Calreticulin Couples Calcium Release and Calcium Influx in Integrin-mediated Calcium Signaling

Abstract

The engagement of integrin α7 in E63 skeletal muscle cells by laminin or anti-α7 antibodies triggered transient elevations in the intracellular free Ca²⁺ concentration that resulted from both inositol triphosphate-evoked Ca²⁺ release from intracellular stores and extracellular Ca²⁺ influx through voltage-gated, L-type Ca²⁺ channels. The extracellular domain of integrin α7 was found to associate with both ectocalreticulin and dihydropyridine receptor on the cell surface. Calreticulin appears to also associate with cytoplasmic domain of integrin α7 in a manner highly dependent on the cytosolic Ca²⁺ concentration. It appeared that intracellular Ca²⁺ release was a prerequisite for Ca²⁺ influx and that calreticulin associated with the integrin cytoplasmic domain mediated the coupling of between the Ca²⁺ release and Ca²⁺ influx. These findings suggest that calreticulin serves as a cytosolic activator of integrin and a signal transducer between integrins and Ca²⁺ channels on the cell surface.

INTRODUCTION

Integrins are crucial for mediating cell–cell and cell–matrix adhesions, and their regulation is involved in such biological phenomena as cell proliferation, cell differentiation, tissue repair, gene expression, and cell death (Albelda and Buck, 1990; Helmer, 1990; Damsky and Werb, 1992; Dustin et al., 1992; Hynes, 1992; Ginsberg et al., 1992; Boudreau et al., 1995). The interaction between integrins and the extracellular matrix (ECM) activates intracellular signaling pathways (outside-in signaling) and results in recruitment of a number of signal molecules into focal adhesion sites. The interaction of these molecules with the integrin cytoplasmic domain elicits immediate feedback via a Ca²⁺ signal that regulates integrin affinity and modulates cell behavior (inside-out signaling; Marks et al., 1991; Hartfield et al., 1993).

Evidence suggests that the cell adhesion and migration mediated by integrins are regulated in part by changes in the free intracellular calcium concentration ([Ca²⁺]_i). For example, increases in [Ca²⁺]_i have been observed upon cell attachment to ECM or the binding of ligands or integrin antibodies to platelets, macrophages, neutrophils, osteoclasts, epithelial cells, or embryonic stem cells (Jaconi et al., 1991; Schwartz, 1993; Shankar et al., 1993; Coppolino et al., 1997). Furthermore, it has been generally accepted that the elevation of [Ca²⁺]_i results from a combination of inositol triphosphate (IP₃)-mediated Ca²⁺ release from intracellular stores in the sarcoplasmic reticulum/endoplasmic reticulum (SR/ER) and Ca²⁺ influx through plasma membrane Ca²⁺ channels, processes involving protein kinases, phospholipase Cγ1 (PLCγ1), calcium/calmodulin-dependent protein kinase II, calcineurin, and calreticulin (Kanner et al., 1993; Bastianutto et al., 1995; Camacho and Lechleiter, 1995; Lawson and Maxfield, 1995; Pomies et al., 1995; Hendey et al., 1996; Wrenn et al., 1996; Bouvard et al., 1998). Nonetheless, it has proved difficult to fully characterize the mechanism by which integrin activation is coupled to IP₃-dependent Ca²⁺ release or to Ca²⁺ channel activation.

Integrin cytoplasmic domains are the primary targets of cytoplasmic signals that alter the conformation of integrin extracellular domains, thereby modulating the affinity of integrins for ECM (Timothy et al., 1994). Ca²⁺-binding proteins that associate with integrins include calreticulin (Rojiani et al., 1991), calcineurin (Lawson and Maxfield, 1995; Pomies et al., 1995), calmodulin (Bouvard et al., 1998), and Ca²⁺- and integrin-binding protein (Naik et al., 1997). Among these proteins, calreticulin interacts with the KXGFFKR motif in the cytoplasmic domain of the integrin α chain (Rojiani et al., 1991), making it a good candidate for a modulator of integrin affinity.

The interaction between integrin and calreticulin is dependent on the activation state of the integrin and can be stimulated by both extracellular and intracellular events. The binding of calreticulin to integrin not only is enhanced by integrin activation but appears to be a requirement for the maintenance of the activated state. Thus, association of the KXGFFKR motif with calreticulin may stabilize the active conformation of the integrin and be important for integrin-mediated adhesion (Timothy et al., 1994; Coppolino et al., 1995; Coppolino and Dedhar, 1998). For instance, recently developed calreticulin-null embryonic stem cells exhibit severely impaired integrin-mediated adhesion to ECM and integrin-triggered extracellular Ca²⁺ influx (Coppolino et al., 1997). Thus, calreticulin is apparently not a simple Ca²⁺ storage protein but instead plays an important role in modulating Ca²⁺ signaling. Moreover, a recently isolated cell surface form of calreticulin, ectocalreticulin, is reported to participate in cell spreading as part of an integrin–calreticulin signaling complex (Zhu et al., 1997), suggesting that calreticulin may be functionally associated in integrin-mediated Ca²⁺ signaling. In the present study, therefore, we examined the functional role of calreticulin in the E63 skeletal muscle cell line and found that integrin-evoked Ca²⁺ signaling involves both Ca²⁺ release from SR and influx of extracellular Ca²⁺ via voltage-gated Ca²⁺ channels. Our findings further suggest that calreticulin mediates the coupling between the Ca²⁺ release and Ca²⁺ influx.

