©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Interleukin-1-induced Calcium Flux in Human Fibroblasts Is Mediated through Focal Adhesions (*)

(Received for publication, June 23, 1994; and in revised form, November 28, 1994)

Pamela D. Arora Johnny Ma Weixian Min (1) Tony Cruz (1) Christopher A. G. McCulloch

From the Medical Research Council Group in Periodontal Physiology, Faculty of Dentistry, University of Toronto, Toronto, M5S 1A8 Canada Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, M5G 1X5 Canada

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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 [Ca]responses that were maximal at 10M IL-1beta with time to maximum flux of 50 s. Cells incubated with anti-Type 1-IL-1 receptor antibody exhibited a 45 nM increase in [Ca] above baseline but demonstrated no calcium response after IL-1beta treatment. Incubation with EGTA (5 mM) or thapsigargin (1 µM) caused 75% and 37% reductions, respectively, in the IL-1-induced [Ca] increase, suggesting that extracellular Ca predominates in IL-1-stimulated calcium flux. Cells in suspension did not exhibit [Ca] responses to IL-1beta. The relationship between [Ca]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) in focal adhesions and demonstrated [Ca]responses with 10M IL-1beta. Cells on poly-L-lysine or cells in suspension did not exhibit co-distribution of pp125, IL-1 receptor, and focal adhesion proteins and did not exhibit calcium flux. The dependence of IL-1-stimulated [Ca] responses on tyrosine kinases was examined first by treating cells with genistein, a selective inhibitor of tyrosine kinases. Genistein (100 µM) completely blocked [Ca]responses to 10M 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 antibody. Cells grown on fibronectin and stimulated with IL-1 exhibited tyrosine phosphorylation of pp125 whereas untreated cells or cells grown on poly-L-lysine and treated with IL-1 showed no reaction. Fibroblasts electroinjected with anti-pp125monoclonal antibody showed no [Ca] response, whereas cells treated with an irrelevant antibody exhibited a normal [Ca] response. Collectively, these data indicate that fibroblasts require substrate attachment and clustering of IL-1 receptors to focal adhesions for IL-1-induced [Ca] responses. Calcium fluxes are mediated through tyrosine kinases whose substrates include pp125. These studies therefore demonstrate that activation of intracellular signaling pathways by IL-1 is dependent on IL-1 receptor-cytoskeletal protein interactions.


INTRODUCTION

Interleukins-1 (IL-1) (^1)are a group of monocyte-derived peptides that play a pivotal role in regulating the host response to infection and injury. Two related forms of IL-1 (alpha and beta) exhibit 26% identity at the amino acid sequence level(1) . These cytokines mediate many features of inflammation such as fever, the acute phase response, leukocyte accumulation, and bone resorption (2) as well as connective tissue degradation and remodelling(3) . IL-1 affects collagen synthesis by osteoblasts(4) , proteoglycan synthesis by chondrocytes(5) , and induces proliferation (6) and collagenase secretion in fibroblasts(7) .

IL-1alpha and -beta share a common, high affinity cell surface receptor which is thought to mediate their biological effects(8, 9, 10) . Two types of receptors for IL-1 have been cloned and characterized biochemically (11) but only the type 1 (80-kDa) IL-1 receptor appears to mediate biological responses to IL-1(12) . The function of the type II (60-kDa) IL-1 receptor is not as well understood but does not appear to transduce signals(13) . Studies of I-labeled IL-1alpha and -beta binding to human fibroblasts reveal high numbers (5,000-15,000) of IL-1 receptors per cell(14) . Evidence from internalization and localization studies in fibroblasts indicates that IL-1 receptors are concentrated at focal adhesions(15) . These data suggest that IL-1 may affect the interactions of fibroblasts with the extracellular matrix by modulating cell-matrix interactions at focal adhesions(16) . Indeed, IL-1 causes a transient increase in phosphorylation and redistribution of talin by rapid post-translational modification(17) .

The detailed mechanism of action of IL-1 is unknown. Although the initial signaling step appears to involve binding to plasma membrane receptors(18, 19) , the mechanisms by which the occupied receptor generates intracellular signals and the nature of these signals are not well understood. For example, the cytoplasmic domain of IL-1 receptor (8) shows no sequence similarity to other protein tyrosine kinase receptors such as platelet-derived growth factor(20) . Although there have been conflicting reports of changes in second messengers(21, 22, 23) , there is some evidence from early changes in protein phosphorylation (24, 25) that binding of IL-1 to its receptors induces protein kinase activity(26) . There is also a possible involvement of G-proteins in signal transduction(27, 28) .

Calcium is an important second messenger that mediates a large number of cellular processes. An increase in intracellular Ca concentration ([Ca]) is critical for signal transduction in many cell types(29, 30) . For example, Ca flux has been implicated in the initial action of another interleukin, IL-2(31) . However, there are very few reports on IL-1 regulation of [Ca]. IL-1 does not appear to induce [Ca] responses in UMR-160 cells, an osteoblastic cell line(32) , in human neutrophils(33) , in a T lymphoma cell line(34) , or in a pre-B cell line(35) . However, one report on foreskin fibroblasts indicated a very slow increase in [Ca] 45-60 min after incubation with IL-1 (36) , the physiological significance of which is unclear.

