INTERLEUKIN-1-INDUCED CALCIUM FLUX IN HUMAN FIBROBLASTS IS MEDIATED THROUGH FOCAL ADHESIONS

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 mu M) 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 (pp125(fak)) 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 pp125(fak), 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 mu M) completely blocked [Ca2+](i) responses to 10(-8) M IL-1, whereas its inactive analogue genistin was not inhibitory. Second, fibroblast lysates were immunoprecipitated with an antiphosphotyrosine antibody and the lysates were Western-blotted with an anti-pp125(fak) antibody. Cells grown on fibronectin and stimulated with IL-1 exhibited tyrosine phosphorylation of pp125(fak) whereas untreated cells or cells grown on poly-L-lysine and treated with IL-1 showed no reaction. Fibroblasts electroinjected with anti-pp125(fak) monoclonal antibody showed no [Ca2+](i) 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 pp125(fak). These studies therefore demonstrate that activation of intracellular signaling pathways by IL-1 is dependent on IL-1 receptor-cytoskeletal protein interactions.