MATERIALS AND METHODS

Materials

Normal mouse serum (NMS), normal rabbit serum (NRS), horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody (Ab), HRP-conjugated goat anti-rabbit Ab, TRITC-conjugated donkey anti-rabbit immunoglobulin, and FITC-conjugated donkey anti-mouse immunoglobulin were all obtained from Jackson ImmunoResearch (West Grove, PA); polyclonal calreticulin Ab (PA3-900) was from Affinity Bioreagents (Golden, CO), and polyclonal calreticulin Ab (LAR090) was kindly provided by Dr. Luis A. Rokeach (University of Montreal, Montreal, Quebec, Canada); dihydropyridine receptor (DHPR) α1 Ab was from Upstate Biotechnology (Lake Placid, NY); Dulbecco's modified Eagle's medium (DMEM), antibiotic antimycotic, and the TRANSPORT transient cell permeabilization kit were from Life Technologies (Grand Island, NY); horse serum was from Gemini Bioproducts (Calabasas, CA); U73122 and neomycin were from Calbiochem (La Jolla, CA); nifedipine, thapsigargin (TG), heparin, and chondroitin sulfate A were from Sigma (St. Louis, MO); fluo-3/AM was from Molecular Probes (Eugene, OR); and Na¹²⁵I was from New England Nuclear (Boston, MA; 100mCi/ml).

Cell Culture

E63 cells, a myogenic clone of L8 rat skeletal myoblasts, were grown in DMEM supplemented with 10% horse serum, 100 U/ml penicillin G, 100 μg/ml streptomycin sulfate, and 250 μg/ml amphotericin under a humidified atmosphere of 90% air and 10% CO₂ at 37°C as previously described (Kaufman and Parks, 1977).

Measurement of [Ca²⁺]_i by Confocal Microscopy

E63 cells grown on 0.2% (wt/vol) gelatin-coated coverslips for 5 d were rinsed twice with bath solution (140 mM NaCl, 5.0 mM KCl, 0.5 mM MgCl₂, 20 mM glucose, 2.5 mM CaCl₂, 5.5 mM HEPES, pH 7.4) and then incubated in the dark for 1 h at 25°C in bath solution containing 5 μM fluo-3/AM. The coverslips were then rinsed twice with bath solution and mounted in a tissue chamber containing 250 μl of bath solution. Ca²⁺ measurements in single cells were made using a Leica (Nussloch, Germany) TCS 4D laser scanning microscope equipped with an argon–krypton laser to excite the dye at 488 nm. Cells were imaged with a 40× (numerical aperture 1.0) oil immersion objective. Before activating integrin in each experiment, areas of interest were selected for analysis. Integrin activation was then initiated by adding 50 μl of laminin (100 μg/ml) or the appropriate anti-α7 antibodies (15 μg/ml) to the tissue chamber. To avoid changes in physical disturbance attributable to the application of reagents, the reagents were added through the chamber wall, and cells were immediately scanned. Images (512 × 512 pixels) were obtained at a rate of one image per 3 s. To quantify fluorescence, pixel intensities within the selected single-cell areas of interest were measured and averaged. The independent experiment was repeated more than five times with the same gain. In a cell viability test using the ionophore A23187, the cells that elicited calcium influx by treatment with A23187 were counted as viable cells. The acquired data were analyzed using Microsoft (Redmond, WA) Excel version 4.0. Mean intensity (I_mean) was defined as an average of fluorescence intensity obtained from each pixel in the selected area, whereas average I_mean (Av. I_mean) was calculated from I_mean (Figure 1).

Figure 1.

Measurement of fluorescence intensity in the selected single-cell area using confocal microscope. Cells preloaded with fluo-3/AM were treated with laminin (100 μg/ml), and the fluorescence intensity was measured every 3 s in the selected area using a confocal microscope (n = 21; SD, ±1.94 ∼ 3.66). (1) A resting cell before laminin treatment elicits the basal level of fluorescence intensity. (2) The selected cell elicits the maximum fluorescence intensity after laminin treatment. (3) Fluorescence intensity drops down to the basal level at 90 s after laminin treatment. (4) Fluorescence intensity is increased by ionophore A23187 treatment, indicating that the cell is viable. n, cell number; SD, SD of Av. I_mean.

Permeabilization

E63 cells were washed twice with PBS, pH 7.4, and permeabilized to selected concentrations of KLGFFKR or KLRFGFK for 10 min using the TRANSPORT transient cell permeabilization kit. The cells were then washed with PBS and immediately added to serum-containing media. After incubation for 3 h, the relative change in [Ca²⁺]_i as reflected by changes in fluo-3 fluorescence was measured by confocal microscopy.

Fluorometric Analysis

For conjugation of FITC to KLGFFKR peptides, 2 mg of KLGFFKR peptides were incubated at 4°C for 8 h with 50 μl of FITC (1 mg/ml) in 1 ml of sodium carbonate buffer (0.1 mM sodium carbonate, pH 9.0). This solution was treated with NH₄Cl to 50 mM, followed by incubation at 4°C for 2 h. FITC-KLGFFKR conjugates were purified by SCL-10A reverse-phase HPLC (Shimadzu, Kyoto, Japan).

For fluorometric analysis, E63 cells cultured in a 35-mm dish were permeabilized using the TRANSPORT transient cell permeabilization kit and then treated with FITC-conjugated KLGFFKR peptides or with KLGFFKR peptides alone. After incubation in serum-containing DMEM for 3 h in 10% CO₂, the cells were extracted for 1 h at 4°C with 0.1 ml of lysis buffer (PBS and 1% Triton X-100). The lysates were centrifuged for 10 min at 12,000 × g, and then the supernatant was loaded into a 96-well microplate and applied to an FL-600 microplate fluorometer (Bio-Tech Instrument, Winooski, VT) equipped with a standard filter set for FITC (excition, 485 nm; emission, 538 nm). For quantification of fluorescence intensity produced by the FITC-conjugated KLGFFKR peptide, natural fluorescence (autofluorescence) of cell lysates was subtracted from the fluorescence of FITC-KLGFFKR peptides. This experiment was repeated at least five times.

Immunofluorescence

E63 cells were grown on coverslips coated with 0.2% gelatin. After washing twice in PBS, the cells were incubated for 80 min at room temperature with calreticulin Ab (PA3-900 or LAR090) diluted in PBS containing 1% (wt/vol) BSA and finally incubated for 40 min at room temperature with TRITC-conjugated donkey anti-rabbit immunoglobulin. The labeled cells were then rinsed with PBS and fixed in 0.1% paraformaldehyde for 10 min. For double staining, the fixed cells were then incubated for 80 min with O26 monoclonal antibody (mAb; 15 μg/ml), followed by incubation for 40 min with FITC-conjugated donkey anti-mouse immunoglobulin. The cells were then dehydrated for 10 min with 95% ethanol and mounted with 90% glycerol and 0.1% o-phenylenediamine in PBS. For clustering of integrin, cells were first incubated with integrin α7 Ab (O26 mAb) and FITC-conjugated donkey anti-mouse immunoglobulin, followed by incubation with calreticulin antibodies and TRITC-conjugated donkey anti-rabbit immunoglobulin. Immunofluorescence was analyzed under a Leica DMRBE microscope equipped with a 63× objective lens and filters for epifluorescence. Fluorescence micrographs were taken on T-max P3200 film (Eastman Kodak, Rochester, NY).

Immunoprecipitation and Immunoblotting

E63 cells were washed three times with PBS and extracted for 1 h at 4°C in 1 ml of extraction buffer containing 200 mM n-octyl-β-d glucopyranoside, 50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM CaCl₂, 1 mM MgCl₂, 2 mM PMSF, 20 μg/ml aprotinin, and 12.5 μg/ml leupeptin. The resultant lysate was centrifuged for 10 min at 12,000 × g. Protein concentrations were determined by BCA assay. For immunoprecipitation, 1 mg of total protein was incubated overnight at 4°C with 15 μg/ml H36 mAb or 4 μg/ml DHPR α1 Ab, followed by further incubation with protein G-Sepharose beads (Amersham Pharmacia Biotech, Uppsala, Sweden) for 4 h at 4°C. The beads were then washed three times with extraction buffer to remove nonspecifically bound proteins. Immune complexes were treated with SDS-sample buffer (5% glycerol, 100 mM DTT, 2% SDS, 0.01% bromphenol blue, and 125 mM Tris, pH 6.8) and subjected to SDS-PAGE. After electrophoresis, proteins were transferred to polyvinylidene difluoride membranes and blocked for 2 h at room temperature in 5% nonfat dry milk in 0.1% Tween 20, 150 mM NaCl, and 50 mM Tris, pH 7.5, incubated for 1 h at room temperature with primary antibodies in 0.1% Tween 20, 150 mM NaCl, and 50 mM Tris, pH 7.5, followed by incubation with HRP-coupled anti-rabbit or anti-mouse immunoglobulin, and detected using ECL according to the manufacturer's protocol. In some cases, the membranes were then stripped by heating at 65°C for 1 h in stripping buffer (100 mM β-mercaptoethanol, 2% SDS, and 62.5 mM Tris, pH 6.7) and reprobed.

Microinjection

E63 cells grown on gelatin-coated coverslips for 5 d were microinjected with 1 mg/ml solution of heparin or chondroitin sulfate A in buffer containing 10 mM Tris, pH 7.4, and 25 mM KCl using an Eppendorf microinjection system (model 5246) attached to a Leica DMIRB microscope. Each cell was injected for 0.2 s at a constant pressure of 150 hectopascals. After microinjection, cells were rinsed with serum-free DMEM and then incubated in DMEM supplemented with 10% horse serum for 3 h, after which relative changes in [Ca²⁺]_i were assessed.

Iodination of Cell Surface Proteins

E63 cells were washed three times with HBSS and then iodinated for 20 min at 24°C in 1 ml of HBSS containing 0.6% glucose, 0.625 U of lactoperoxidase, 0.125 U of glucose oxidase, and 1 mCi of Na¹²⁵I (100 mCi/ml; New England Nuclear). The iodinated cells were lysed for 1 h at 4°C with 1 ml of radioimmunoprecipitation assay buffer (0.1% SDS, 1% deoxycholate, 1% Triton X-100, 100 mM Tris, pH 7.0, 1 mM EDTA, 150 mM NaCl, 2 mM PMSF, 20 μg/ml aprotinin, and 12.5 μg/ml leupeptin). The lysate was centrifuged for 10 min at 12,000 × g, and then supernatant was incubated overnight at 4°C with calreticulin Ab (PA3-900), followed by further incubation with protein A-Sepharose beads. The beads were subjected to SDS-PAGE, and the radiolabeled calreticulin was visualized by autoradiography.

RESULTS

Transient Elevation of [Ca²⁺]_i Evoked by Laminin or Integrin α7 Ab in E63 Muscle Cells

The expression of integrin α7 is known to increase during differentiation of E63 cells from undifferentiated myoblasts into terminally differentiated myotubes (Song et al., 1992). To better understand the function of integrin α7, we investigated some of the intracellular events associated with integrin α7 activation, particularly those related to changes in [Ca²⁺]_i.

Integrin α7 was engaged by exposing E63 cells to 100 μg/ml laminin, and relative changes in [Ca²⁺]_i were assessed as a function of changes in fluo-3 fluorescence, as described in MATERIALS AND METHODS (Figure 1). [Ca²⁺]_i in undifferentiated myoblasts (2 d old) was unaffected by laminin (our unpublished data); however, once the cells had elongated after 5 d in culture, laminin evoked transient elevations in [Ca²⁺]_i within ∼100 s of its application (Figure 1). To obtain more specific information about the role of integrin α7 in the laminin-evoked responses, fluo-3 fluorescence was measured in cells exposed to O26 and H36 mAbs (15 μg/ml), which bind integrin α7 by targeting the extracellular domain. Like laminin, O26 and H36 mAbs elicited transient [Ca²⁺]_i elevations in 5-d-old E63 cells, although Ca²⁺ transients developed more rapidly in response to the Abs than to laminin (Figure 2A). This is also true of promoting association with the cytoskeleton and in producing a change in the α7 integrin cytoplasmic domain (Song et al., 1993). O26 and H36 mAbs also had no effect on 2-d-old undifferentiated myoblasts. This is likely due to the concentration of integrin. It will not be cross-linked with Ab or laminin if integrin α7 expresses at a low level on the cell surface (Song et al., 1992). Given the similarity between the responses elicited by the integrin α7 mAbs and laminin, the former were used to activate integrin α7 in subsequent experiments. As a control, 5-d-old E63 cells were exposed to NMS (25 μg/ml) or NRS (25 μg/ml), which had no effect on [Ca²⁺]_i (Figure 2A). At the end of the experiment, cells were treated with A23187 to trigger a calcium influx, thereby demonstrating that the cells were still viable (Kao et al., 1989).

Figure 2.

Elevation of [Ca²⁺]_i elicited by integrin α7 mAbs in E63 cells. (A) Transient elevations in [Ca²⁺]_i elicited by H36 mAb (red; n = 21; SD, ±0.76 ∼ 4.02) and O26 mAb (blue; n = 56; SD, ±0.43 ∼ 2.74) in 5-d-old E63 cells. Exposure to buffer (black; n = 21; SD, ±0.42 ∼ 3.02), NMS (green; n = 21; SD, ±1.14 ∼ 3.53), or NRS (pink; n = 28; SD, ±0.59 ∼ 3.63) had no effect on [Ca²⁺]_i. (B) Cells were preincubated for 5 min in either normal bath solution (red; n = 56; SD, ±1.07 ∼ 3.04) or bath solution containing 25 μM nifedipine (blue; n = 48; SD, ±0.40 ∼ 2.04), 10 mM EGTA (green; n = 28;SD, ±0.83 ∼ 1.32), or 200 μM Cd²⁺ (black; n = 28; SD, ±0.51 ∼ 1.81). After preincubation, cells were treated with O26 mAb (15 μg/ml), and the change of [Ca²⁺]_i was measured. (C) Elevations in [Ca²⁺]_i elicited by treatment with calreticulin polyclonal Ab. After preincubation for 5 min in either bath solution (red; n = 40; SD, ±1.13 ∼ 6.46) or bath solution containing 25 μM nifedipine (blue; n = 28; SD, ±1.83 ∼ 5.19) or 200 μM Cd²⁺ (black; n = 28; SD, ±0.93 ∼ 2.76), cells were exposed to calreticulin Ab (PA3-900). Crk-associated substrate (Cas) polyclonal Ab, a nonspecific Ab, did not elicit [Ca²⁺]_i. The values shown are averages of fluorescence intensity obtained from at least 20 single cells in at least five independent experiments conducted under the same experimental conditions. n, cell number; SD, SD of Av. I_mean.

To investigate the source of the Ca²⁺ serving to elevate [Ca²⁺]_i, extracellular Ca²⁺ was depleted by adding 10 mM EGTA to the bath solution before integrin α7 engagement by O26 mAb. The addition of the EGTA completely blocked the evoked Ca²⁺ transients (Figure 2B). Moreover, in pretreating cells for 5 min with 200 μM Cd²⁺ or 25 μM nifedipine, a nonspecific calcium channel blocker and a specific L-type calcium channel antagonist, respectively (Juberg et al., 1995; Reid et al., 1997), O26 mAb-evoked Ca²⁺ transients were completely blocked (Figure 2B).

According to Vazquez et al. (1998), calcium influx by a store-operated channel is insensitive to L-type calcium channel antagonists such as nifedipine and verapamil in skeletal muscle cells. In our experiment, calcium influx induced by integrin α7 Ab was completely inhibited by nifedipine in L8E63 skeletal muscle cells, indicating that L-type calcium channels are mediating this influx.