IL-1 strongly affects periodontal connective tissue metabolism(37, 38) . Cells from these tissues exhibit large numbers of high affinity receptors (15) and have been used extensively to study IL-1 regulatory mechanisms(15, 17, 39) . Therefore, we have used human gingival fibroblasts as a model to examine the role of focal adhesions and associated tyrosine kinases in IL-1-induced calcium signaling in fibroblasts.


EXPERIMENTAL PROCEDURES

Materials

Recombinant human IL-1alpha and -beta (40) and IL-1B-PE were obtained from R& Systems (Minneapolis, MN). The protein was purified by sequential chromatography (to >97% purity), and the endotoxin level was determined to be leq0.1 ng/µg IL-1. Fura2 and fura2/AM were obtained from Molecular Probes. Ionomycin was from Calbiochem. Mouse monoclonal antibody to the human IL-1 type I receptor was from Genzyme. Rabbit and mouse antifocal adhesion kinase antibodies were from UBI, mouse monoclonal antibodies to vinculin (clone hvin-1) and talin (clone cd4) were from Sigma and anti-human CD4 antibody was from Coulter. TRITC- and FITC-conjugated antibodies, cytochalasin D, and PGE(2) were from Sigma. SM-2 Bio-Beads and electroporation cuvettes and columns were from Bio-Rad. Protein G beads were from Pharmacia Biotech Inc., and the ECL system was from Amersham. Genistein was from Life Technologies, Inc., genistin was from Extrasynthase (France), and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) was from Toronto Research Biochemicals.

Cell Culture

Human periodontal ligament and human gingival fibroblasts obtained as described (41) were grown in T-75 flasks containing minimal essential medium supplemented with antibiotics (0.17% penicillin V, 0.1% gentamycin sulfate, and 0.01 µg/ml amphotericin) and 15% fetal bovine serum. The cells were cultured at 37 °C in a humidified atmosphere of 95% air and 5% CO(2) and passaged after detachment with 0.01% trypsin. Prior to experiments with IL-1, cells were detached by gentle scraping with a rubber policeman or by incubation for 8 min at 37 °C with 0.5 M EDTA after washing with phosphate-buffered saline. Fibroblasts were plated on 60-mm Petri dishes 2 days before performing experiments. Cells between 4th and 12th passages were used in all experiments.

Dye Loading

Cells in suspension were loaded with 1 µM fura2/AM for 15 min at 37 °C followed by 15 min at room temperature to obtain uniform dye loading. Visual inspection of loaded cells by fluorescence microscopy showed even distribution of dye throughout the cytoplasm with no evidence of discrete vesicular labeling. The cells were washed twice to remove extracellular dye and finally suspended in buffer containing 145 mM NaCl, 5 mM KCl, 5 mM MgCl(2), 10 mM glucose, 10 mM HEPES, 1 mM CaCl(2), with pH adjusted to 7.4. Osmolality of the buffer was measured by freezing point depression and adjusted to 291 mosm. Attached cells were loaded with 3 µM fura2/AM at 25 °C for 35 min. CaCl(2) was omitted from the buffer solution where indicated.

[Ca](i) Measurement

The relative fluorescence intensity of suspended cells was estimated by using single-wavelength excitation (380 nm; Hitachi-F2000) or by dual wavelength excitation (355 nm/380 nm; Photon Technology International, London, ON) with emission at 510 nm. The slit widths were set at 5 and 10 nm for the Hitachi system and 2 and 5 nm for the PTI system. Estimates of [Ca](i) independent of the precise intracellular concentration of fura2 were calculated from emitted fluorescence according to the equation of Grynkiewicz et al.(42) where [Ca](i) (in nM) = K(d) times Sf(2)/Sb(2) times (R - R(min))/(R(max) - R). The K(d) (224 nM) and Sf(2)/Sb(2) ratio were calculated at 380 nM from 11 excitation wavelength scans of 1 µM fura2 free acid in buffers with [Ca](i) ranging from 0-39.8 µM. The maximal 346/380 ratio (R(max)) was measured after saturation of intracellular fura2 with Ca by adding 3 µM ionomycin to allow equilibration with extracellular calcium ions. The minimal 346/380 ratio (R(min)) measured during complete disassociation of fura2 from Ca was obtained by adding 2 mM EGTA to the bathing buffer for each measured cell. In each experiment, background was estimated by incubation of cells with 10 mM Mn, and this value was subtracted from the cellular fluorescence values before the 346/380 ratios were calculated. Intracellular calcium measurements on attached cells were obtained by a dual excitation epifluorescence illumination system (PTI, London, Canada) using a Nikon 40times oel objective (NA 1.32). A variable aperture mask (50 µm^2) within the optical path was used to restrict measurements to single cells. The emission signal was filtered by a 510 nm long pass barrier filter and detected by photon counting. The output of the photon counter was digitized and recorded on the system computer.

Flow Cytometry

Single cell suspensions were prepared by treating cell cultures with 0.5 mM EDTA and washing twice with saline buffer. The cells were subsequently stained with IL-1beta conjugated to phycoerythrin (IL-1beta-PE). Nonspecific IL-1beta-PE staining was evaluated by preincubation with 100-fold molar excess of unlabeled IL-1beta. Measurements of fluorescent intensity for individual cells due to specific binding of IL-1beta-PE were obtained by comparing cells stained with IL-1beta-PE and cells stained with streptavidin-PE as controls to determine fluorescence channel thresholds. To assess the relative abundance of IL-1 receptors, cells in suspension were labeled with monoclonal antibody to IL-1 type 1 receptor (1:10 dilution) for 1 h followed by FITC-conjugated goat anti-mouse (1:100 dilution). Background fluorescence of cells due to nonspecific staining by second antibody was estimated by measuring cells without primary antibody. Samples were analyzed on a FACSTAR Plus flow cytometer (Becton-Dickinson) at 200 cells/s with 488 nm laser excitation. Emission signals were obtained with a 590/30 nm band pass filter. Ten thousand cells were analyzed in each sample, and data were collected in list mode using logarithmic amplifiers. To eliminate signals due to cellular debris, particles with forward light scatter comparable to previously established threshold values for fibroblasts were assessed.

