Clinical Pharmacology, National Heart and Lung Institute, Imperial College of Science, Technology & Medicine, QEQM Wing, St Mary's Hospital, Paddington, London W2 1NY, UK
* Author for correspondence (e-mail: j.lymn{at}ic.ac.uk)
Accepted 22 August 2002
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Summary |
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Key words: Thrombospondin-1, Chemotaxis, DNA synthesis, Vascular smooth muscle
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Introduction |
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Data from our laboratory has previously demonstrated that soluble TSP-1 exerts a growth factor-like action on human vascular smooth muscle cells (HVSMC) in vitro, inducing both chemotaxis and proliferation to levels similar to that seen with platelet-derived growth factor (PDGF) (Patel et al., 1997). Similarly we have shown that both TSP-1-induced chemotaxis and DNA synthesis are tyrosine kinase-dependent processes (Patel et al., 1997
), and that TSP-1-induced chemotaxis occurs through the sequential activation of phosphatidylinositol 3-kinase (PI3K) and focal adhesion kinase (FAK) (Lymn et al., 1999
). The receptor-binding mechanisms leading to the generation of these intracellular signals however remains obscure.
The identification of receptor(s) for TSP-1 on HVSMC is complicated by the presence of multiple domains within the sequence of TSP-1, which interact with distinct cellular receptors (Chen et al., 2000). The N-terminal heparin-binding domain binds to heparan sulphate proteoglycans (Sun et al., 1989
) while the type 1 repeats, containing the peptide sequence CSVTCG, bind to CD36 (Vogel et al., 1993
). The Arg-Gly-Asp (RGD) sequence in the type III calcium-binding repeat, on the other hand, binds to integrin receptors of the ß3 subclass (Lawler et al., 1988
), while the C-terminal cell-binding domain binds to a 52 kDa receptor identified as integrin-associated protein (IAP) (Gao et al., 1996b
). This in turn associates with, and regulates the activity of, a number of integrins (Gao et al., 1996a
; Chung et al., 1997
; Wang and Frazier, 1998
). Coupled with this plethora of putative receptors the nature of the interaction between TSP-1 and the cell surface is further complicated by other factors including cell type, activation status and presentation of TSP-1. Indeed CD36-transfected fibroblasts bind soluble TSP-1 through the CSVTCG sequence in the type 1 repeats, but attach to immobilised TSP-1 through the N-terminal type 3 repeats and the C-terminal domain (Magnetto et al., 1998
). Bowes melanoma cells, on the other hand, attach to immobilised TSP-1 through the type 1 repeats, while NRK, endothelial and smooth muscle cell attachment is blocked by peptides containing an RGD motif (Magnetto et al., 1998
; Lawler et al., 1988
). Similarly TSP has been demonstrated to bind to a single receptor in resting platelets, but to at least two other distinct receptors in activated platelets (Dorahy et al., 1997
).
Previous data from our laboratory have demonstrated that TSP-1-induced chemotaxis of HVSMC is an RGD-dependent process (Patel et al., 1997). We report here that TSP-1-induced DNA synthesis in HVSMC however, is RGD-independent. Similarly tyrosine phosphorylation of FAK and the p85 regulatory subunit of PI3K (p85PI3K) are inhibited by RGD-containing peptides, while tyrosine phosphorylation, and activation, of extracellular regulated kinase (Erk2) are unaffected. Both pertussis toxin (PTX) and a function-blocking antibody to integrin
vß3, markedly inhibited TSP-1-induced chemotaxis but did not affect DNA synthesis. Neutralising antibodies to integrin ß1-subunits on the other hand did not affect cell chemotaxis but significantly inhibited TSP-1-induced DNA synthesis. The specific integrin involved in this inhibition was determined to be
3ß1. Similarly TSP-1-induced Erk2 activity was completely inhibited by both the integrin ß1 subunit antibody and heparin.
These data demonstrate that TSP-1-stimulated DNA synthesis and chemotaxis of HVSMC are independent, and separable events, which occur following binding of soluble TSP-1 to different cellular receptors, and involve activation of distinct signalling mechanisms.
