Interleukin 1-induced calcium signalling in chondrocytes requires focal adhesions

Abstract

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.

The Recruitment of the Interleukin-1 (IL-1) Receptor-associated Kinase (IRAK) into Focal Adhesion Complexes Is Required for IL-1β-induced ERK Activation

“…Our findings that IL-1 receptors are enriched in FACs and that FACs are required for IL-1-induced ERK activation and calcium responses (44,51) indicate a prominent role for FACs in restricting IL-1-generated signals.…”

“…Although the precise requirement for FACs in IL-1-induced signaling and, more specifically, the functional relationship between FACs and the IL-1 receptor complex remains unclear, we showed previously that FACs are necessary for IL-1-induced ERK activation and calcium responses (44,51). In this paper we show that it was the lack of FACs and not the altered cell morphology or the presence of poly-L-lysine that inhibited ERK activation; we were able to partially restore IL-1-induced ERK activation in rounded fibroblasts plated on poly-L-lysine by inducing the formation of FACs with collagen-coated beads.…”

“…Previously, fibronectin-coated microbeads and fluorescence microscopy have been used to show FAC assembly at sites of microbead-cell contact (50,51). We used a modification of this technique to demonstrate the recruitment of IRAK into the FACs of IL-1 stimulated cells.…”

SHP-2 Modulates Interleukin-1-induced Ca2+ Flux and ERK Activation via Phosphorylation of Phospholipase Cγ1

“…No significant changes in Ca 2ϩ were detected at sites remote from the beads, and no evidence for IL-1-induced Ca 2ϩ signaling was found in cells plated on poly-L-lysine and incubated with BSA-coated beads. Therefore, as shown previously (10,12), integrin-dependent focal adhesion formation is essential for IL-1-induced Ca 2ϩ signals. The novel aspect of the current observations is that Ca 2ϩ fluxes induced by IL-1 originate from and are strictly localized to the vicinity of focal adhesions.…”

Interleukin-1β Regulates the Human Brain Natriuretic Peptide Promoter via Ca 2+ -Dependent Protein Kinase Pathways

“…6,8,10,[12][13][14] Another important component of the IL-1␤ response is increased intracellular calcium (Ca 2ϩ ) concentration. IL-1␤-induced changes in intracellular Ca 2ϩ have been documented in chondrocytes, 15 11 This might account for the abnormal cardiac myocyte function in the infarcted heart; myocytes isolated from infarcted rat hearts have a lower peak cytosolic Ca 2ϩ concentration during contraction than do control myocytes. 18 IL-1␤ signaling has been shown to involve Ca 2ϩ -regulated kinases, such as protein kinase C (PKC).…”