Immunofluorescence Staining

Multichamber glass slides were coated with bovine plasma fibronectin (1 µg/ml) or poly-L-lysine (1 mg/ml) when required and allowed to dry for 3 h under sterile conditions before plating the cells. Cells were plated for 24 h, fixed in 3.7% buffered formaldehyde for 15 min at room temperature, permeabilized with 0.3% Triton X-100, and thoroughly rinsed with phosphate-buffered saline. Immunofluorescence staining for the focal adhesion kinase (pp125) was performed using a rabbit antifocal adhesion kinase polyclonal antibody (1:20 dilution) or a mouse monoclonal antifocal adhesion kinase antibody (1:10 dilution) for 1 h at 37 °C followed by a TRITC-conjugated donkey anti-rabbit antibody (1:100 dilution) for 1 h at 37 °C. Mouse monoclonal anti-human talin, anti-human vinculin, anti-human IL-1 type I receptor, or anti-pp125 antibodies were used at 1:10 or 1:20 dilutions followed by FITC-conjugated goat anti-mouse antibody (1:100 dilution). Nonspecific labeling associated with TRITC-conjugated reagents was reduced by incubating TRITC-conjugated antibodies with gel filtration beads followed by centrifugation, a procedure that removes free dye. Preimmune goat and donkey sera were used to block nonspecific staining prior to each antibody incubation. Control staining was performed on the same slide using secondary antibody only. All slides were counterstained with 4`,6-diamidino-2-phenylindole (1 µg/ml). Preparations of attached cells were mounted with an anti-fade solution (43) . To immunolabel single cell suspensions, T-75 cell culture flasks were washed with phosphate-buffered saline followed by 12-min incubation in 0.5 mM EDTA. Cells were fixed by 3.7% formaldehyde, permeabilized in 0.02% Triton X-100, double-stained for pp125 and IL-1 receptor or vinculin or talin as described above, and suspended in phosphate-buffered saline.

Electroporation

Mouse monoclonal anti-human pp125 or an irrelevant antibody (CD4, human lymphocyte surface marker) was electroinjected into cells by electroporation as described(44) . Briefly, a Bio-Rad gene pulser with a capacitance extender and sterile cuvettes were used for experiments. An 800-µl aliquot of 2 times 10^6 cells/ml was placed into an electroporation cuvette (0.4-cm interelectrode distance). The cells were electroporated in a buffer containing 1.26 mM CaCl(2), 5.37 mM KCl, 0.52 mM KH(2)PO(4), 0.64 mM MgCl(2), 0.63 mM MgSO(4), 85.5 mM NaCl, 5.8 mM NaHCO(3), 0.50 mM NaH(2)PO(4), and 12.5 mM HEPES at a field strength of 250 V/cm and capacitance of 500 µF. The cells were collected by centrifugation, suspended in growth medium, allowed to attach, and subsequently loaded with fura2/AM for [Ca](i) measurements. Previous detailed assessments of this protocol have demonstrated that 10^8 antibody molecules per cell can be introduced into viable cells (44) and that electroinjected antibodies can specifically bind and inactivate specific functional proteins(41) .

Confocal Microscopy and Fluorescence Spectrophotometry

The spatial distribution of probes for talin, vinculin, IL-1 receptor, and pp125 in focal adhesions was imaged in single cells on a confocal microscope (Leica CLSM). For FITC-labeled probes, excitation was set at 488 nm and emission at 530 nm. For TRITC, excitation was set at 530 nm and emission at 620 nm. Cells were imaged with a 63times oil immersion lens (N.A. 1.4), and transverse optical sections (nominal thickness = 0.5 µm) were obtained at the level of cell attachment to the substratum.

Comparison of IL-1 binding in well-spread, attached cells and in detached cells was evaluated by affinity labeling with IL-1beta-PE. The fluorescence associated with labeled cells was measured with a fluorescence spectrophotometer (MVP-SP, Leitz, Wetzlar, Germany) equipped with a X63 PlanApo objective (N.A. 1.4, Leitz). Excitation light was obtained from a 100-watt, voltage-stabilized (±1%) mercury arc lamp and a 530/20 filter cube. Emission due to IL-1beta-PE labeling was collected with an emission monochromator set to 610/3 nm. The photomultiplier tube voltage was set to 781 V and gain to 4times. Corrections for background fluorescence intensity and dark current were made by subtraction of separate unlabeled samples from previously determined measurements of labeled. Each cell was measured five times, and the average fluorescence intensity per cell was calculated. Nonspecific binding was evaluated by incubation with streptavidin-PE and subtraction from the signals obtained with IL-1beta-PE staining.