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Materials and Methods |
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Vascular smooth muscle cell culture
Human saphenous vein was obtained from patients undergoing cardiovascular surgery. Tissues were surplus to requirements, and their use conformed to local ethics committee guidelines. VSMC were cultured using an explant technique as previously described (Chan et al., 1993), and were routinely used at third passage unless otherwise stated. VSMC were cultured in DMEM buffered with 25 mmol/l HEPES and supplemented with 15% (vol/vol) FCS, 4 mmol/l l-alanyl-l-glutamine (Glutamax-I), penicillin (100 U/ml), streptomycin (100 µg/ml), and gentamicin (25 µg/ml). Cell cultures were maintained in a humidified atmosphere of 5% CO2 (vol/vol) in air at 37°C. VSMC purity was regularly checked by immunocytochemical studies for
-actin staining.
Cell stimulation
HVSMC were subcultured and plated onto petri dishes at a known density (1,000,000 cells/dish), and allowed to attach for 24 hours in DMEM supplemented with 15% (vol/vol) FCS. Cells were then washed twice with phosphate buffered saline and maintained in serum-free medium for a period of seven days. Quiescent cells were then stimulated with 1 µg/ml TSP-1 for a period of 30 minutes at 37°C. Following stimulation cells were washed twice with ice-cold PBS, and scraped into 1 ml of ice-cold lysis buffer (50 mmol/l Tris (pH 7.4); 150 mmol/l NaCl; 1 mmol/l EGTA; 1% (vol/vol) NP-40; 0.25% sodium deoxycholate; 1 mmol/l sodium fluoride, sodium orthovanadate, phenymethylsulphonyl fluoride; 1 µg/ml aprotinin, pepstatin, leupeptin). This lysate was allowed to stand on ice for 10 minutes prior to centrifugation (15,800 g, 15 minutes, 4°C). The resulting supernatants were used for protein measurement.
Immunoprecipitation
A known concentration of cellular protein was removed from all samples and pre-cleared for 1 hour with albumin-agarose, prior to incubation with antibody (1 µg Ab/100 µg protein) for 3 hours at 4°C. Immunoprecipitates were captured on protein A-agarose in the manner previously described (Lymn et al., 1999).
Immunoblotting
Immunoblotting was performed in the manner previously described by us (Lymn et al., 1999). Briefly proteins were separated on 10% SDS-polyacrylamide gels prior to transfer to nitrocellulose using a Bio-Rad wet gel transfer system. Following successful transfer nitrocellulose blots were blocked in 5% BSA for 1 hour and washed three times in Tris buffered saline containing 0.05% Tween 20 (TTBS), prior to probing with primary antibody for 1 hour. Blots were washed well in TTBS before being probed with the appropriate secondary antibody and developed using enhanced chemiluminesence.
DNA synthesis assays
DNA synthesis assays were conducted as described previously (Patel et al., 1997). Quiescent cells were stimulated with an approximately 50% maximal (EC50) concentration of TSP-1 (5 µg/ml), in the presence or absence of functional integrin antibodies. [methyl 3H]-Thymidine was added (1 µCi per well, 5 µCi/ml) 24 hours after stimulation, for six hours, and the experiment was terminated with 10% (w/v) trichloroacetic acid.
Cell chemotaxis assays
These assays were performed using blind well chemotaxis chambers as previously described (Patel et al., 1997; Clunn et al., 1997
). In brief, the cell suspension (2.25x105 cells/ml) was preincubated for 60 minutes in the presence or absence of functional blocking antibodies, prior to migration assay. TSP-1 (10 µg/ml in serum-free medium) was added to the lower chamber and acted as the chemoattractant. The upper and lower compartments of the blind well chambers were separated by either gelatin-coated or collagen-coated 13 mm polycarbonate filters as stated. The cell suspension was then added to the upper chamber, and migration was allowed to proceed for 5 hours at 37°C. The filters were then removed, fixed in ethanol, and stained in toluidine blue (1%). The migrated cells were counted under a light microscope.
Statistical analysis of data
Unless otherwise stated TSP-1 stimulated data was defined as 100%, and experimental data were expressed as mean±s.e.m. in relation to this response. Statistical differences between groups of means were compared using a Friedman and Conover's Multirange Test for paired non-parametric data. All other data was compared using a Wilcoxon matched pairs signed ranks test.
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Results |
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This suggests that the RGD sequence is critical for TSP-1-induced chemotaxis, over and above any role in cell attachment. Further, TSP-1 may signal for human VSMC chemotaxis and DNA synthesis through separate, RGD-dependent and RGD-independent, receptor mechanisms respectively.