Elevation of [Ca²⁺]_i Induced by Calreticulin Antibodies

Although it was originally characterized as a Ca²⁺-binding protein, calreticulin and its recently identified cell surface isoform ectocalreticulin have emerged as regulators of integrin-mediated Ca²⁺ signaling and cell adhesion (Coppolino et al., 1997; Coppolino and Dedhar, 1998; Zhu et al., 1997). Therefore, to assess the extent to which ectocalreticulin regulates the cytosolic Ca²⁺ transients elicited by activation of integrin α7, the relative changes in [Ca²⁺]_i evoked in E63 cells by exposure to calreticulin Ab were examined. Like O26 mAb, calreticulin Ab elicited Cd²⁺- and nifedipine-sensitive Ca²⁺ transients (Figure 2C). However, the time courses of the responses elicited by calreticulin Ab were quite different from those elicited by O26 mAb (Figure 2, compare A and C). Whereas Ca²⁺ transients elicited by O26 mAb developed within 30–40 s and had a duration of ∼40–55 s, responses to calreticulin Ab developed within ∼10–20 s and then slowly declined over the next 210–280 s.

Cellular Localization of Integrin α7and Calreticulin in E63 Cells

To determine the cellular localization of integrin α7 and calreticulin on the cell surface, 5-d cultured cells were subjected to double immunofluorescence analysis. When cells were first reacted with calreticulin Ab followed by addition of integrin α7 antibody, ectocalreticulin appeared to be diffusely distributed on the surface of E63 cells (Figure 3A, a and b). In contrast, the prior incubation of integrin α7 Ab before addition of calreticulin Ab dramatically promoted change of ectocalreticulin distribution on the cell surface, in which colocalization of integrin α7 and calreticulin occurred throughout the cells (Figure 3A, c–f). The colocalization of these molecules was more apparent at high magnification (Figure 3A, g and h). This suggests that the clustering of integrin α7 by antibodies may promote a change in association with ectocalreticulin on the cell surface. These results are consistent with the previous findings that reactivity of the integrin α7 with primary and secondary antibodies promotes the association of the integrin with the cell cytoskeleton, as noted by colocalization with actin filaments (Kaufman and Robert-Nicoud, 1985, Song et al., 1993).

Figure 3.

Association of integrin α7 with ectocalreticulin and DHPR α1 subunit in E63 cells. (A) The unfixed E63 cells (5 d old) were first immunostained with calreticulin Ab (PA3-900) and then stained with integrin α7 Ab (O26 mAb) again. Ectocalreticulin was diffusely distributed on the cell surface (a), whereas integrin α7 displayed a stress fiber-like distribution (b). In contrast, prior incubation of integrin α7 Ab (c–h) before staining of calreticulin Ab (PA3-900 [c and g] or LAR090 [e]) induced cellular localization of ectocalreticulin (c, e, and g), in which ectocalreticulin appears to be colocalized with integrin α7 (d, f, and h). In control experiments, cells were first stained with normal mouse immunoglobulin and then stained with normal rabbit immunoglobulin (i and j). Cells shown in the left panels (a, c, e, g, and i) were stained with TRITC-conjugated donkey anti-rabbit immunoglobulin, whereas those in the right panels (b, d, f, h, and j) were stained with FITC-conjugated donkey anti-mouse immunoglobulin. Bars: in f (for a–f, i, and j), 10 μm; in h (for g and j), 5 μm. (B) Cell surface proteins in 5-d cultured cells were iodinated with Na¹²⁵I and immunoprecipitated with calreticulin Ab (PA3-900). Autoradiography of cell surface proteins (a) and imunoblot analysis (b) of cell lysates with calreticulin Ab (PA3-900) are presented. Iodinated ectocalreticulin is indicated by ¹²⁵I-CRT. (C) Cell lysates were immunoprecipitated (IP) using integrin α7 Ab (H36 mAb) and DHPR α1 Ab and then immunoblotted (IB) with calreticulin Ab: PA3-900 (left panel) and LAR090 (right panel). (D) After stripping, the same membrane was reprobed with DHPR α1 Ab. Note that integrin α7 was associated with both calreticulin and DHPR α1.

To further confirm that ectocalreticulin is present on the cell membrane, cell surface proteins in 5-d cultured E63 cells were labeled using Na¹²⁵I and lactoperoxidase. Immunoprecipitation with calreticulin Ab (PA3-900) and autoradiography revealed the 62-kDa membrane calreticulin (Figure 3B, a). In contrast, immunoblot analysis of cell lysates with the calreticulin Ab identified two proteins, the 62-kDa ectocalreticulin and the 52 kDa cytoplasmic endocalreticulin (Figure 3B, b). Immunoblot analysis using two different calreticulin antibodies, LAR090 and PA3-900, revealed that the 62-kDa ectocalreticulin is associated with both the integrin α7 and the DHPR α1 subunits (Figure 3, C and D).

Integrin α7-mediated Ca²⁺ Influx Is Dependent on the Cytosolic Ca²⁺ Concentration

