Skip to main content
NCBI home page
Immunology logoLink to Immunology
. 1998 Jul;94(3):356–362. doi: 10.1046/j.1365-2567.1998.00528.x

Immunolocalization of protease-activated receptor-2 in skin: receptor activation stimulates interleukin-8 secretion by keratinocytes in vitro.

L Hou 1, S Kapas 1, A T Cruchley 1, M G Macey 1, P Harriott 1, C Chinni 1, S R Stone 1, G L Howells 1
PMCID: PMC1364253  PMID: 9767417

Abstract

The protease-activated receptor-2 (PAR-2) is a seven transmembrane domain receptor related to the thrombin receptor, which is activated in vitro by cleavage by trypsin. Affinity-purified rabbit IgG raised against a peptide corresponding to the trypsin cleavage site of PAR-2 was used for an immunohistochemical study of skin. The expression of PAR-2 in epidermis was striking, with keratinocytes showing abundant intercellular and cytoplasmic staining. Basal cells showed the strongest staining intensity and the stratum corneum was negative. Staining with control IgG used at the same concentration was consistently negative. The functional expression of PAR-2 by the simian virus transformed human skin keratinocyte cell line SVK14 was demonstrated by Northern blot analysis, flow cytometric analysis and the measurement of intracellular calcium. Treatment of SVK14 with trypsin or a receptor agonist peptide (SLIGKV-NH2) caused a dose-dependent increase in the secretion of the chemokine interleukin-8 (IL-8) in vitro. The effect of the peptide was specific, since control acetylated peptide was without activity. We conclude that PAR-2 is highly expressed by epidermal keratinocytes and receptor activation in vitro leads to increased IL-8 secretion by keratinocytes. These data raise the possibility that PAR-2 may play a role in epidermal homeostasis and inflammatory conditions.