Effect of integrin antibodies on functional responses to TSP-1 stimulation
In order to further elucidate the nature of the interactions between TSP-1 and HVSMC the effect of neutralising antibodies to both the vß3 (LM609) and ß1 (CD29) integrin receptors on the functional effects of TSP-1 stimulation were investigated. In order to ensure that the effects seen with LM609 were specific, rather than the result of inhibition of cell attachment, experiments were conducted using collagencoated filters. These data demonstrate that LM609 exhibited a dose-dependent inhibitory effect on TSP-1-induced chemotaxis (20 µg/ml=59±8%; 50 µg/ml=38±16% of the stimulated response), while PDGF-BB-induced cell chemotaxis on collagen filters was unaffected (Fig. 2A). There was no effect of the function blocking ß1 antibody (20 µg/ml CD29) on TSP-1-induced cell chemotaxis (111±26% of stimulated response).
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In contrast LM609 (20 µg/ml) did not affect TSP-1-induced DNA synthesis, while CD29 (20 µg/ml) induced a significant reduction in DNA synthesis (20±7% of the stimulated response). Intriguingly the ß1integrin-neutralising antibody (CD29) had no effect on PDGF-BB-induced DNA synthesis in these HVSMC (Fig. 2B). Further investigation of the specific ß1 integrin involved revealed a significant inhibition of TSP-1-induced DNA synthesis with a function-blocking antibody to 3ß1 (20 µg/ml P1B5=25±9% of the stimulated response). Function-blocking antibodies to both
5ß1 and
2ß1 on the other hand had no effect on TSP-1-induced DNA synthesis (Fig. 2C).
Role of IAP and Pertussis toxin in mediating functional responses to TSP-1
It has been suggested that TSP-1 associates with the Vß3 integrin dimer via the adapter molecule, Integrin-Associated Protein (IAP), consequently we investigated the effects of an IAP function-neutralising antibody (B6H12) on TSP-induced chemotaxis and DNA synthesis. At 20 µg/ml B6H12 resulted in a partial, but significant, inhibition of TSP-1-induced cell chemotaxis to 67±10% (P<0.05) of the TSP-1-stimulated response. This was not significantly affected by increasing the concentration of B6H12 (50 µg/ml=65±6% of the stimulated response, P<0.05). In contrast B6H12 did not inhibit stimulated DNA synthesis in response to TSP-1 (B6H12 50 µg/ml=111±12% stimulated response, Fig. 3A).
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Recent data have suggested that IAP may couple (directly or indirectly), to a pertussis toxin (PTX)-sensitive G-protein, and that PTX can inactivate this G-protein, thus inhibiting TSP-1-mediated responses (Gao et al., 1996a; Chung et al., 1997
; Guo et al., 1998
). Therefore we investigated the effect of PTX on TSP-1-stimulated cell chemotaxis and DNA synthesis. Pre-incubation with 0.5 µg/ml PTX for 18 hours prior to TSP-1 stimulation resulted in a marked inhibition of TSP-1-induced chemotaxis (PTX=26±8% of the TSP-1-stimulated response, Fig. 3B). In contrast PTX treatment resulted in only a modest inhibition of PDGF-BB-induced chemotaxis (73±23% of stimulated response, n=6). In keeping with the B6H12 data, PTX treatment did not have any significant effect on the TSP-1-stimulated DNA synthetic response (Fig. 3B).
TSP-1-induced cell signalling
Immunoprecipitation of phosphotyrosine proteins, followed by immunoblotting with specific antibodies, demonstrated that TSP-1 stimulation induces tyrosine phosphorylation of FAK (305±79% of basal level, n=12, P<0.01), p85PI3K (338±63% of basal level, n=9, P<0.01) and Erk2 (407±104% of basal level, n=11, P<0.05). As TSP-1-induced chemotaxis and DNA synthesis are differentially affected by the RGD peptide, this peptide should differentially affect the tyrosine phosphorylation, and/or activation, of the signalling molecules involved in these processes.
Indeed the RGD peptide (0.1 mM) inhibited TSP-induced tyrosine phosphorylation of both FAK and p85P13K, reducing them to levels not significantly different from control cells (FAK=170±79%; p85P13K=213±77 of basal response respectively). TSP-1-stimulated tyrosine phosphorylation of Erk2 (407±104% of basal response) however, remained significantly elevated, even in the presence of the RGD peptide (Fig. 4). In contrast, treatment of unstimulated cells with the RGD peptide did not significantly affect basal levels of tyrosine phosphorylation of either p85PI3K (138±47%, n=6) or FAK (133±50%, n=6).