It is now known that integrin activation is coupled to tyrosine phosphorylation-dependent activation of PLC (Kanner et al., 1993; Morimoto and Tachibana, 1996; Wrenn et al., 1996) and the resultant generation of IP₃ (Somogyi et al., 1994; Hellberg et al., 1996). In that context, our observation that the differing lag times between activation of integrin α7 or calreticulin and the development of Ca²⁺ transients led to us to investigate the mechanism by which these molecules regulate Ca²⁺ channel opening. We initially observed that Ca²⁺ transients elicited by integrin α7 activation were blocked by genistein, a tyrosine kinase inhibitor, whereas Ca²⁺ transients evoked by calreticulin Ab were insensitive to genistein (our unpublished data). In addition, neomycin, which is an aminoglycoside antibiotic that binds polyphosphoinositides, making them unavailable to PLC, completely blocked integrin α7-mediated elevations in [Ca²⁺]_i, as did U73122, an inhibitor of PLC (De Boland et al., 1996; Hellberg et al., 1996) (Figure 4A). Ca²⁺ transients elicited by calreticulin Ab were unaffected by either neomycin or U73122 (Figure 4B), however, suggesting that whereas responses mediated by integrin α7 are dependent on PLC activation and Ca²⁺ release from SR, those mediated by calreticulin are independent of PLC activation. As reported previously (Thastrup et al., 1990), depletion of SR Ca²⁺ stores using TG elicited a significant increase in [Ca²⁺]_i that was followed by a sustained plateau (Figure 4A, green). This effect was independent of PLC and was therefore not blocked by U73122 (Figure 4A, red). In the presence of TG, O26 mAb still evoked nifedipine-sensitive Ca²⁺ transients regardless of the presence U73122 (Figure 4A). In a parallel experiment, TG-pretreated cells exposed to 20 μM ATP did not show the additional calcium release, indicating that calcium was completely depleted from internal calcium stores (our unpublished data). This result suggests that the O26 mAb-evoked calcium peak in the presence of TG is not due to the additional calcium release from internal calcium stores. Also, this suggests that Ca²⁺ release from intracellular stores is a prerequisite for O26 mAb-mediated Ca²⁺ influx. Consistent with that idea, microinjection of heparin, an IP₃ receptor antagonist (Mohri et al., 1995), into E63 cells blocked O26 mAb-evoked Ca²⁺ transients, whereas microinjection of buffer or chondroitin sulfate A had no effect (Table 1).

Figure 4.

Effect of PLC inhibitors on integrin α7-mediated Ca²⁺ signaling. (A) Pretreatment with 10 μM U73122 (pink; n = 35; SD, ±1.85 ∼ 2.11) or 2 mM neomycin (blue; n = 35; SD, ±0.17 ∼ 1.68) completely blocked α7-mediated [Ca²⁺]_i transient. Addition of 1 μM TG (green; n = 42; SD, ±0.76 ∼ 4.37) elicited the characteristic [Ca²⁺]_i plateau because of the calcium release from internal calcium stores and restored Ca²⁺ transients even in U73122-pretreated cells (red; n = 28; SD, ±1.32 ∼ 4.74), but not in nifedipine-pretreated cells (black; n = 28; SD, ±1.83 ∼ 5.49). (B) Pretreating cells for 5 min with bath solution alone (red; n = 35; SD, ±0.71 ∼ 3.29) or bath solutioncontaining 10 μM U73122 (pink; n = 35; SD, ±1.37 ∼ 5.02), 2 mM neomycin (blue; n = 35; SD, ±1.80 ∼ 4.23), or 1 μM TG (green; n = 35; SD, ±1.29 ∼ 4.63) did not inhibit calreticulin-mediated Ca²⁺ transients. (C) Cells were exposed to U73122 for 5 min in the absence or presence of TG, followed by exposure to integrin α7 Ab (H36 mAb) for 5 min. The lysate was immunoprecipitated (IP) with H36 mAb and then immunoblotted (IB) with calreticulin Ab (PA3-900). Note that the association of integrin α7 with calreticulin was highly dependent on increased cytosolic Ca²⁺. n, cell number; SD, SD of Av. I_mean.

Table 1.

Effects of microinjected heparin in E63 cells

	Total injected cells (n)	A23187-responsive cells	O26 mAb-responsive cells	Inhibition (%)a
Buffer	244	190	175	7.9
0.1 mg/ml heparin	272	231	108	53.2
1 mg/ml heparin	288	213	12	94.4
1 mg/ml CSA	323	252	225	10.7

Cells were microinjected with buffer solution, 1 mg/ml heparin, or 1 mg/ml chondroitin sulfate A (CSA). Heparin inhibited integrin induced Ca²⁺ transients, whereas the same concentration of CSA or buffer had no effect. 

^aPercent inhibition was calculated by considering the ratio of the number of O26 mAb-responsive cells to A23187-responsive cells. 

Association of Integrin α7 with Calreticulin Is Dependent on the Cytosolic Ca²⁺ Concentration

Calreticulin was previously shown to modulate integrin–ligand affinity through an effect on the cytoplasmic domain of integrin α chain (Rojiani et al., 1991; Leung-Hagesteijn et al., 1994; Coppolino et al., 1995). Our present findings indicate that integrin α7 associates with ectocalreticulin on the cell surface. It seems reasonable, therefore, that calreticulin may function to modulate the coupling between Ca²⁺ extracellular influx and Ca²⁺ release from intracellular stores. Immunoprecipitation carried out to assess the effects of U73122 and TG on the interaction between integrin α7 and calreticulin confirmed that integrin α7 interacted directly with calreticulin in U73122-untreated cells, but it did not resolve whether the binding occurs at the intracellular or extracellular domain. There was no interaction between integrin α7 and calreticulin when PLC was blocked with U73122, although after TG-evoked depletion of SR, integrin α7 was bound to calreticulin regardless of the presence of U73122 (Figure 4C).

Inhibition of Integrin α7-mediated Ca²⁺ Influx by KLGFFKR Peptide

Calreticulin is known to interact with the KXGFFKR motif in the cytoplasmic domain of the integrin α subunit (Krause and Michalak, 1997). To further characterize the interaction between integrin α7 and calreticulin, KLGFFKR peptide was introduced into transiently permeabilized 5-d-old E63 cells to compete with the integrin α subunit sequence. By itself, permeabilization had no effect on cell viability. In addition, to test the membrane permeability of the peptides, FITC-KLGFFGR peptides were introduced into the permeabilized cells, and fluorescence intensity was measured. Cells loaded with FITC-KLGFFKR peptides elicited the increase of fluorescence intensity in a dose-dependent manner up to 100 μg/ml, indicating that cells are permeable to the peptides (Figure 5B).

