like factor binding elements in activating the interleukin-8 gene by pro-inflammatory cytokines. J Biol Chem. 1990 Dec 5;265(34):21128–21133. [PubMed] [Google Scholar]
  18. Mukaida N., Morita M., Ishikawa Y., Rice N., Okamoto S., Kasahara T., Matsushima K. Novel mechanism of glucocorticoid-mediated gene repression. Nuclear factor-kappa B is target for glucocorticoid-mediated interleukin 8 gene repression. J Biol Chem. 1994 May 6;269(18):13289–13295. [PubMed] [Google Scholar]
  19. Ohtsuka T., Kubota A., Hirano T., Watanabe K., Yoshida H., Tsurufuji M., Iizuka Y., Konishi K., Tsurufuji S. Glucocorticoid-mediated gene suppression of rat cytokine-induced neutrophil chemoattractant CINC/gro, a member of the interleukin-8 family, through impairment of NF-kappa B activation. J Biol Chem. 1996 Jan 19;271(3):1651–1659. doi: 10.1074/jbc.271.3.1651. [DOI] [PubMed] [Google Scholar]
  20. Perkins N. D., Felzien L. K., Betts J. C., Leung K., Beach D. H., Nabel G. J. Regulation of NF-kappaB by cyclin-dependent kinases associated with the p300 coactivator. Science. 1997 Jan 24;275(5299):523–527. doi: 10.1126/science.275.5299.523. [DOI] [PubMed] [Google Scholar]
  21. Ray A., Prefontaine K. E. Physical association and functional antagonism between the p65 subunit of transcription factor NF-kappa B and the glucocorticoid receptor. Proc Natl Acad Sci U S A. 1994 Jan 18;91(2):752–756. doi: 10.1073/pnas.91.2.752. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ray A., Prefontaine K. E., Ray P. Down-modulation of interleukin-6 gene expression by 17 beta-estradiol in the absence of high affinity DNA binding by the estrogen receptor. J Biol Chem. 1994 Apr 29;269(17):12940–12946. [PubMed] [Google Scholar]
  23. Ray K. P., Kennard N. Interleukin-1 induces a nuclear form of transcription factor NF kappa B in human lung epithelial cells. Agents Actions. 1993;38(Spec No):C61–C63. doi: 10.1007/BF01991138. [DOI] [PubMed] [Google Scholar]
  24. Sakai D. D., Helms S., Carlstedt-Duke J., Gustafsson J. A., Rottman F. M., Yamamoto K. R. Hormone-mediated repression: a negative glucocorticoid response element from the bovine prolactin gene. Genes Dev. 1988 Sep;2(9):1144–1154. doi: 10.1101/gad.2.9.1144. [DOI] [PubMed] [Google Scholar]
  25. Scheinman R. I., Cogswell P. C., Lofquist A. K., Baldwin A. S., Jr Role of transcriptional activation of I kappa B alpha in mediation of immunosuppression by glucocorticoids. Science. 1995 Oct 13;270(5234):283–286. doi: 10.1126/science.270.5234.283. [DOI] [PubMed] [Google Scholar]
  26. Scheinman R. I., Gualberto A., Jewell C. M., Cidlowski J. A., Baldwin A. S., Jr Characterization of mechanisms involved in transrepression of NF-kappa B by activated glucocorticoid receptors. Mol Cell Biol. 1995 Feb;15(2):943–953. doi: 10.1128/mcb.15.2.943. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Schüle R., Evans R. M. Cross-coupling of signal transduction pathways: zinc finger meets leucine zipper. Trends Genet. 1991 Nov-Dec;7(11-12):377–381. doi: 10.1016/0168-9525(91)90259-s. [DOI] [PubMed] [Google Scholar]
  28. Schüle R., Rangarajan P., Kliewer S., Ransone L. J., Bolado J., Yang N., Verma I. M., Evans R. M. Functional antagonism between oncoprotein c-Jun and the glucocorticoid receptor. Cell. 1990 Sep 21;62(6):1217–1226. doi: 10.1016/0092-8674(90)90397-w. [DOI] [PubMed] [Google Scholar]
  29. Stein B., Cogswell P. C., Baldwin A. S., Jr Functional and physical associations between NF-kappa B and C/EBP family members: a Rel domain-bZIP interaction. Mol Cell Biol. 1993 Jul;13(7):3964–3974. doi: 10.1128/mcb.13.7.3964. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Stein B., Yang M. X. Repression of the interleukin-6 promoter by estrogen receptor is mediated by NF-kappa B and C/EBP beta. Mol Cell Biol. 1995 Sep;15(9):4971–4979. doi: 10.1128/mcb.15.9.4971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Whelan J., Ghersa P., Hooft van Huijsduijnen R., Gray J., Chandra G., Talabot F., DeLamarter J. F. An NF kappa B-like factor is essential but not sufficient for cytokine induction of endothelial leukocyte adhesion molecule 1 (ELAM-1) gene transcription. Nucleic Acids Res. 1991 May 25;19(10):2645–2653. doi: 10.1093/nar/19.10.2645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Whelan J. Selectin synthesis and inflammation. Trends Biochem Sci. 1996 Feb;21(2):65–69. [PubMed] [Google Scholar]
  33. Wingfield P., Payton M., Tavernier J., Barnes M., Shaw A., Rose K., Simona M. G., Demczuk S., Williamson K., Dayer J. M. Purification and characterization of human interleukin-1 beta expressed in recombinant Escherichia coli. Eur J Biochem. 1986 Nov 3;160(3):491–497. doi: 10.1111/j.1432-1033.1986.tb10066.x. [DOI] [PubMed] [Google Scholar]
  34. van de Stolpe A., Caldenhoven E., Raaijmakers J. A., van der Saag P. T., Koenderman L. Glucocorticoid-mediated repression of intercellular adhesion molecule-1 expression in human monocytic and bronchial epithelial cell lines. Am J Respir Cell Mol Biol. 1993 Mar;8(3):340–347. doi: 10.1165/ajrcmb/8.3.340. [DOI] [PubMed] [Google Scholar]
  35. van de Stolpe A., Caldenhoven E., Stade B. G., Koenderman L., Raaijmakers J. A., Johnson J. P., van der Saag P. T. 12-O-tetradecanoylphorbol-13-acetate- and tumor necrosis factor alpha-mediated induction of intercellular adhesion molecule-1 is inhibited by dexamethasone. Functional analysis of the human intercellular adhesion molecular-1 promoter. J Biol Chem. 1994 Feb 25;269(8):6185–6192. [PubMed] [Google Scholar]
  36. van den Hoff M. J., Moorman A. F., Lamers W. H. Electroporation in 'intracellular' buffer increases cell survival. Nucleic Acids Res. 1992 Jun 11;20(11):2902–2902. doi: 10.1093/nar/20.11.2902. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES