title: Interleukin-1-induced calcium flux in human fibroblasts is mediated through focal adhesions.
abstract: Interleukin-1 (IL-1) is an important mediator of inflammation and also modulates fibroblast metabolism. To assess mechanisms of IL-1-induced signal transduction and calcium flux, early passage human fibroblasts were loaded with fura2/AM. Cells grown on coverslips exhibited dose-dependent [Ca2+]i responses that were maximal at 10(-8) M IL-1 beta with time to maximum flux of 50 s. Cells incubated with anti-Type 1-IL-1 receptor antibody exhibited a 45 nM increase in [Ca2+]i above baseline but demonstrated no calcium response after IL-1 beta treatment. Incubation with EGTA (5 mM) or thapsigargin (1 microM) caused 75% and 37% reductions, respectively, in the IL-1-induced [Ca2+]i increase, suggesting that extracellular Ca2+ predominates in IL-1-stimulated calcium flux. Cells in suspension did not exhibit [Ca2+]i responses to IL-1 beta. The relationship between [Ca2+]i signaling and focal adhesions was examined by plating cells on fibronectin or poly-L-lysine, conditions that either permitted or blocked the formation of focal adhesions. Cells on fibronectin exhibited co-distribution of immunostaining for talin, vinculin, IL-1 receptor, and focal adhesion kinase (pp125fak) in focal adhesions and demonstrated [Ca2+]i responses with 10(-8) M IL-1 beta. Cells on poly-L-lysine or cells in suspension did not exhibit co-distribution of pp125fak, IL-1 receptor, and focal adhesion proteins and did not exhibit calcium flux. The dependence of IL-1-stimulated [Ca2+]i responses on tyrosine kinases was examined first by treating cells with genistein, a selective inhibitor of tyrosine kinases. Genistein (100 microM) completely blocked [Ca2+]i responses to 10(-8) M IL-1, whereas its inactive analogue genistin was not inhibitory. Second, fibroblasts lysates were immunoprecipitated with an antiphosphotyrosine antibody and the lysates were Western-blotted with an anti-pp125fak antibody. Cells grown on fibronectin and stimulated with IL-1 exhibited tyrosine phosphorylation of pp125fak whereas untreated cells or cells grown on poly-L-lysine and treated with IL-1 showed no reaction. Fibroblasts electroinjected with anti-pp125fak monoclonal antibody showed no [Ca2+], response, whereas cells treated with an irrelevant antibody exhibited a normal [Ca2+]i response. Collectively, these data indicate that fibroblasts require substrate attachment and clustering of IL-1 receptors to focal adhesions for IL-1-induced [Ca2+]i responses. Calcium fluxes are mediated through tyrosine kinases whose substrates include pp125fak. These studies therefore demonstrate that activation of intracellular signaling pathways by IL-1 is dependent on IL-1 receptor-cytoskeletal protein interactions.
authors.affiliation: Medical Research Council Group in Periodontal Physiology, Faculty of Dentistry, University of Toronto, Canada.
authors.forename: P D
authors.initials: PD
authors.lastname: Arora
authors.forename: J
authors.initials: J
authors.lastname: Ma
authors.forename: W
authors.initials: W
authors.lastname: Min
authors.forename: T
authors.initials: T
authors.lastname: Cruz
authors.forename: C A
authors.initials: CA
authors.lastname: McCulloch
mesh_terms: D002118:Calcium / Q000378:metabolism*; D002448:Cell Adhesion / Q000187:drug effects*; D015815:Cell Adhesion Molecules / Q000032:analysis / Q000378:metabolism; D002478:Cells, Cultured; D018274:Electroporation; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D016257:Fura-2 / Q000031:analogs & derivatives; D005881:Gingiva / Q000378:metabolism*; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology*; D007700:Kinetics; D018613:Microscopy, Confocal; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000032:analysis / Q000378:metabolism; D017472:Receptors, Interleukin-1 / Q000187:drug effects / Q000502:physiology*; D011994:Recombinant Proteins / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D013050:Spectrometry, Fluorescence; D016608:Talin / Q000032:analysis; D013997:Time Factors; D016596:Vinculin / Q000032:analysis
chemical_list: D015815:Cell Adhesion Molecules; D005456:Fluorescent Dyes; D007375:Interleukin-1; D017472:Receptors, Interleukin-1; D011994:Recombinant Proteins; D016608:Talin; C049925:fura-2-am; D016596:Vinculin; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; D002118:Calcium; D016257:Fura-2