Tyrosine Phosphorylation

If focal adhesion proteins are involved in IL-1 signal transduction, we sought to determine if IL-1 would increase the tyrosine phosphorylation of the focal adhesion kinase, pp125. Cells were grown on 120-mm dishes coated with either fibronectin or poly-L-lysine as described above and depleted of fetal bovine serum overnight in regular medium to decrease endogenous phosphorylation of pp125. Cells were either stimulated with IL-1 (1 nM) or were unstimulated, immediately washed twice with phosphate-buffered saline and 100 µM NaVO(4), harvested with 0.8 ml of 0.25% SDS (w/v) in phosphate-buffered saline containing 100 µM NaVO(4), and put on ice for 10 min. The lysates were boiled for 3 min, put on ice for 15 min, and centrifuged, and the supernatant was collected and made up to a volume of 4 ml. Supernatants were incubated with a rabbit polyclonal anti-phosphotyrosine antibody (4 µg/ml), gently shaken at 4 °C for 1 h, and passed three times through a 0.5 times 10 cm column (Bio-Rad) containing protein G beads (Pharmacia). The column was washed with phosphate-buffered saline plus 100 µM NaVO(4), and bound proteins were eluted with 1 ml of 40 mM phenyl phosphate containing 100 µM NaVO(4), 90 mM NaCl, 0.05% SDS, and 1 mM NaF. Fractions (75-80 µl) were collected, run on 7.5% SDS-polyacrylamide gel electrophoresis, and immunoblotted. Blots were probed with mouse monoclonal anti-human pp125 (4.2 µg/5 ml for 1 h) and detected with second antibodies (1:1000 for 1 h) and ECL supplied by Amersham.

PGE(2) Studies

To assess whether PGE(2) like IL-1 may require substrate attachment for calcium flux, we conducted PGE(2) dose-response studies. Attached or suspended fibroblasts (5 times 10^6M cells per ml) loaded with fura2/AM were stimulated with PGE(2). We also examined if IL-1-induced calcium flux may be mediated indirectly through PGE(2) release. Cells were incubated with indomethacin to block PGE(2) release.

Statistical Analysis

For [Ca](i), fluorescence spectrophotometry and flow cytometry data, means and standard errors were computed and comparisons between two groups were evaluated with unpaired Student's t test.


RESULTS

Calcium Response

Initial work with flow cytometric analysis of IL-1beta-PE binding to gingival and periodontal fibroblasts demonstrated that gingival fibroblasts exhibited 25% higher mean fluorescence compared to periodontal ligament fibroblasts. Therefore, subsequent work was performed on a gingival fibroblast line only (PA-4; (41) ). Initial experiments were also performed to compare rIL-1beta and rIL-1alpha [Ca](i) responses in fibroblasts. rIL-1beta exhibited 20% higher and more consistent [Ca](i) responses than rIL-1alpha at the same doses. Hence, for this study, all experiments used rIL-1beta.

In substrate-attached cells grown on glass, there was a dose-dependent increase of [Ca](i) with increasing doses of IL-1beta (Fig. 1). The dose of IL-1beta required to generate a maximal [Ca](i) response was 10M with a time to maximum flux of 50 s (Fig. 2A). There was no detectable change in [Ca](i) observed in suspended fibroblasts (5 times 10^6 cells/ml) even at very high concentrations (10M) of IL-1alpha or IL-1beta (Fig. 2A), a thousandfold higher than the concentration at which biological responses have been reported previously(3, 45, 46) . Cells stimulated with anti-type 1 IL-1 receptor antibody (1:10 dilution) exhibited a small (45 nM) increase in [Ca](i) above baseline, but these cells did not respond subsequently to 10M IL-1beta (Fig. 2B), indicating that the IL-1beta-induced calcium flux was mediated through the type 1 receptor. Cells incubated in 5 mM EGTA and stimulated with 10M IL-1beta exhibited a 120 ± 10 nM increase of [Ca](i) above baseline (n = 3), which was a 75% reduction of the IL-1-induced increase of [Ca](i) compared to cells in medium with calcium (Fig. 2B; p < 0.01). Release of calcium from intracellular stores was examined by preincubation with 1 µM thapsigargin for 30 min. Thapsigargin is a tumor-promoting sesquiterpene lactone which blocks the ATPase required for Ca uptake into intracellular stores. Cells that were then stimulated with 10M IL-1beta exhibited a 305 ± 30 nM [Ca](i) increase (n = 3), which was a 37% reduction (Fig. 2B; p < 0.01). When cells were incubated with thapsigargin (1 µM) for 30 min, reapplication of thapsigargin 10 min later produced no further increase of [Ca](i), indicating that this protocol effectively depleted releasable intracellular stores. Thus, in substrate-attached cells, IL-1-induced calcium flux originated predominantly from Ca in the extracellular medium.


Figure 1: Dose-response data of peak [Ca] in attached fibroblasts after IL-1beta stimulation. Cells were plated on glass and formed focal adhesions. Cells were stimulated with either vehicle (water) or with indicated dosages of IL-1beta. Figure is a log (IL-1 concentration) versus a linear ([Ca]) plot.




Figure 2: Sample tracings showing the [Ca] response of suspended and attached fibroblasts to IL-1beta, the dependence of calcium flux on external and internal calcium stores, and signaling through the type 1 IL-1 receptor. A, ratio fluorimetry of fura2-loaded fibroblasts was used to measure [Ca]. Attached cells exhibited a sharp increase of [Ca] when stimulated with 10M IL-1beta, whereas no response was detected in suspended cells, even when stimulated with 10M IL-1beta. B, top trace shows typical [Ca] response to IL-1beta (10M) of attached cells pretreated with thapsigargin (1 µM) to deplete internal calcium stores. Middle trace shows representative response of cells incubated in buffer without calcium ions and with 5 mM EGTA. Bottom trace shows [Ca]response of attached cells to anti-type 1-IL-1 receptor antibody and subsequently stimulated with IL-1beta (10M). Traces have been offset vertically for clarification, but the actual baseline [Ca] before each treatment was not significantly different from that of the untreated cells. The marker above each trace indicates the time of addition of IL-1beta.



IL-1 Receptor Expression

As the absence of a detectable calcium flux in suspended cells could be explained by down-regulation of IL-1 receptors, flow cytometry was used to estimate IL-1 receptor expression and whether suspended cells bound IL-1beta. Cells stained with streptavidin-PE exhibited fluorescence (mean channel number = 10.2 ± 0.3) which was not significantly different from cells that were preincubated with unlabeled 10M IL-1beta and then stained with 10M IL-1beta-PE (mean channel number = 11.8 ± 0.3; p > 0.5). Cells stained with 10M IL-1beta-PE exhibited 25-fold higher fluorescence (mean channel number = 295.6 ± 9.8; p < 0.001). We next compared the binding of IL-1beta-PE to cells in suspension or in populations of attached cells by microfluorimetry. The mean fluorescence of attached cells was 18.0 ± 0.44 fluorescence units and for suspended cells was 17.2 ± 1.67 fluorescence units (p > 0.5), indicating that IL-1beta binding to cells was not affected by attachment to substrate. Assessment of IL-1 receptor expression by flow cytometry showed that cells stained with antibody to IL-1 type 1 receptor exhibited 12-fold higher fluorescence (mean channel number = 117.104 ± 12.5) than cells stained with second antibody only (mean channel number = 10.49 ± 3.25; p < 0.001), indicating the presence of abundant type I receptors.

Regulation by Extracellular Matrix

A large body of evidence suggests that integrins, as cell adhesion receptors, can transduce biochemical signals from the extracellular matrix to the cell interior (47) . To examine if IL-1-induced [Ca](i) signaling was dependent on focal adhesions, cells were plated for 24 h on glass, fibronectin, or poly-L-lysine. Immunolocalization of focal adhesions with antivinculin antibody and imaging by confocal microscopy showed discrete localization of focal adhesions at the substrate-cell interface but only in cells plated on glass or fibronectin (Fig. 3A). Cells grown on poly-L-lysine exhibited diffuse staining throughout the cell, and staining could be visualized only when photomultiplier tube voltages were increased (Fig. 3B). Cells plated on fibronectin exhibited a resting [Ca](i) = 97.6 ± 5.9 nM (n = 4) and, when stimulated with IL-1beta (10M), exhibited a four-fold increase of [Ca](i) (400 ± 37.0 nM; n = 4; Fig. 3C) which was not significantly different from the calcium flux for cells plated on glass (Fig. 1; p > 0.2). In contrast, cells plated on poly-L-lysine showed no calcium flux, even at higher IL-1 concentrations (10M; Fig. 3C). Cells in suspension incubated with soluble fibronectin (10 µg/ml) and stimulated with IL-1beta (10M) exhibited no [Ca](i) response (Fig. 3C), demonstrating a requirement for substrate attachment and not just occupation of fibronectin receptors for promotion of an IL-1beta-induced calcium flux (Fig. 3C).


Figure 3: Fluorescence micrographs and measurements of [Ca] to illustrate the dependence of IL-1-induced calcium flux on focal adhesions. A and B, fluorescence confocal micrographs of fibroblasts stained for vinculin when grown on either fibronectin (A) or on poly-L-lysine (B). Note that vinculin labeling is concentrated at the cell attachment sites on fibronectin, whereas on poly-L-lysine there is diffuse, uniform staining throughout the cell. In the cells plated on poly-L-lysine, the photomultiplier tube voltages of the confocal microscope were sharply increased so that vinculin staining could be visualized. C, representative tracings demonstrating [Ca]responses to IL-1beta (10M) of attached fibroblasts plated on fibronectin (1 µg/ml) or on poly-L-lysine (1 mg/ml). Sample tracings show responses of cells treated with cytochalasin D (1 µM) and then stimulated with IL-1beta (10M) or of cells in suspension incubated with soluble fibronectin (10 µg/ml) and stimulated with IL-1beta (10M). Note that sample tracings have been offset vertically for clarity.



As cytoskeletal components interact with the cytoplasmic domains of integrins at focal adhesions, cytoskeletal organization may orchestrate signals from the extracellular matrix. To probe the role of actin filaments in [Ca](i) signal transduction, fibroblasts were treated with cytochalasin D (1 µM) for 10 min and then stimulated with 10M IL-1beta after the cytochalasin was washed out with fresh buffer. This protocol is known to completely disrupt cortical actin filaments in fibroblasts (48) . Cytochalasin D-treated cells exhibited no [Ca](i) response to IL-1beta (Fig. 3C) indicating that calcium flux may be mediated through filamentous actin inserting into focal adhesions.

Role of Focal Adhesion Kinases

As IL-1 receptors are clustered at focal adhesions in substrate-attached cells(15) , we reasoned that IL-1 signal transduction occurs through focal adhesions and possibly involves focal adhesion-associated kinases(49) . First we used genistein as a selective inhibitor of tyrosine kinases as it does not inhibit other kinases such as protein kinase A and protein kinase C (50) . Incubation with genistein (100 µM; 10 min) followed by stimulation with 10M IL-1beta showed complete inhibition of the calcium response. An inactive analogue of genistein that lacks anti-tyrosine kinase activity, genistin (100 µM), did not markedly inhibit IL-1beta-induced [Ca](i) responses (Table 1). In Western blots of cell lysates that were probed with a rabbit anti-phosphotyrosine polyclonal antibody, there was complete blockade of activity with 100 and 50 µM genistein but not with the genistin, indicating that this protocol was indeed effective.



As protein kinase C is also localized to focal adhesions(51) , we employed the specific inhibitor H-7 (9 µM; 30 min; (52) ) to evaluate the role of protein kinase C in IL-1beta-induced calcium flux. Cells treated with H-7 showed a 37% reduction of [Ca](i) responses to IL-1 compared to untreated controls, indicating that the IL-1-induced calcium flux is mediated partly through protein kinase C (Table 1).

Immunolocalization of pp125 and vinculin, or talin or IL-1beta receptor showed co-distribution of probes in focal adhesions of double-labeled cells. In well-spread fibroblasts, confocal optical sections showed bright staining of arrowhead-shaped structures reminiscent of focal adhesions at the substratum-cell interface (Fig. 4, A-D). However, we were unable to detect co-distribution of pp125, vinculin, or IL-1 receptor in suspended cells (not shown).


Figure 4: Fluorescence micrographs and intracellular calcium measurements showing dependence of IL-1-induced calcium flux on focal adhesion kinases. A and B, paired fluorescence confocal micrographs of single fibroblasts double-stained for talin (A) and for pp125 (B) demonstrating co-distribution at the focal adhesions. C and D, paired confocal micrographs of fibroblasts double-stained for IL-1 receptor (C) and for pp125(D) showing co-distribution of receptor and focal adhesion kinase in the focal adhesions. E, confocal micrograph of a fibroblast stained with FITC-labeled goat anti-mouse antibody after electroporation in the presence of pp125 monoclonal antibody. The micrograph demonstrates specific binding of the antibody to focal adhesions and shows that cells electroinjected with pp125 antibody are fully capable of attaching and spreading. F, [Ca] responses of attached fibroblasts to IL-1beta (10M) after electroporation with pp125 antibody or an irrelevant antibody.



We examined the role of focal adhesion kinases in [Ca](i) signal transduction directly by electroporating cells in the presence of monoclonal pp125 antibody (250 µg/ml) or in controls electroporated with an irrelevant antibody that does not bind to any known fibroblast antigenic determinants (anti-CD4, a human lymphocyte surface marker; 250 µg/ml). Cells electroinjected with pp125 antibody were fixed and stained with FITC-labeled goat anti-mouse antibody to determine if the electroporation protocol resulted in antibody binding to pp125. Optical sectioning with the confocal microscope showed discrete localization of staining in focal adhesions (Fig. 4E). Cells electroporated with anti-pp125 or with the irrelevant antibody showed similar patterns of spreading on glass and fibronectin. The surface areas of attachment to the substrate as measured by confocal microscopy were not detectably different, indicating that the pp125 antibody did not interfere with cell attachment, and immunoprecipitates of tyrosine-phosphorylated proteins that were immunoblotted with pp125 antibodies showed inhibition of phosphorylation in cells electropored with pp125 antibodies. In separate experiments, electroporated cells were allowed to attach, loaded with fura2/AM, and subsequently challenged with rIL-1beta (10M). Cells electroporated with antibody to pp125 and stimulated with IL-1beta exhibited no significant change in [Ca](i) (resting [Ca](i) = 102.6 ± 4.3 nM; n = 4; IL-1-stimulated [Ca](i) = 121.5 ± 6.8 nM; n = 4) whereas cells electroporated with the irrelevant antibody showed a 3.5-fold higher [Ca](i) response (Fig. 4F; resting [Ca](i) = 105.6 ± 4.3 nM; n = 4; IL-1-stimulated [Ca](i) = 348.3 ± 36.5 nM; n = 4).

We used another, more direct approach to assess the role of pp125 in IL-1 signal transduction by immunoprecipitating cells with an antiphosphotyrosine antibody and then probing the cell lysates with a monoclonal antibody to pp125. Cells grown on poly-L-lysine did not exhibit tyrosine phosphorylation of pp125 either before or after IL-1 stimulation (Fig. 5). In contrast, cells grown on fibronectin exhibited increased phosphorylation after IL-1 stimulation, indicating that pp125 is itself phosphorylated after IL-1 binds to its receptor but only if it is aggregated into focal adhesions. The exact time for detection of pp125 phosphorylation after IL-1 stimulation was difficult to assess because of the cell preparation steps required for preparation of cell lysates but we estimate it to be less than 1 min.


Figure 5: Increased phosphorylation of pp125after IL-1 stimulation of fibroblasts on fibronectin but not poly-L-lysine substrates. Fibroblasts were grown on fibronectin (F, FC) or poly-L-lysine (P, PC), depleted of fetal bovine serum overnight to reduce endogenous phosphorylation of pp125 and either stimulated (F, P) or not stimulated (FC, PC) with IL-1 (1 nM). Cell lysates were immunoprecipitated with antiphosphotyrosine antibodies, and two different column fractions (P(1), PC(1), F(1), FC(1) or P(2), PC(2), F(2), FC(2) were blotted and probed with anti-pp125).



Comparison with PGE(2)-induced Calcium Flux

As IL-1induced calcium flux in fibroblasts appeared to be dependent on substrate attachment, we asked if other agents may also require cell attachment in order to induce a calcium flux. PGE(2) was used as a model cytokine to stimulate calcium flux in attached cells or in cell suspensions. Cells were incubated with PGE(2) at doses between 10 and 10M. For cells in suspension, maximal [Ca](i) responses were obtained at 2.5 times 10M PGE(2) with time to maximum flux of 40 s whereas for substrate-attached cells, the maximal response was obtained at 1.5 times 10M PGE(2) with time to maximum flux of 20 s. As IL-1 stimulates PGE(2) release from fibroblasts(28) , we determined if the IL-1-induced calcium flux may be mediated through PGE(2). Cells were pretreated with 10M indomethacin to block PGE(2) release. Previous studies on periodontal fibroblasts have shown that this protocol abrogates PGE(2) secretion(53) . Indomethacin-treated cells exhibited normal IL-1-induced calcium responses (resting [Ca](i) = 106.9 ± 11.2 nM; n = 4; IL-1-stimulated [Ca](i) = 345.5 ± 29.2 nM; n = 4) indicating that the IL-1 response was not likely mediated through PGE(2).

We examined the calcium response of cells spread on fibronectin that were preincubated with cytochalasin D and then treated with PGE(2) (10M) to determine if the disruption of actin in focal adhesions and stress fibers would also inhibit calcium flux. In contrast to IL-1, cells exhibited [Ca](i) responses albeit at an attenuated amplitude ([Ca](i) baseline = 115 ± 7.3 nM; stimulated with PGE(2) = 282 ± 35.5 nM; pretreated with cytochalasin and stimulated with PGE(2) = 168 ± 32.1 nm).

To determine the relative specificity of the genistein block on PGE(2)-induced calcium flux, cells in suspension were preincubated with 10, 50, or 100 µM genistein and then stimulated with PGE(2) (10M). Genistein reduced but did not completely inhibit the calcium response, even at 100 µM ([Ca](i) values: at 10 µM genistein, baseline = 83 ± 4.0 nM, stimulated = 211 ± 20.2 nM; at 50 µM genistein, baseline = 87 ± 7.8 nM, stimulated = 140 ± 15.0 nM; at 100 µM genistein, baseline = 87 ± 5.8 nM, stimulated = 132 ± 9.4 nM).


DISCUSSION

Calcium Flux

We have demonstrated an absolute requirement of substrate attachment and focal adhesion formation for IL-1beta-induced calcium flux in fibroblasts. A thousandfold higher than the required dose of IL-1 for maximal Ca response in attached cells failed to induce calcium flux in cell suspensions. This result was not simply due to dye leakage or cell death as the cells responded to low doses of ionomycin and also exhibited calcium fluxes to PGE(2). Thus, the methods employed to detect changes in [Ca](i) were of adequate sensitivity. The failure to induce calcium flux in suspended cells was also not because of stripping receptors from the cell surface as studies with flow cytometry and microfluorimetry indicated the presence of IL-1 receptors and specific IL-1beta binding in detached and attached fibroblasts. Notably, previous reports have also failed to show IL-1alpha- or IL-1beta-induced calcium flux in suspended UMR-106 cells(32) , T lymphoma cells(34) , pre-B cells(35) , or in human neutrophils(33) . However, Bouchelouche et al.(36) reported a very delayed (45 min) calcium flux in response to rIL-1alpha and -beta treatment of fibroblasts. These results were not suggestive of a classical receptor-mediated calcium response.

Our findings show that the dose-dependent calcium flux induced by IL-1 was mediated through the type I IL-1 receptor and was due predominantly to extracellular Ca and to a lesser extent originated from intracellular stores. These data indicate that IL-1 may regulate a Ca-permeable ion channel and to a lesser extent may activate inositol 1,4,5-trisphosphate-dependent Ca release from internal stores. In T lymphoma cells, both external and internal calcium sources contributed significantly to calcium flux after IL-1 treatment but only when cells were preincubated with phytohemagglutinin (34) and there was no calcium flux with IL-1beta alone. In view of these reports, it is conceivable that, depending on the type of cell attachment to the substrate, IL-1 might differentially activate target cells depending on the predominant intracellular signaling pathway for the particular cell type(16) . In the cells studied here, IL-1 alone was able to trigger a classical, receptor-dependent calcium flux within 50 s of incubation.

