Pd. Arora et al., INTERLEUKIN-1-INDUCED CALCIUM FLUX IN HUMAN FIBROBLASTS IS MEDIATED THROUGH FOCAL ADHESIONS, The Journal of biological chemistry, 270(11), 1995, pp. 6042-6049
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-d
ependent [Ca2+](i) responses that were maximal at 10(-8) M IL-1 beta w
ith 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 base
line but demonstrated no calcium response after IL-1 beta treatment. I
ncubation with EGTA (5 mM) or thapsigargin (1 mu M) caused 75% and 37%
reductions, respectively, in the IL-1-induced [Ca2+](i) increase, sug
gesting that extracellular Ca2+ predominates in IL-1-stimulated calciu
m flux. Cells in suspension did not exhibit [Ca2+](i) responses to IL-
1 beta. The relationship between [Ca2+](i) signaling and focal adhesio
ns was examined by plating cells on fibronectin or poly-L-lysine, cond
itions that either permitted or blocked the formation of focal adhesio
ns. Cells on fibronectin exhibited co-distribution of immunostaining f
or talin, vinculin, IL-1 receptor, and focal adhesion kinase (pp125(fa
k)) 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 e
xhibit co-distribution of pp125(fak), IL-1 receptor, and focal adhesio
n proteins and did not exhibit calcium flux. The dependence of IL-1-st
imulated [Ca2+](i) responses on tyrosine kinases was examined first by
treating cells with genistein, a selective inhibitor of tyrosine kina
ses. 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 antiphosp
hotyrosine 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 i
rrelevant antibody exhibited a normal [Ca2+](i) response. Collectively
, these data indicate that fibroblasts require substrate attachment an
d clustering of IL-1 receptors to focal adhesions for IL-1-induced [Ca
2+](i) responses. Calcium fluxes are mediated through tyrosine kinases
whose substrates include pp125(fak). These studies therefore demonstr
ate that activation of intracellular signaling pathways by IL-1 is dep
endent on IL-1 receptor-cytoskeletal protein interactions.