Interleukin 1-induced calcium signalling in chondrocytes requires focal adhesions.
The cytokine interleukin 1 (IL-1) is an important mediator of connective-tissue destruction in arthritic joints but the mechanisms by which IL-1 mediates signal transduction in chondrocytes is poorly understood. Previous results have indicated that IL-1 receptors co-localize with focal adhesions [Qwarnstrom, Page, Gillis and Dower (1988) J. Biol. Chem. 263, 8261-8269], discrete adhesive domains of cells that function in cell attachment and possibly in signal transduction. We have determined whether focal adhesions restrict IL-1-induced Ca2+ signalling in primary cultures of bovine chondrocytes. In cells grown for 24 h on fibronectin, the basal intracellular Ca2+ ion concentration ([Ca2+]i) was 100+/-3 nM. Optimal increases of [Ca2+]i above baseline were induced by 10 nM IL-1 (183+/-30 nM above baseline). There was no significant difference between cells plated on fibronectin or type II collagen (P>0.2; 233+/-90 nM above baseline). Ca2+ transients were significantly decreased by the inclusion of 0.5 mM EGTA in the bathing buffer (74+/-11 nM above baseline), and 1 microM thapsigargin completely blocked Ca2+ transients. Cells plated on poly-(l-lysine) or suspended cells showed no Ca2+ increases, whereas cells grown on fibronectin exhibited IL-1-induced Ca2+ responses that corresponded temporally to the time-dependent cell spreading after plating on fibronectin. Cells plated on poly-(l-lysine) and incubated with fibronectin-coated beads exhibited vinculin staining in association with the beads. In identical cell preparations, IL-1 induced a 136+/-39 nM increase of [Ca2+]i above baseline in response to 10 nM IL-1beta. There were no IL-1-induced Ca2+ increases when cells on poly-(l-lysine) were incubated with fibronectin-coated beads for only 15 min at 37 degrees C, in cells maintained for 3 h at 4 degrees C, in cells incubated with BSA beads for 3 h at 37 degrees C, or in cells pretreated with cytochalasin D. Labelling of IL-1 receptors with 125I-IL-1beta showed 3-fold more specific labelling of focal adhesion complexes in cells incubated with fibronectin-coated beads compared with cells incubated with BSA-coated beads, indicating that IL-1 receptor binding or the number of IL-1 receptors was increased in focal adhesions. These results indicate that, in chondrocytes, IL-1-induced Ca2+ signalling is dependent on focal adhesion formation and that focal adhesions recruit IL-1 receptors by redistribution in the cell membrane.
Luo
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MRC Group in Periodontal Physiology, Medical Sciences Building, 8 Taddle Creek Road, Toronto, Ontario, Canada M5G 1G6.
Cruz
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McCulloch
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D000818:Animals; D015703:Antigens, CD / Q000032:analysis; D002118:Calcium / Q000502:physiology*; D002358:Cartilage, Articular / Q000166:cytology / Q000187:drug effects*; D002417:Cattle; D002448:Cell Adhesion; D002478:Cells, Cultured; D015638:Cytochalasin D / Q000494:pharmacology; D005109:Extracellular Matrix / Q000502:physiology; D005353:Fibronectins; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D039482:Integrin alpha5; D007375:Interleukin-1 / Q000494:pharmacology*; D018613:Microscopy, Confocal; D011107:Polylysine; D017472:Receptors, Interleukin-1 / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects*; D016608:Talin / Q000032:analysis; D016596:Vinculin / Q000032:analysis
D015703:Antigens, CD; D005353:Fibronectins; D039482:Integrin alpha5; D007375:Interleukin-1; D017472:Receptors, Interleukin-1; D011994:Recombinant Proteins; D016608:Talin; D016596:Vinculin; D015638:Cytochalasin D; D011107:Polylysine; D002118:Calcium