Full text

PDF
356

Images in this article

359Figure 2
on p.359
359Figure 3
on p.359
360Figure 5
on p.360

Selected References

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

  1. Alkemade J. A., Molhuizen H. O., Ponec M., Kempenaar J. A., Zeeuwen P. L., de Jongh G. J., van Vlijmen-Willems I. M., van Erp P. E., van de Kerkhof P. C., Schalkwijk J. SKALP/elafin is an inducible proteinase inhibitor in human epidermal keratinocytes. J Cell Sci. 1994 Aug;107(Pt 8):2335–2342. doi: 10.1242/jcs.107.8.2335. [DOI] [PubMed] [Google Scholar]
  2. Bohm S. K., Kong W., Bromme D., Smeekens S. P., Anderson D. C., Connolly A., Kahn M., Nelken N. A., Coughlin S. R., Payan D. G. Molecular cloning, expression and potential functions of the human proteinase-activated receptor-2. Biochem J. 1996 Mar 15;314(Pt 3):1009–1016. doi: 10.1042/bj3141009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bootman M. D., Berridge M. J., Taylor C. W. All-or-nothing Ca2+ mobilization from the intracellular stores of single histamine-stimulated HeLa cells. J Physiol. 1992 May;450:163–178. doi: 10.1113/jphysiol.1992.sp019121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Coughlin S. R. Protease-activated receptors start a family. Proc Natl Acad Sci U S A. 1994 Sep 27;91(20):9200–9202. doi: 10.1073/pnas.91.20.9200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Derian C. K., Eckardt A. J., Andrade-Gordon P. Differential regulation of human keratinocyte growth and differentiation by a novel family of protease-activated receptors. Cell Growth Differ. 1997 Jul;8(7):743–749. [PubMed] [Google Scholar]
  6. Fräki J. E., Schechter N. M., Lazarus G. S. Human skin proteinases as inflammatory mediators. Br J Dermatol. 1983 Jul;109 (Suppl 25):72–76. doi: 10.1111/j.1365-2133.1983.tb11627.x. [DOI] [PubMed] [Google Scholar]
  7. Gillitzer R., Ritter U., Spandau U., Goebeler M., Bröcker E. B. Differential expression of GRO-alpha and IL-8 mRNA in psoriasis: a model for neutrophil migration and accumulation in vivo. J Invest Dermatol. 1996 Nov;107(5):778–782. doi: 10.1111/1523-1747.ep12371803. [DOI] [PubMed] [Google Scholar]
  8. Grynkiewicz G., Poenie M., Tsien R. Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985 Mar 25;260(6):3440–3450. [PubMed] [Google Scholar]
  9. Howells G. L., Macey M. G., Chinni C., Hou L., Fox M. T., Harriott P., Stone S. R. Proteinase-activated receptor-2: expression by human neutrophils. J Cell Sci. 1997 Apr;110(Pt 7):881–887. doi: 10.1242/jcs.110.7.881. [DOI] [PubMed] [Google Scholar]
  10. Howells G. L., Macey M., Curtis M. A., Stone S. R. Peripheral blood lymphocytes express the platelet-type thrombin receptor. Br J Haematol. 1993 May;84(1):156–160. doi: 10.1111/j.1365-2141.1993.tb03039.x. [DOI] [PubMed] [Google Scholar]
  11. Ishihara H., Connolly A. J., Zeng D., Kahn M. L., Zheng Y. W., Timmons C., Tram T., Coughlin S. R. Protease-activated receptor 3 is a second thrombin receptor in humans. Nature. 1997 Apr 3;386(6624):502–506. doi: 10.1038/386502a0. [DOI] [PubMed] [Google Scholar]
  12. Jenkins A. L., Howells G. L., Scott E., Le Bonniec B. F., Curtis M. A., Stone S. R. The response to thrombin of human neutrophils: evidence for two novel receptors. J Cell Sci. 1995 Sep;108(Pt 9):3059–3066. doi: 10.1242/jcs.108.9.3059. [DOI] [PubMed] [Google Scholar]
  13. Li J., Ireland G. W., Farthing P. M., Thornhill M. H. Epidermal and oral keratinocytes are induced to produce RANTES and IL-8 by cytokine stimulation. J Invest Dermatol. 1996 Apr;106(4):661–666. doi: 10.1111/1523-1747.ep12345482. [DOI] [PubMed] [Google Scholar]
  14. McKay I. A., Leigh I. M. Altered keratinocyte growth and differentiation in psoriasis. Clin Dermatol. 1995 Mar-Apr;13(2):105–114. doi: 10.1016/0738-081x(95)93817-8. [DOI] [PubMed] [Google Scholar]
  15. Mirza H., Yatsula V., Bahou W. F. The proteinase activated receptor-2 (PAR-2) mediates mitogenic responses in human vascular endothelial cells. J Clin Invest. 1996 Apr 1;97(7):1705–1714. doi: 10.1172/JCI118597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Molino M., Barnathan E. S., Numerof R., Clark J., Dreyer M., Cumashi A., Hoxie J. A., Schechter N., Woolkalis M., Brass L. F. Interactions of mast cell tryptase with thrombin receptors and PAR-2. J Biol Chem. 1997 Feb 14;272(7):4043–4049. doi: 10.1074/jbc.272.7.4043. [DOI] [PubMed] [Google Scholar]
  17. Molino M., Woolkalis M. J., Reavey-Cantwell J., Praticó D., Andrade-Gordon P., Barnathan E. S., Brass L. F. Endothelial cell thrombin receptors and PAR-2. Two protease-activated receptors located in a single cellular environment. J Biol Chem. 1997 Apr 25;272(17):11133–11141. doi: 10.1074/jbc.272.17.11133. [DOI] [PubMed] [Google Scholar]
  18. Nickoloff B. J., Mitra R. S., Varani J., Dixit V. M., Polverini P. J. Aberrant production of interleukin-8 and thrombospondin-1 by psoriatic keratinocytes mediates angiogenesis. Am J Pathol. 1994 Apr;144(4):820–828. [PMC free article] [PubMed] [Google Scholar]
  19. Nystedt S., Emilsson K., Wahlestedt C., Sundelin J. Molecular cloning of a potential proteinase activated receptor. Proc Natl Acad Sci U S A. 1994 Sep 27;91(20):9208–9212. doi: 10.1073/pnas.91.20.9208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Posnett D. N., Tam J. P. Multiple antigenic peptide method for producing antipeptide site-specific antibodies. Methods Enzymol. 1989;178:739–746. doi: 10.1016/0076-6879(89)78048-4. [DOI] [PubMed] [Google Scholar]
  21. Santulli R. J., Derian C. K., Darrow A. L., Tomko K. A., Eckardt A. J., Seiberg M., Scarborough R. M., Andrade-Gordon P. Evidence for the presence of a protease-activated receptor distinct from the thrombin receptor in human keratinocytes. Proc Natl Acad Sci U S A. 1995 Sep 26;92(20):9151–9155. doi: 10.1073/pnas.92.20.9151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Sower L. E., Froelich C. J., Carney D. H., Fenton J. W., 2nd, Klimpel G. R. Thrombin induces IL-6 production in fibroblasts and epithelial cells. Evidence for the involvement of the seven-transmembrane domain (STD) receptor for alpha-thrombin. J Immunol. 1995 Jul 15;155(2):895–901. [PubMed] [Google Scholar]
  23. Wakita H., Furukawa F., Takigawa M. Thrombin and trypsin induce granulocyte-macrophage colony-stimulating factor and interleukin-6 gene expression in cultured normal human keratinocytes. Proc Assoc Am Physicians. 1997 Mar;109(2):190–207. [PubMed] [Google Scholar]

Articles from Immunology are provided here courtesy of British Society for Immunology

RESOURCES