©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Thrombospondin Mediates Calcium Mobilization in Fibroblasts via Its Arg-Gly-Asp and Carboxyl-terminal Domains (*)

(Received for publication, February 17, 1995; and in revised form, July 18, 1995)

Peter W. Tsao Shaker A. Mousa (§)

From the DuPont Merck Pharmaceutical Company, Cardiovascular Diseases Division, Wilmington, Delaware 19880-0400

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Thrombospondin is a matrix glycoprotein found in various cells that can modulate cell attachment, migration, and proliferation. We now show that intact soluble thrombospondin causes a transient [Ca] increase in IMR-90 fibroblasts. This [Ca] increase is mediated partly by the RGD-containing domain of thrombospondin that binds to the integrin alphavbeta3 as demonstrated by inhibitor studies using anti-alphavbeta3 antibody and RGD-containing peptides. A non-RGD and non-alphavbeta3 component of this [Ca] increase is mediated by the carboxyl-terminal domain of thrombospondin through an unidentified receptor on fibroblasts as shown by the antibody to the carboxyl-terminal of thrombospondin, C6.7. In addition, the carboxyl-terminal derived peptide, RFYVVMWK, also triggers [Ca] increase in 35% of fibroblasts. Both EGTA and Ni block the entire [Ca] increase indicating that this is due to an influx of extracellular Ca. B6H12, an antibody to the integrin-associated protein, blocks this [Ca]increase by 50%, suggesting that some of the Ca might be entering through an integrin-associated calcium channel. The current findings demonstrate that multiple domains on thrombospondin can trigger signal transduction events by increasing [Ca] through their interactions with different cell receptors.


INTRODUCTION

Thrombospondin (TSP) (^1)is a 450-kDa trimeric matrix glycoprotein. It was first described as an alpha-granular protein secreted by platelets upon thrombin activation. TSP is also synthesized by fibroblasts, endothelial cells, smooth muscle cells, monocytes, macrophages, osteoblasts, and tumor cells(1, 2, 3, 4, 5, 6) . TSP has been shown to modulate cell attachment, migration, and proliferation and has an important role in wound healing, hemostasis, and angiogenesis(2, 3, 5, 7, 8, 9, 10) . Like other matrix proteins such as fibrinogen and fibronectin, TSP possesses multiple cell binding domains (6, 11) . Four known cell binding domains have been described for thrombospondin: (i) the heparin binding domain at the amino-terminal (12) , (ii) the type I repeats containing the CSVTCG sequence(13) , (iii) the Arg-Gly-Asp (RGD) sequence in the type III calcium binding repeat(7) , and (iv) the carboxyl-terminal cell binding domain(14, 15) . Each of these domains was found to bind to distinct cell surface receptors: (i) heparan sulfate proteoglycans(16) , (ii) CD36 or GPIV (17) , (iii) integrin alphavbeta3(7, 18) , and (iv) the 105/80-kDa receptor (4) or the 52-kDa receptor(19) , respectively.

Recently there has been major interest in the integrin alphavbeta3 because of its role in angiogenesis and apoptosis(20, 21) . An initial step in these processes often involves the binding of a matrix protein to alphavbeta3. There has been extensive work on the role of TSP on cell attachment involving alphavbeta3; however, little is known about the signal transduction events(4, 7, 18) . Increasing evidence from various groups clearly shows that the role of integrins extends beyond the binding of ligands or matrix proteins(22, 23, 24) . The binding of various matrix proteins to their receptors, especially integrins, often triggers different signals such as tyrosine phosphorylation and changes in intracellular pH or Ca(25, 26, 27) . These outside-in signaling events serve as important regulatory mechanisms to mediate subsequent molecular events such as modulation of integrin affinity (28, 29) or gene transcription(30) .

In this study, we investigated the role of TSP in triggering Ca mobilization in a lung fibroblast cell line, IMR-90. In addition, we also used known inhibitors of both the cell binding domains and the receptors of TSP to elucidate the structural basis of TSP-induced transmembrane signaling. The findings suggest that intact TSP can mediate Ca mobilization via both the RGD and carboxyl-terminal domains.