Our data show that IL-1beta-stimulated [Ca](i) responses and phosphorylation of pp125 in attached fibroblasts were dependent on the previous formation of focal adhesions (Fig. 5). Attached fibroblasts on poly-L-lysine could not form focal adhesions, did not exhibit calcium fluxes, and did not demonstrate increased phosphorylation of pp125 after IL-1 treatment, whereas cells on glass or fibronectin-coated glass did form focal adhesions, did exhibit calcium flux, and did exhibit increased phosphorylation of pp125. Immunolocalization studies with antibody to type I IL-1 receptor and affinity labeling with IL-1beta-PE demonstrated that the receptors were present at focal adhesions and that there was avid binding of IL-1 to receptors in both attached and suspended cells. These findings are consistent with reports that human gingival fibroblasts have high numbers (11,000 ± 100) of receptors per cell which bind IL-1beta with high affinity (10 ± 10^9M; (16) ) and that 70% of radiolabeled IL-1beta localizes to focal adhesions(15, 39) . As IL-1 receptors are concentrated in focal adhesions, it is conceivable that signal transduction is not so much dependent on the absolute number of receptors per cell but rather upon the concentration of receptors to localized regions where receptor density is high. The actual mechanism for signal transduction through the receptor is not known, but previous data have shown that IL-1 binding induces phosphorylation via a protein serine/threonine kinase(19, 25) . Consequently, it is possible that upon IL-1 binding to its receptor, the receptor phosphorylates calcium-permeable ion channels on serine and threonine residues and alters channel-opening probability, a phenomenon that has been described in other ion channels (for review, see (54) ).

Focal Adhesions

Increasing evidence has shown that the integrin family of cell adhesion receptors can transduce signals from the extracellular matrix to the cell interior(47) . There are also convincing data to show that cytokines can regulate intracellular metabolism by inducing integrin clustering after ligand binding, which in turn regulates the assembly of cytoplasmic plaques and stress fibers (55, 56, 57) . Our data indicated that the formation of focal adhesions can also regulate a separate cell signaling system, the IL-1 receptor. Thus, fibroblasts plated on fibronectin or on glass and stimulated with IL-1beta exhibited elevations of [Ca](i), whereas cells on poly-L-lysine showed no response. Further, preincubation of suspended cells with soluble fibronectin was not sufficient to permit IL-1induced calcium flux, apparently because spreading and focal adhesion formation do not occur in suspended cells. This finding is consistent with integrin-mediated cell spreading and regulation of [Ca](i) in human endothelial cells (58) , and the generation of spontaneous or chemoattractant-triggered [Ca](i) elevations in neutrophils adherent to fibronectin-coated surfaces(59) . Selective disruption of actin filaments with cytochalasin D completely inhibited the ability of ILl-1beta to increase [Ca](i), consistent with the inhibition of bombesininduced signaling in Swiss 3T3 cells by cytochalasin(60) . Thus, substrate attachment, focal adhesion formation, and actin filaments were necessary conditions for IL-1-induced calcium flux in fibroblasts. Notably, depolymerization of actin filaments by cytochalasin D also reduced the amplitude of [Ca](i) elevations after PGE(2) treatment, but there was not the complete inhibition as seen with IL-1. Thus, actin filaments are important for function of ligand-activated calcium-permeable channels, but the degree of sensitivity appears to be ligand-specific.

Tyrosine kinase activity in focal adhesion proteins is an important signaling system for integrin-dependent pathways(57, 61) . As shown by the genistein blockade experiment, tyrosine kinase activity was also essential for the IL-1-induced calcium flux, an observation that is supported by the increased phosphorylation of pp125 after IL-1 stimulation. However, the IL-1 receptor contains no amino acid sequences in the cytoplasmic domain that are suggestive of tyrosine kinase activity (16) nor do the cytoplasmic tails of the alpha or beta chains of the integrins exhibit such sequences(62) . Therefore, the increased phosphorylation of pp125 that we observed is probably not a result of activated IL-1 receptor directly phosphorylating pp125 but instead may be a reflection of either intermediate kinase cascades or autophosphorylation.

Several kinases have been identified in focal adhesions including protein kinase C, pp60, and pp125. We immunolocalized vinculin, talin, IL-1 receptors, and pp125 to common sites, indicating that these proteins are concentrated in focal adhesions and may be involved in IL-1-induced signal transduction. Plating cells on poly-L-lysine prevented the localization of pp125 to the focal adhesions, an observation consistent with the inhibition of pp125 phosphorylation when NIH 3T3 cells were plated on poly-L-lysine(63) . Electroinjection of a blocking antibody to the pp125 completely inhibited calcium flux, suggesting that the pp125 is an essential component of the IL-1-induced calcium flux. In contrast, inhibition of protein kinase C by H-7 pretreatment of cells only partly inhibited IL-1-induced calcium flux, indicating that this enzyme is not absolutely essential for the IL-1 signaling pathway. These data suggest that pp125 phosphorylation is an important component of IL-1-induced signal transduction and help to explain the dependence of IL-1-induced calcium flux on focal adhesion formation. Further, these findings in fibroblasts are consistent with data on ion channels in carbachol-stimulated cardiac muscle and neurons indicating that calcium flux is dependent on tyrosine kinases (54) and that bombesin-stimulated signaling in Swiss 3T3 cells is mediated through pp125(60) . Collectively, the data support the notion that IL-1 signal transduction in fibroblasts is dependent on the nature of the substrate and of the cellular attachments to the substrate and also suggest a mechanism by which fibroblasts can only respond to certain agonists when the conditions of their matrix attachment are appropriate. Thus, degradation of matrix proteins in inflammatory lesions may lead to significant alterations in cellular attachments and in the responses of cells to cytokines like IL-1.


FOOTNOTES

*
This work was supported by a Medical Research Council of Canada Group grant (to C. A. G. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

(^1)
The abbreviations used are: IL-1, interleukin-1; PE, phycoerythrin; TRITC, tetramethylrhodamine B isothiocyanate; FITC, fluorescein isothiocyanate; PG, prostaglandin.


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