Figure 5.

Inhibition of integrin α7-mediated Ca²⁺ signaling by KLGFFKR peptide. (A) E63 cells were transiently permeabilized in the absence (b, g, and l) or presence of 25 μg/ml (c, h, and m), 50 μg/ml (d, i, and n), or 75 μg/ml (e, j, and o) KLGFFKR peptide or, as a control, 75 μg/ml (a, f, and k) of KLRFGFK (scrambled peptide). The top row (a–e) was obtained before O26 mAb (15 μg/ml) treatment; the middle row (f–j) shows the elevated [Ca²⁺]_i within 40 s after exposure to O26 mAb; and the bottom row (k–o) was obtained within 10 s after exposing cells to the Ca²⁺ ionophore A23187 as a positive control. (B) Five-day cultured cells were permeabilized and treated with FITC-KLGFFKR peptides or with KLGFFKR peptides. The supernatant of lysates was applied to an FL-600 microplate fluorometer equipped with a standard filter set for FITC (excitation, 485 nm; emission, 538 nm) for fluorometric analysis. For quantification of fluorescence intensity, natural fluorescence (autofluorescence) by cell lysates was subtracted from the fluorescence intensity of FITC-KLGFFKR peptides. This experiment was repeated at least five times. (C) Cell lysates were immunoprecipitated (IP) with integrin α7 Ab (H36 mAb) in the absence or presence of 75 μg/ml KLGFFKR or 75 μg/ml KLRFGFK and immunoblotted (IB) with calreticulin Ab (PA3-900). Note that the integrin–calreticulin interaction was partially blocked by the KLGFFKR peptide.

When cells were loaded with a scrambled peptide (KLRFGFK), typical Ca²⁺ transients were elicited by O26 mAb. Ca²⁺ transients elicited in cells loaded with up to 75 μg/ml KLGFFKR peptide, on the other hand, were dose-dependently attenuated (Figure 5A and Table 2). In addition, immunoprecipitation demonstrated that the KLGFFKR peptide completely blocked the interaction between calreticulin and integrin α7, whereas the scrambled peptide (KLRFGFK) had a minor effect in their association (Figure 5C). Thus, the binding of calreticulin to the KLGFFKR motif in the cytoplasmic domain of integrin α7 appears to be prerequisite for integrin α7-mediated Ca²⁺ influx.

Table 2.

Effects of intracellular KLGFFKR peptide on E63 cells

	A23187-responsive cells	O26 mAb-responsive cells	Inhibition (%)
75 μg/ml KLRFGFK	231	203	12.1
0 μg/ml KLGFFKR	203	197	3.0
25 μg/ml KLGFFKR	217	105	51.6
50 μg/ml KLGFFKR	280	84	70.0
75 μg/ml KLGFFKR	252	21	91.7

^aPercent inhibition was calculated by considering the ratio of the number of O26 mAb-responsive cells to A23187-responsive cells. 

DISCUSSION

Changes in [Ca²⁺]_i during integrin-mediated cell adhesion have been reported in a variety of cell types (Richardson and Parsons, 1995), although the mechanism responsible is not yet fully understood. We demonstrate here that in a skeletal muscle cell line, integrin-mediated Ca²⁺ signaling requires both Ca²⁺ release from IP₃-sensitive SR Ca²⁺ stores and extracellular Ca²⁺ influx through L-type Ca²⁺ channels. Moreover, calreticulin appears to serve as a mediator coupling Ca²⁺ release from IP₃-sensitive calcium stores and Ca²⁺ influx.

Calcium release from the SR/ER (less than micromolar range) was not detected because of the limitation of confocal microscopy to measure changes in intracellular calcium concentration in that range. Measurements were therefore limited to changes in intracellular calcium (more than micromolar range). However, our findings indicate that O26 mAb binding to integrin α7 stimulates phosphatidylinositol 4,5-bisphosphate hydrolysis to IP₃, which in turn triggers release of Ca²⁺ from SR/ER. Neomycin and U73122, two inhibitors of IP₃ synthesis (De Boland et al., 1996; Hellberg et al., 1996), each completely blocked O26 mAb-evoked Ca²⁺ transients, as did microinjected heparin, which inhibits IP₃ binding to its receptor (Mohri et al., 1995). These results are consistent with findings made in pancreatic acinar cells and Madin–Darby canine kidney cells, where cell adhesion to the RGD sequence stimulates IP₃ synthesis (Somogyi et al., 1994; Sjaastad et al., 1996). Furthermore, TG-induced release of SR Ca²⁺ restored responsiveness to U73122-treated cells, suggesting that Ca²⁺ influx is highly dependent on prior elevation of the cytosol calcium concentration.

Store-operated Ca²⁺ entry, a model of Ca²⁺ influx activated by depletion of Ca²⁺ from internal stores, has been found in a wide variety of cell types and may be the primary mechanism for Ca²⁺ entry in nonexcitable cells (Montell, 1997). Store-operated channels or Ca²⁺ release-activated Ca²⁺ channels are a family of nonselective cation channels and are insensitive to L-type Ca²⁺ channel antagonists, such as nifedipine and verapamil (Vazquez et al., 1998). Therefore, blockage of [Ca²⁺]_i after nifedipine treatment suggests that Ca²⁺ transients evoked by the O26 mAb resulted from an influx of extracellular Ca²⁺ through L-type calcium channels.

