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Hoffmann, R. A wiki for the life sciences where authorship matters. Nature Genetics (2008)
 
 
 
 
 

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 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.[1]

References

  1. Interleukin-1-induced calcium flux in human fibroblasts is mediated through focal adhesions. Arora, P.D., Ma, J., Min, W., Cruz, T., McCulloch, C.A. J. Biol. Chem. (1995) [Pubmed]
 
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