Thrombin induces mast cell adhesion to fibronectin: evidence for involvement of protease-activated receptor-1.
Thrombin activates mast cells to release inflammatory mediators through a mechanism involving protease-activated receptor-1 (PAR-1). We hypothesized that PAR-1 activation would induce mast cell adhesion to fibronectin (FN). Fluorescent adhesion assay was performed in 96-well plates coated with FN (20 microg/ml). Murine bone marrow cultured mast cells (BMCMC) were used after 3-5 wk of culture (>98% mast cells by flow cytometry for c-Kit expression). Thrombin induced beta-hexosaminidase, IL-6, and matrix metalloproteinase-9 release from BMCMC. Thrombin and the PAR-1-activating peptide AparafluoroFRCyclohexylACitY-NH(2) (cit) induced BMCMC adhesion to FN in a dose-dependent fashion, while the PAR-1-inactive peptide FSLLRY-NH(2) had no effect. Thrombin and cit induced also BMCMC adhesion to laminin. Thrombin-mediated adhesion to FN was inhibited by anti-alpha(5) integrin Ab (51.1 +/- 6.7%; n = 5). The combination of anti-alpha(5) and anti-alpha(4) Abs induced higher inhibition (65.7 +/- 7.1%; n = 5). Unlike what is known for FcepsilonRI-mediated adhesion, PAR-1-mediated adhesion to FN did not increase mediator release. We then explored the signaling pathways involved in PAR-1-mediated mast cell adhesion. Thrombin and cit induced p44/42 and p38 phosphorylation. Pertussis toxin inhibited PAR-1-mediated BMCMC adhesion by 57.3 +/- 7.3% (n = 4), indicating that G(i) proteins are involved. Wortmannin and calphostin almost completely inhibited PAR-1-mediated mast cell adhesion, indicating that PI-3 kinase and protein kinase C are involved. Adhesion was partially inhibited by the mitogen-activated protein kinase kinase 1/2 inhibitor U0126 (24.5 +/- 3.3%; n = 3) and the p38 inhibitor SB203580 (25.1 +/- 10.4%; n = 3). The two inhibitors had additive effects. Therefore, thrombin mediates mast cell adhesion through the activation of G(i) proteins, phosphoinositol 3-kinase, protein kinase C, and mitogen-activated protein kinase pathways.
Vliagoftis
Harissios
H
Pulmonary Research Group, Department of Medicine, 550 HMRC, University of Alberta, Edmonton, Alberta, Canada T6G 2S2. harissios.vliagoftis@ualberta.ca
D000818:Animals; D001798:Blood Proteins / Q000378:metabolism; D029742:Caenorhabditis elegans Proteins; D002448:Cell Adhesion / Q000502:physiology; D002478:Cells, Cultured; D005353:Fibronectins / Q000378:metabolism*; D006636:Histamine Release / Q000502:physiology; D015850:Interleukin-6 / Q000378:metabolism; D008297:Male; D008407:Mast Cells / Q000201:enzymology* / Q000378:metabolism / Q000502:physiology; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin / Q000096:biosynthesis / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology; D013917:Thrombin / Q000502:physiology*; D001619:beta-N-Acetylhexosaminidases / Q000378:metabolism
D001798:Blood Proteins; D029742:Caenorhabditis elegans Proteins; D005353:Fibronectins; D015850:Interleukin-6; D044463:Receptor, PAR-1; D018179:Receptors, Thrombin; C016308:serum P-component; D017346:Protein Serine-Threonine Kinases; D001619:beta-N-Acetylhexosaminidases; D013917:Thrombin; D020780:Matrix Metalloproteinase 9