Integrin-mediated cell adhesion requires both outside-in and inside-out signaling. The former is integrated with other intracellular signaling pathways and usually elicits feedback via inside-out signaling. We suggest that Ca²⁺ release elicits positive feedback, promoting further Ca²⁺ influx, which is a key factor for integrin-mediated cell adhesion. Many cytosolic regulatory proteins including calreticulin, β-calnexin, and calcium- and integrin-binding protein have been shown to bind to the integrin cytoplasmic domain (Lenter and Vestweber, 1994; Naik et al., 1997), but among them, calreticulin appears to mediate Ca²⁺ influx.

Calreticulin has been localized to ER/SR, to the cell surface, and to perinuclear areas (Michalak et al., 1992; Roderick et al., 1997; Zhu et al., 1997). Zhu et al. (1997) showed that calreticulin can exists in an ecto (62-kDa) form on the cell surface in association with integrin or in an endo (52-kDa) form found in the interior of cells. We observed that integrin α7, ectocalreticulin, and DHPR are clustered on surface of E63 cells, and our immunoprecipitation analysis demonstrated that integrin α7 binds to the 62-kDa calreticulin but not to the 52-kDa calreticulin. Therefore the 62-kDa calreticulin seems to be a membrane-bound calreticulin even if it exists either on cell surface or in association with the cytoplasmic GFFKR sequence of integrin α chain.

The interaction between calreticulin and the KLGFFKR motif in the integrin α subunit is dependent on the activation state of the integrin (Leung-Hagesteijn et al., 1994). For example, when Jurkat cells, a T-lymphoblastoid cell line, were exposed to activating Abs raised against the integrin α2 and β1 subunits, there was an increased association between integrin α2β1 and calreticulin and increased cell adhesion to collagen (Coppolino et al., 1995). In addition, calreticulin-null embryonic stem cells exhibit severely impaired adhesion to ECM and reduced influx of extracellular Ca²⁺ influx (Coppolino et al., 1997; Coppolino and Dedhar, 1998). Our finding that loading cells with the KLGFFKR peptide antagonized integrin α7-evoked Ca²⁺ influx further confirms that Ca²⁺ release from SR/ER promotes the binding of calreticulin to the KLGFFKR motif, thereby mediating extracellular Ca²⁺ influx. The inhibition of Ca²⁺ transients by introduction of KLGFFKR peptides is consistent with an earlier report in which introduction of calreticulin Ab into Jurkat cells inhibited activation of integrin α2β1 by integrin antibodies (Coppolino et al., 1995). Zhu et al. (1997) postulated that the binding of calreticulin to integrin cytoplasmic domains might propagate a signal to the ligand binding site, increasing its affinity.

Taken together, we propose that calreticulin plays a mediator to couple calcium release and calcium influx, and calcium release is a prerequisite for calcium influx. However, the possibility should be considered that the opening of the calcium channel is mediated by the change of membrane potential during integrin activation. Even though we have also not proved yet how ectocalreticulin regulates the gating of calcium channels, it is interesting to note that addition of polyclonal calreticulin antibodies elicited the immediate extracellular Ca²⁺ influx compared with a delayed response upon addition of integrin α7 Ab. This suggests that ectocalreticulin, but not the integrin, may directly modulate channel opening. The colocalization of the integrin α7 and ectocalreticulin suggests that ectocalreticulin may modulate signaling from the integrin α7 to the DHPR. However, the exact molecular mechanism remains to be elucidated further.

Calreticulin contains two Ca²⁺ binding domains: the C domain is a low-affinity (K_d, ∼2 mM), high-capacity domain (B_max, >25 mol Ca²⁺/mol of protein), whereas conversely, the P domain is a high-affinity (K_d, ∼1 μM), low-capacity domain (B_max, 1 mol Ca²⁺/mol of protein; Baksh and Michalak, 1991). Consequently, Ca²⁺ release (less than micromolar concentration) may at first partially activate calreticulin by binding to the P domain, thereby promoting the association with the integrin cytoplasmic domain but not affecting the extracellular ligand binding domains. Similar results were observed in Madin–Darby canine kidney cells in which inhibition of Ca²⁺ influx reduced adhesion to RGD beads, and prior release of Ca²⁺ from IP₃-sensitive stores by ATP or TG had little effect on adhesion (Sjaastad et al., 1996). Thus, it may be that Ca²⁺ influx via Ca²⁺ channels causes a large increase in local Ca²⁺ concentration in the vicinity of the cell membrane that is sufficient to fully activate calreticulin, to increase integrin activation, and to mediate cell adhesion to ECM.

Integrin-mediated increases in intracellular calcium may have additional physiological consequences during the development of skeletal muscle. Clustering of acetylcholine receptors on the surface of myoblasts is an early step in the formation of neuromuscular junctions, and this is a calcium-dependent process. Specific spliced variants of the integrin α7β1, in response to laminin, have an important role in the aggregation of these receptor clusters (Burkin et al., 1998). Whereas engaging the integrin with laminin (or with concentrations of integrin α7 Ab that cross-link the integrin) promotes acetylcholine receptor clustering, it is highly likely that the increase in intracellular calcium concentration induced by engaging the integrin described herein underlies this calcium-dependent step in the formation of neuromuscular junctions.