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IL-1 induced release of Ca²⁺ from internal stores is dependent on cell-matrix interactions and regulates ERK activation¹

QIN WANG^*, GREGORY P. DOWNEY, CHRISTINE CHOI^*, ANDRÁS KAPUS and CHRISTOPHER A. MCCULLOCH^*,2

^* CIHR Group in Matrix Dynamics, University of Toronto, Toronto, Ontario M5S 3E2, Canada;
Division of Respirology, Department of Medicine, University of Toronto and the Research Institute, Toronto General Division of the University Health Network Research Institute, Toronto, Ontario M5G 2C4, Canada; and
Department of Surgery, University of Toronto and the Division of Surgery, Toronto General Hospital, Toronto, Ontario M5G 1G6, Canada

²Correspondence: Room 244, Fitzgerald Bldg., 150 College St., University of Toronto, Toronto, Ontario, Canada M5S 3E2. E-mail: christopher.mcculloch{at}utoronto.ca.

SPECIFIC AIM

The cellular mechanisms that modulate IL-1 signaling and lead to production of inflammatory mediators are not defined. The aim of this study was to determine the role of cell adhesion in regulating specific steps of Ca²⁺ handling in the IL-1 signals that lead to ERK activation in human fibroblasts.

PRINCIPAL FINDINGS

1. Hep-I, SPARC peptides, and tenascin-C inhibit focal adhesion formation and block IL-1-induced Ca²⁺ signaling
We used human gingival fibroblasts as an in vitro cell model to study IL-1 signaling. When spread on surfaces coated with fibronectin, these cells cluster IL-1 signaling receptors into focal adhesions. Treatment of cells with focal adhesion dispersing agents (Hep-I, SPARC peptides or tenascin-C for 45 min) induced dose-dependent loss of focal adhesions as measured by immunostaining and immunoblotting for vinculin and talin. When the cells were subsequently stimulated with IL-1, the peptides reduced the amplitude of IL-1-induced Ca²⁺ signals by 50% (P<0.05; n=4) compared with controls (Fig. 1 A, C). This inhibition was reversed when cells were incubated with fibronectin-coated beads in the absence of peptides, but much less so with BSA-coated beads (Fig. 1B, C ).

View larger version (23K): [in this window] [in a new window]   Figure 1. Disassembly of focal adhesion complexes by Hep-I, SPARC, and tenascin-C diminish IL-1-induced Ca2+ flux. A) Fibroblasts plated on fibronectin for 24-48 h were preincubated with Hep-I, SPARC or tenascin-C at 37°C for 45 min, then treated with IL-1. Intracellular calcium concentration ([Ca2+]i) was measured in fura-2-loaded cells by ratio fluorometry. Cells were pretreated with vehicle as control. B) Fibroblasts pretreated with Hep-I and SPARC were incubated with fibronectin- and BSA-coated beads, respectively, and stimulated with IL-1. C) Summary data (mean±SE) from n=4–6 experiments. Data show increased [Ca2+]i above baseline in control, Hep-I, SPARC, and tenascin-C-pretreated cells.

2. The ability of IL-1 to release calcium from internal stores is dependent on focal adhesions
IL-1 induced a precipitous reduction of the mag-fura-2 ratio, followed by a rapid recovery to baseline in cells plated on fibronectin. This indicated that cells with focal adhesions readily release calcium from ER stores in response to IL-1. In contrast, there was no change of the mag-fura-2 ratio in IL-1-treated cells plated on poly-L-lysine, culture conditions that prevent focal adhesion formation. The addition of fibronectin-coated beads but not BSA-coated beads restored the IL-1-induced Ca²⁺ transients in cells grown on poly-L-lysine, presumably by the formation of focal adhesions on the dorsal cell surface. In cells plated on fibronectin, the block of release from the endoplasmic reticulum could be replicated by treatment with jasplakinolide that induces subcortical actin filament assembly.

3. Activation of store-operated calcium channels requires intact focal adhesions
Cells were pretreated with thapsigargin to deplete endoplasmic reticulum calcium stores. Subsequent addition of Ca²⁺ to the external medium induced a sustained increase of [Ca²⁺]_i indicative of store-operated calcium entry through the plasma membrane. The maximum entry of Ca²⁺ was markedly diminished in cells grown on poly-L-lysine in which focal adhesions were dissipated. The effect of poly-L-lysine on store-mediated calcium entry was reversed in cells incubated with fibronectin-coated beads, which restored focal adhesions. Depolymerization of actin filaments with latrunculin B also overcame the block to store refilling. The significance of this finding for IL-1 signaling is that refilling of endoplasmic reticulum stores is mediated by store-operated channels that are activated by Ca²⁺ depletion of the stores.

4. IL-1-induced ERK phosphorylation is dependent on [Ca²⁺]_i and focal adhesions
In cells plated on fibronectin to permit focal adhesion formation, ERK activity was blocked by pretreatment with an intracellular calcium buffer (BAPTA/AM), suggesting that Ca²⁺ signals were important for ERK stimulation by IL-1. No significant IL-1-induced ERK activation occurred in cells plated on poly-L-lysine (Fig. 2 A), indicating that focal adhesions are required for IL-1 activation of ERK. In a second, related approach, we used Hep-I, SPARC and tenascin-C to examine the importance of focal adhesion formation on IL-1-induced ERK phosphorylation. Fibroblasts were plated on tissue culture plastic in normal growth medium and cells were pretreated with Hep-I or SPARC peptides or tenascin-C before incubation with IL-1. In peptide-treated cells, IL-1-induced ERK phosphorylation was markedly reduced compared with controls (Fig. 2C, D ). Treatment of cells with increasing concentrations of Hep-I, SPARC, and tenascin-C resulted in a dose-response inhibition of ERK phosphorylation.

View larger version (40K): [in this window] [in a new window]   Figure 2. IL-1-induced ERK phosphorylation is dependent on [Ca2+]i and focal adhesions. A) Fibroblasts plated on fibronectin or poly-L-lysine were preincubated in either normal or 100 nM Ca2+ medium with 3 µM BAPTA/AM for 15 min before addition of IL-1 (20 ng/mL) or pervanadate (50 µM). Phospho-ERK activity was assessed by separating lysates with SDS-PAGE and probing blots with mouse monoclonal anti-phospho-ERK. Total ERK was assessed probing separate blots with rabbit polyclonal anti-ERK. B) Quantification by densitometry of immunoblots are shown as histograms of mean density ± SE (n=5). C) Cells plated on tissue culture plastic in normal growth medium were pretreated with Hep-I (0.5, 1.0, 1.5 µg/mL), SPARC (0.5, 1.0, 1.5 µg/mL), or tenascin-C (15, 30, 45 µg/mL), respectively, for 45 min before stimulation with IL-1, BSA (7 µg/mL) -pretreated cells as controls. Phospho-ERK and total ERK were assessed as described in panels A, D. Quantification by densitometry of immunoblots are shown as histograms of mean density ± SE (n=3).

CONCLUSIONS

The novel findings of this study are 1) focal adhesion dispersing peptides block IL-1-induced calcium signaling and activation of ERK by mediating disassembly of focal adhesions; 2) the ability of IL-1 to release calcium from internal stores is dependent on the formation of focal adhesions and the organization of the actin cytoskeleton; and 3) IL-1-induced release of calcium from internal stores mediates activation of store-operated calcium channels and this process requires intact focal adhesions. To our knowledge, this is the first description of the specific mechanisms by which focal adhesions regulate calcium handling in cells and provide a structural context by which the organization of the cytoskeleton affects calcium signaling and subsequently MAP kinase activation in response to an inflammatory cytokine.

We had previously demonstrated that IL-1-induced calcium signals are dependent on focal adhesions, but until now we had no insight into how intracellular calcium handling would affect downstream inflammatory mediators such as ERK activation. Regeneration of focal adhesions by application of fibronectin-coated beads onto the dorsal surface of rounded cells plated on poly-L-lysine was sufficient to restore responsiveness to IL-1 without inducing cell spreading. Thus, fibronectin-induced cross-linking of integrins in small, discrete sites was sufficient for formation of the adhesive cellular domains required for IL-1 receptor clustering and IL-1 signal transduction.

Our new observations show that the nature of the adhesions providing cellular attachment to the extracellular matrix strongly affects the organization of the actin cytoskeleton. The organization of actin filaments in the vicinity of the endoplasmic reticulum stores in turn regulates release of calcium from the stores (Fig. 3 ). The failure to release Ca²⁺ from the endoplasmic reticulum has important consequences. Because the stores cannot be depleted (either by plasma membrane agonists or directly by thapsigargin), the store-operated channels will not open; this leads to complete loss of both the intra- and extracellular components of the Ca²⁺ signal. These data support the notion that cellular adhesions with matrix ligands regulate the generation of signals following IL-1 receptor ligation (Fig. 3) and provide a direct link between actin cytoskeletal organization and the responsiveness of cells to proinflammatory cytokines.

View larger version (27K): [in this window] [in a new window]   Figure 3. Segregation of IL-1 receptors to focal adhesions. A model showing how the organization of the actin cytoskeleton affects the ability of the ER to release calcium after IL-1 stimulation. As a consequence the store-operated channels will not activate and allow entry of calcium. The failure to release calcium from the endoplasmic reticulum or to permit entry of calcium through the plasma membrane blocks the activation of ERK that would normally occur after IL-1 stimulation. This model provides a structural context by which downstream calcium and MAP kinase signals are restricted when cells do not form appropriately organized actin filaments and IL-1 receptors into focal adhesions.

Our findings highlight how interactions of cell surface receptors with integrins and related proteins in focal adhesions are crucial in regulating MAP kinases such as ERK and, downstream of ERK, the expression of genes that regulate cell behavior such as the matrix metalloproteinases. The biological significance of our data are that focal adhesion-dependent increases of intracellular calcium in response to IL-1 provide a link between IL-1 signaling, the organization of the actin cytoskeleton, and activation of the MAP kinase ERK (Fig. 3) .

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0069fje; doi: 10.1096/fj.03-0069fje

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This Article Full Text (PDF) All Versions of this Article: 17/13/1898 03-0069fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by WANG, Q. Articles by MCCULLOCH, C. A. Search for Related Content PubMed PubMed Citation Articles by WANG, Q. Articles by MCCULLOCH, C. A.