EXPERIMENTAL PROCEDURES

Materials

Human platelet-derived thrombospondin, TSP-1, was from Haematologic Technologies Inc. (Essex Junction, VT). Fura-2/AM ester was from Molecular Probes. Echistatin and the RFYVVMWK peptide were purchased from Bachem (King of Prussia, PA). Peptides GRGDSP, GRGESP, and CSVTCG were synthesized by Dr. Ram Seetharam of the DuPont Merck Pharmaceutical Co. Monoclonal antibodies used in this study include anti-alphavbeta3 (LM609, Chemicon, CA), anti-alphavbeta5 (P1F6, Becton Dickinson), and anti-CD36 (FA6.152, Immunotech, ME). Anti-TSP antibodies, C6.7 and A4.1, were from Life Technologies, Inc. Anti-integrin-associated protein antibody, B6H12, was a gift from Dr. Eric Brown (Washington University, St. Louis)(31) . All antibodies were purified IgG protein except C6.7 and A4.1 which were in ascites form. Lung fibroblast cell line IMR-90 (American Type Culture Collection) was grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum (Life Technologies, Inc.). To minimize the effect of cell passage, cells were used between passages 12 and 15 and were in culture 4-6 days before each experiment.

Cell Surface Expression of Thrombospondin Receptors

Expression of thrombospondin receptors on IMR-90 was determined by indirect immunofluorescence and analyzed by flow cytometry. IMR-90 were removed from culture flasks using trypsin/EDTA. Cells were washed and resuspended in Ca-containing phosphate-buffered saline. They were then incubated with saturating amounts of primary antibodies followed by phycoerythrin-conjugated goat anti-mouse IgG F(ab`)(2) (Chemicon). Labeled cells were analyzed on a FACScan flow cytometer (Becton Dickinson).

Indirect Immunofluorescence Microscopy

IMR-90 fibroblasts were cultured in chamber slides using the same conditions described above. For immunofluorescence staining, cells were washed with phosphate-buffered saline with 0.15% bovine serum albumin and fixed in 4% paraformaldehyde/phosphate-buffered saline for 15 min. The slides were incubated overnight at 4 °C with the primary antibody, LM609 (2 µg/ml), followed by a 1:400 dilution of Cy3-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) for 2 h at 4 °C. To induce integrin clustering, fibroblasts were pretreated with TSP for 1-2 min and fixed immediately with paraformaldehyde followed by antibody staining. Cells were visualized using a 63times/N.A. 1.4 oil immersion objective on a Zeiss Axiovert microscope (Carl Zeiss). Fluorescence images were collected through a dichroic mirror and 580 nm long pass filters onto a CCD camera (Zeiss ZVS-47DEC). Images were stored and processed on a Macintosh workstation using Oncor Image software (Oncor Imaging, Gaithersburg, MD) and Adobe Photoshop v.3.

Intracellular Calcium [Ca](i)Measurements

Intracellular calcium in single cells was measured using Fura-2 ratio imaging. Briefly, IMR-90 fibroblasts were grown on fibronectin-coated glass coverslips and loaded with 2 µM Fura/AM ester for 1 h at 37 °C. Cells were then washed three times with buffer containing 130 mM NaCl, 5 mM KCl, 1 mM MgCl(2), 1.8 mM CaCl(2), 10 mM glucose, and 20 mM HEPES, at pH 7.4. The coverslip was mounted on the stage of a Nikon Diaphot microscope using an open coverslip chamber (Warner Instruments, Hamden, CT), and the cells (10-20 cells per field) were visualized using a Nikon Fluor 40times/N.A. 1.3 oil immersion objective. Dual excitation was provided from a 150-watt xenon arc lamp through a Ludl filter wheel system with band pass filters at 340 and 380 nm. Epifluorescence images were captured by an intensified CCD camera (QX-100, Quantex), and emitted light was collected through a dichroic mirror and a 520 nm long pass filter. The system was operated by a Dell 386 Dimension computer using the Image1/FL software (Universal Imaging Corp, West Chester, PA) for both data acquisition and analysis. The 340/380 ratio was calculated and exported to a Microsoft Excel spreadsheet, and intracellular calcium was estimated using the equation:

R(max) was determined by 2-4 µM ionomycin (Calbiochem) and R(min) by 10 µM EGTA. S and S were determined by procedures published by Grynkiewicz et al.(32) . All single-cell fluorescence recordings were done at room temperature with background subtracted. Background was determined using a field on the coverslip with no cells. For each experiment, the responses of 5-10 individual cells were averaged. In inhibition experiments, peptides or antibodies were preincubated with cells for 2-5 min before addition of TSP. Increases in [Ca](i) due to TSP varied among different cell preparations in the range of 91-569 nM. To normalize for this interexperimental difference, % control or % inhibition was calculated from the TSP response of each experiment. Cell morphology was monitored by phase contrast microscopy. None of the antagonists or antibodies used in these experiments significantly affected the morphology of these cells within the time period of the experiment.


RESULTS

Addition of TSP to IMR-90 cells caused a concentration-dependent transient rise in [Ca](i) ranging from 55-1000 nM in >90% of the cells (Fig. 1). This [Ca](i) increase peaked within 10-30 s and returned to resting levels after 60-180 s depending on the concentration of TSP (Fig. 1). With 0.08 µM TSP, the mean increase in [Ca](i) was 220 ± 34 nM (n = 20) above the resting level of 93 ± 8 nM (n = 18) (Fig. 2A). This increase in [Ca](i) was eliminated completely in the absence of extracellular Ca by the addition of 5 mM EGTA (Fig. 2B). It is possible that the blocking of the [Ca](i) increase is due to EGTA's effect on Ca-dependent ligand binding to TSP receptors rather than depletion of the source of Ca available for transmembrane fluxes(4, 33) . Chelation of Ca has been shown to dissociate the integrin heterodimer and alter the tertiary structure of TSP(7, 18, 34, 35) . This, in turn, could indirectly affect ligand binding and thus the ability to trigger an increase in [Ca](i). To verify that the attenuated Ca was due to inhibition of Ca influx, cells were pretreated with Ni, which is known to block Ca fluxes through many types of Ca channels. Ni at 10 mM abolished the rise in [Ca](i) induced by TSP similar to that of EGTA (Fig. 2C). Neither EGTA nor Ni affected the baseline [Ca](i) under these conditions. These results indicate that the increase in [Ca](i) from TSP is dependent on extracellular Ca fluxes into the cells.


Figure 1: Thrombospondin-induced transient increase in [Ca] in IMR-90 lung fibroblasts. TSP-induced increase in [Ca] in a concentration-dependent manner. Representative [Ca] tracings of single cell recordings from IMR-90.




Figure 2: Thrombospondin-induced transient increase in [Ca] in IMR-90 is dependent on extracellular Ca. Representative [Ca] tracings of recordings from cells stimulated with 0.08 µM TSP (A) and pretreatment with 5 mM EGTA (B) or 10 mM Ni (C) before TSP addition. Note the inhibition of this increase in [Ca] by EGTA or Ni pretreatment. Representative tracings of single cell recordings from at least four separate experiments. Assay was carried out as described under ``Experimental Procedures.''



Role of alphav Integrins in Mediating TSP-induced [Ca](i)Increase

TSP is known to have multiple cell binding domains and multiple cell surface receptors. To address the issue of structural specificity, experiments were carried out to study the effects of known inhibitors on these cell binding domains and receptors. One of the major cell surface receptors for TSP belongs to the alphav integrin family(6, 7) . Previous studies have shown that other soluble ligands to the alphav integrin were able to raise [Ca](i)(36, 37) . The IMR-90 cells express both alphavbeta3 and alphavbeta5 on their cell surface as evident from the flow cytometry data (Fig. 3). To investigate the possibility that the TSP-induced increase in [Ca](i) was mediated via the alphav integrin, the effect of an antibody to alphavbeta3 (LM609) was studied(38) . LM609, at 4 and 10 µg/ml, inhibited TSP-induced increases in [Ca](i) by 54 and 63%, respectively (Fig. 4A and Fig. 5).


Figure 3: Cell surface expression of thrombospondin receptors in IMR-90 fibroblasts by flow cytometry. Cells were gated by forward and side light scatter, and 5,000 gated events were collected. IMR-90 expressed both alphavbeta3 and alphavbeta5 but not CD36 on their cell surface. Assay was carried out as described under ``Experimental Procedures.''




Figure 4: [Ca] tracings showing the effect of various anti-alphav antibodies on the thrombospondin-induced rise in [Ca]. Cells were pretreated with 4 µg/ml LM609 (anti-alphavbeta3) (A), 4 µg/ml B6H12 (anti-IAP) (B), or 10 µg/ml P1F6 (anti-alphavbeta5) (C) before addition of 0.08 µM TSP. Representative tracings of single cell recordings from at least four separate experiments. Note the partial inhibition of TSP-induced increases in [Ca] with LM609 and B6H12 but not with P1F6. Assay was carried out as described under ``Experimental Procedures.''




Figure 5: Effect of anti-alphav antibodies on the thrombospondin-induced rise in [Ca]. Antibodies LM609 and B6H12 (4 and 10 µg/ml) partially inhibited TSP-induced increases in [Ca]. P1F6 at a similar concentration has minimal effect. Mean ± S.E. (n = 4). Assay was carried out as described under ``Experimental Procedures.''



Since this Ca increase was apparently due to a calcium influx, we investigated further whether it was via the integrin-associated protein (IAP) which has been shown to mediate integrin-regulated Ca flux into cells(39) . We tested the effect of a recently described antibody to IAP, B6H12(31) , on TSP-induced increases in [Ca](i). B6H12, at 4 and 10 µg/ml, inhibited TSP-induced increases in [Ca](i) by 42% and 50%, respectively (Fig. 4B and Fig. 5). On the other hand, the TSP-induced increase in [Ca](i) was not inhibited by the control antibody to alphavbeta5 (Fig. 4C and Fig. 5). In addition, LM609 and B6H12 do not block non-TSP-, i.e. thrombin, mediated increases in [Ca](i), which indicates they specifically block alphavbeta3 integrin-mediated Ca flux. (^2)None of these monoclonal antibodies raised the baseline [Ca](i) at the concentrations tested.

Earlier studies have reported that integrin clustering is very important in signal transduction(25, 40, 41) . Here we investigated the effect of TSP and inhibitors of the TSP-alphavbeta3 interaction on integrin clustering. As shown in Fig. 6, TSP induces significant integrin clustering in IMR-90. This is not seen in cells that were not exposed to TSP or those that were pretreated with LM609, suggesting a specific interaction between the integrin and the matrix protein.


Figure 6: Immunolocalization of alphavbeta3 integrin on IMR-90 using indirect fluorescence. Fluorescence images showing very diffuse distribution of alphavbeta3 integrin on the surface of IMR-90 (A). With pretreatment of 0.17 µM TSP for 2 min (B), cells showed significant integrin clustering as indicated by the white arrows. A 2-min preincubation with 20 µg/ml LM609, which prevents the interaction of alphavbeta3 with TSP, showed significantly less clustering (C). Cells incubated with isotype match antibody did not stain (not shown). The distribution of alphavbeta3 was observed using LM609 followed by Cy3-conjugated goat anti-mouse IgG as described under ``Experimental Procedures'' (magnification, times 1000).



Role of the RGD Domain in TSP-induced[Ca](i)Increase

Many of the integrins, including alphavbeta3, recognize the RGD tripeptide sequence as a binding motif. The RGD domain is found in the type III calcium binding repeats and has been shown to support cell attachment. Two RGD-containing peptides, GRGDSP and echistatin, were used to test the RGD dependence of the TSP-induced increase in [Ca](i). At 0.8 mM GRGDSP, the increase is inhibited by 48%, whereas the same concentration of GRGESP only inhibited 7% (Fig. 7, A and B). Echistatin, which is known to be a more potent integrin antagonist(42) , inhibited the increase in [Ca](i) by 72% at 0.8 µM (Fig. 7C). Both GRGDSP and echistatin inhibit the TSP-induced [Ca](i) increase in a concentration-dependent manner (Fig. 8).


Figure 7: Effect of RGD-containing peptides on the thrombospondin-induced rise in [Ca]. Cells were pretreated with 0.8 mM GRGDSP (A), 0.8 mM GRGESP (B), or 0.8 µM echistatin (C) before the addition of 0.08 µM TSP. Note the partial inhibition of TSP-induced increases in [Ca]with GRGDSP and echistatin but not with the control inactive peptide GRGESP. However, GRGDSP at this concentration also increases [Ca]. Representative tracings of single cell recordings from at least four separate experiments. Assay was carried out as described under ``Experimental Procedures.''




Figure 8: Dose-response relationship of the inhibitory effect of RGD-containing peptides. The effect of echistatin (bullet), GRGDSP (circle), or GRGESP (times) on TSP-induced increases in [Ca]. Echistatin showed more potent inhibition of TSP-induced increases in [Ca] than GRGDSP with a shift of 3-4 log concentrations to the left. Each data point is the mean of at least four experiments with S.E. leq 10%. Assay was carried out as described under ``Experimental Procedures.''



In addition to blocking the TSP-induced increase in [Ca](i), GRGDSP by itself, at concentration >0.8 mM, caused a transient increase in [Ca](i) in >90% of the cells. It is possible that the depletion of available Ca due to this GRGDSP-induced increase in [Ca](i) might be responsible for the attenuation of [Ca](i) signal when TSP is added. However, this is unlikely since subsequent stimulation with other agonists, such as thrombin, was able to elicit another transient increase in [Ca](i).^2 This response to GRGDSP was RGD-specific as the control peptide, GRGESP, did not induce an increase in [Ca](i). Also, echistatin, by itself, demonstrated a similar increase in [Ca](i) in 50% of the cells at 5 µM, but no effect was seen at 1 µM.

Role of Non-RGD Cell Binding Domains in [Ca](i)Increase

Since neither anti-alphavbeta3 nor RGD-containing peptides can completely inhibit TSP-induced rises in [Ca](i), it is possible that there may be other components that mediate the Ca signaling in addition to the integrin alphavbeta3. To identify the structural basis for this non-alphavbeta3 component of the intracellular signaling, a series of experiments was carried out using antibodies and peptides directed to either the different cell binding domains or the receptor(s) of TSP.

One of the potential candidates is CD36, which is the receptor for the type I repeat of TSP(17) . This surface glycoprotein CD36 has been shown to mediate signal transduction in platelets by increasing [Ca](i)(43) . However, in this system, it is unlikely that CD36 mediates the increase in [Ca](i) as IMR-90 do not express CD36 on their cell surface (Fig. 3). In addition, the peptide CSVTCG, which corresponds to the CD36 binding motif on TSP and has been shown to inhibit cell adhesion(13) , did not have any effect on TSP-induced rises in [Ca](i) (Fig. 9). CSVTCG by itself had no effect on baseline [Ca](i). Likewise, the heparin binding domain of TSP also did not seem to play a role in generating this [Ca](i) increase because heparin, at a concentration (100 µg/ml) that had been shown to effectively block cell adhesion(15) , did not inhibit TSP-induced rises in [Ca](i) (Fig. 9).


Figure 9: Effect of inhibitors against the non-RGD domains of thrombospondin on thrombospondin-induced rise in [Ca]. Antibodies to TSP cell binding domains, C6.7 (1:100), A4.1 (1:100), heparin (100 µg/ml), and control mouse IgG (50 µg/ml) were preincubated with TSP for 30 min before addition to cells. Peptide CSVTCG (0.1 mM) was added directly to the IMR-90 cells. Of these inhibitors only the antibody to the carboxyl-terminal domain of TSP, C6.7, is effective in inhibiting TSP-induced increase in [Ca]. Assay was carried out as described under ``Experimental Procedures.''



Another possibility is the carboxyl-terminal cell binding domain of TSP which has been described to support non-RGD-dependent cell adhesion in melanoma cell lines(14, 15) . When TSP was preincubated with C6.7, an antibody against this domain, the TSP-induced increase in [Ca](i) is inhibited by 50%. This suggests that part of the Ca signal generated by TSP may be due to the binding of the carboxyl-terminal domain to its receptor (Fig. 9). This effect is apparently specific to the carboxyl-terminal binding domain since antibody A4.1(10, 44) , directed against the amino-terminal half of the central stalk region of TSP, did not block the [Ca](i) increase. Nonspecific antibody did not have any significant effect on [Ca](i) increase. Interestingly, when both LM609 (10 µg/ml) and C6.7 (1:100) were used together, no synergistic or additive effect was seen. (^3)It is unclear, at this point, if this is due to a lack of cooperativity of the two sites. Nevertheless, more direct evidence of the role of the carboxyl-terminal of TSP came from studies with the carboxyl-terminal domain peptide RFYVVMWK(45) . This peptide at 100 µM triggered a transient increase in [Ca](i) that reached geq2 µM in 35% of the cells (Fig. 10). This suggests that the carboxyl-terminal cell binding domain may mediate part of the TSP-induced rise in [Ca](i). Altogether, these data indicate that intact TSP can mediate an increase in [Ca](i) in IMR-90 fibroblasts via at least two pathways: the RGD domain binding to the alphavbeta3 integrin and the carboxyl-terminal domain possibly binding to an unidentified receptor.


Figure 10: [Ca] tracing showing the effect of thrombospondin carboxyl-terminal peptide on [Ca] in IMR-90 cells. The carboxyl-terminal peptide, RFYVVMWK (100 µM), increases [Ca] in IMR-90. Representative tracings of single cell recordings from at least four separate experiments. Assay was carried out as described under ``Experimental Procedures.''




DISCUSSION

In this study we have shown that TSP, a matrix protein which binds to multiple cell surface receptors, increases [Ca](i) in IMR-90 fibroblasts. This increase, which is partially mediated by the RGD domain of TSP binding to the integrin alphavbeta3, is not limited to IMR-90, as we have also observed this in endothelial cells.^3 We have also shown that known inhibitors of alphavbeta3, GRGDSP peptide and echistatin, in addition to blocking TSP-induced signaling, are capable of triggering an increase in [Ca](i) by themselves. This suggests that RGD-containing peptides can function as both competitive antagonists and partial agonists for their receptors. This had been reported previously by several other groups; however, our threshold concentrations are much higher than those reported elsewhere(36, 37) . One possible explanation is that high concentrations of peptides or antibodies could induce clustering of integrins, which could activate the integrins and trigger transmembrane Ca flux not normally seen at the lower concentrations(26, 46) . This clustering is seen in the present study when alphavbeta3 integrins interact with TSP. These findings illustrate that integrin clustering, presumably caused by multimeric interactions between the ligand and its receptors, is important in transmembrane signaling(41) . This is further supported by the observation that RGD peptide can produce an increase in [Ca](i) (>1 mM) in kidney epithelial cells when coupled to beads but not when it is in the soluble monomeric form(29) . In addition, earlier studies with beta2 integrin (CD11b/CD18) demonstrate that antibodies to the alpha and beta can induce Ca signal only if they are cross-linked(40) .

Another confounding factor is that this ligand-induced integrin signaling may be dependent on the type of cell preparation being used. Sjaastad et al.(29) have reported that soluble RGD (0.3 mM) was not able to produce an increase in [Ca](i) in kidney epithelial cells, whereas two other groups both observed an increase in osteoclasts at a similar concentration(36, 37) . Even within the same cell type the data are not consistent. In that regard, echistatin was shown to induce increases in [Ca](i) in rat osteoclasts by one group but not by the other group(36, 37) . This discrepancy indicates the complexity of ligand/integrin-mediated signaling.

Ligand-induced signaling through integrins does not follow the paradigm for traditional signaling receptors. In general, integrins differ from the known signaling receptors in their short cytoplasmic domain, lack of kinase activity, and lack of direct interaction with G-proteins (23) . In this study we have shown that the [Ca](i) signal triggered by the soluble matrix protein, TSP, via the integrin alphavbeta3, is dependent on extracellular calcium. The present data using EGTA or Ni indicates that the increase in [Ca](i) is due primarily to an influx of extracellular Ca, possibly through some type of Ca channel. Additional evidence supporting this hypothesis was demonstrated by our data with the antibody to IAP, B6H12, which was shown to block 50% of [Ca](i) increase. IAP, which has been shown to be closely associated with alphavbeta3(31) , was recently shown by Schwartz et al.(39) to be required specifically for integrin-regulated Ca influx. This suggests that IAP may function as an integrin-associated calcium channel. Moreover, purified integrin alphaIIbbeta3 was reported to function as a calcium channel when reconstituted into liposomes(47) . This suggests that the integrin itself can act as a conduit for transmembrane Ca flux that is distinct from the classical Ca channels. Taken together, it is possible that the alphavbeta3 integrin or its associated proteins, upon ligand binding, can be activated and function as a Ca channel for signal transduction.

The present data also show that there are other components of the transmembrane Ca flux that are non-RGD and non-alphavbeta3-dependent. This notion comes from the inability of the synthetic RGD-peptide and LM609 to completely inhibit TSP-induced rises in [Ca](i) and is verified by the observation that an antibody to carboxyl-terminal domain, C6.7, is also able to inhibit 50% of the Ca signal. The carboxyl-terminal domain of TSP has been reported to support chemotaxis and cell migration(48, 49) . Further studies using the carboxyl-terminal domain peptide, RFYVVMWK, indicate that the carboxyl-terminal domain of TSP is also capable of triggering Ca increases by itself. Since this Ca increase is observed only in a subpopulation of cells, this receptor might not be present on all cells. Two possible receptors for the carboxyl-terminal binding domain have so far been identified, an 80/105-kDa complex on carcinoma cells (4) and a 52-kDa domain on K-562 cells(19) . Even if either of these receptors exists in the IMR-90, it is unclear if they are responsible for the binding of the carboxyl-terminal TSP fragment and the subsequent transduction of the signal across the cell membrane. The carboxyl-terminal peptide and GRGDSP trigger increases in [Ca](i) in the millimolar range at peptide concentrations of 0.1 and 0.8 mM, respectively. With intact TSP, only 1 µM is required to trigger similar increases in [Ca](i). The main reason for this disparity in threshold concentrations is the multivalent and higher affinity binding of the multimeric matrix protein TSP versus the monovalent and lower affinity binding of the peptide fragments(41, 50) . In addition to having high affinity receptors, multimeric ligands are more effective in inducing receptor clustering as shown in the present study; together, these have been shown to work synergistically in transmembrane signaling(41) .

In addition, TSP might potentially mediate signaling via other receptors. One such TSP receptor is CD36 which was not studied here due to the lack of expression on IMR-90 fibroblasts. There is evidence from the literature that CD36 is capable of mediating signal transduction by increasing [Ca](i) in platelets and triggering oxidative bursts in monocytes(43, 51) . However, these two studies utilized monoclonal antibodies against CD36 to trigger the signaling events, and, therefore, it is uncertain if intact TSP or its fragments will produce similar results via CD36.

In summary, we have demonstrated that TSP can trigger transient increases in [Ca](i) in IMR-90 fibroblasts by increasing Ca influx. The response is inhibitable by specific TSP receptor antibodies indicating that this is a receptor-mediated effect. While we have shown this to occur primarily via the RGD and carboxyl-terminal domains of TSP and their corresponding receptors, we cannot rule out the possibility of the other TSP receptors as signaling molecules in other cellular systems. The data from the present study, together with previous reports, indicate that TSP can support cell attachment and signaling through its different cell binding domains(4, 7, 13, 14, 18, 45) . These observations confirm that matrix proteins, with their multiple cell binding domains, can interact with corresponding cell surface receptors to transduce signals across the cell membrane. These outside-in signaling events will be important in understanding the role TSP plays in cell attachment, migration, and proliferation.


FOOTNOTES

*
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.

§
To whom correspondence should be addressed: The DuPont Merck Pharmaceutical Co., The Experimental Station, E400/3456, Wilmington, DE 19880-0400. Tel.: 302-695-8418; Fax: 302-695-4083.

(^1)
The abbreviations used are: TSP, thrombospondin; IAP, integrin-associated protein.

(^2)
Data not shown.

(^3)
P. W. Tsao and S. A. Mousa, unpublished observations.


ACKNOWLEDGEMENTS

We thank Dr. Robert C. Newton for critically reading the manuscript, Dr. Ram Seetharam for peptide synthesis, Dr. Eric J. Brown for his gift of B6H12, Pamela Kidd for her expert technical assistance, and William Lorelli for his editorial assistance.


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