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Analysis of extracellular-regulated kinase activity
Erk2 is a serine/threonine kinase, which is activated by sequential phosphorylation on tyrosine and threonine residues (Yung et al., 1997). Although data presented here demonstrate a significant effect of TSP-1 on tyrosine phosphorylation of Erk2, this does not necessarily reflect an increase in the activity of the enzyme. Consequently the effect of the RGD peptide, heparin and integrin-neutralising antibodies CD29, LM609 and P1B5 on TSP-1-induced Erk2 activity was assessed. The level of tyrosine/threonine phosphorylation, by means of a monoclonal antibody specific for the dual phosphorylated kinase, was utilised as a measure of enzyme activity (Yung et al., 1997
). Cells were stimulated with TSP-1 (1 µg/ml) for 30 minutes and Erk2 was immunoprecipitated prior to immunoblotting with a phosphospecific antibody. These data indicate that TSP-1-induced significant Erk2 activation (TSP-1=330±54% of control cells, P<0.05, n=10), which correlates well with the increased level of Erk1/2 tyrosine phosphorylation induced by TSP-1. The TSP-1-induced activation of Erk2 is completely inhibited by CD29, heparin and P1B5, being reduced to below the levels seen in control cells, but is not significantly affected by either LM609 or the RGD peptide (Fig. 5). This suggests that TSP-1 stimulates Erk2 activity through a ß1 integrin-dependent but RGD-independent, or RGD-redundant pathway.
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Effect of MAP kinase kinase inhibitor on TSP-1-induced cellular responses
To further investigate the differential signalling via TSP-1 receptors we assessed the effect of inhibiting the DNA synthetic pathway using the MAP kinase kinase (MEK) inhibitor, PD98059, on the cellular responses to TSP-1. PD98059 (10 µM) had no significant effect on TSP-1-induced cell chemotaxis but completely inhibited TSP-1-induced DNA synthesis to below unstimulated levels. (Fig. 6).
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Discussion |
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A previous study characterising the integrin species present on the cell surface, from similarly explant-cultured human saphenous vein-derived SMC (Itoh et al., 1997), demonstrated the presence of the
vß3 integrin, along with the ß1 subunit in conjunction with a range of
subunits. Our data indicate that TSP-1 associates specifically with the integrin
vß3, through its RGD sequence, to stimulate cellular chemotaxis. Indeed the function-blocking antibody LM609 significantly inhibited TSP-1-induced chemotaxis, reducing the response to levels similar to that seen with the RGD peptide, while the ß1 (CD29) did not affect TSP-1-induced cell chemotaxis. This receptor-binding mechanism is similar to that described previously for osteopontin-induced chemotaxis of aortic VSMC, being
vß3 sensitive, but ß1 insensitive (Yue et al., 1994
).
Interestingly our data show that TSP-1-induced chemotaxis was only partially inhibited by the function-blocking IAP antibody (B6H12) at both 20 and 50 µg/l, suggesting that this represents a maximum response to this antibody. These data indicate that, although IAP is undoubtedly present on the surface of saphenous vein-derived HVSMC, it does not represent the sole functional receptor-binding site for TSP-1 in these cells. This differs significantly from other published reports, which have shown that TSP-1-induced cell spreading on vitronectin is completely inhibited by both LM609 and B6H12 (Gao et al., 1996a), while TSP-induced chemotaxis of human endothelial and aortic smooth muscle cells is inhibited by B6H12 (Wang and Frazier, 1998
). It is, however, consistent with the idea that IAP regulates a subset of
vß3-mediated functions, and that IAP and
vß3 can be expressed on the plasma membrane in both complexed and independent forms simultaneously (Green et al., 1999
). Although only moderately affected by B6H12, the chemotactic response of HVSMC was completely inhibited by PTX. This is surprising as the role of PTX-sensitive heterotrimeric G-proteins in modulating TSP-1-induced cell chemotaxis has previously been thought to occur solely through association with IAP-integrin complexes (Frazier et al., 1999
; Wang et al., 1999
). Nevertheless our data indicate that the majority of TSP-1-stimulated chemotaxis occurs through binding of the RGD sequence to the integrin
vß3 independently of IAP, suggesting that the integrin itself may be associated in some manner with a PTX-sensitive heterotrimeric G-protein. Interestingly, chemotaxis of CHO cells expressing the N-formyl peptide receptor, is significantly inhibited by RGD, a function-blocking antibody to the
5 integrin subunit, PTX, and the PI3K inhibitor wortmannin (Miettinen et al., 1998
). This pattern of inhibition is similar to that seen in our current and previous data (Lymn et al., 1999
), and indicates that heterotrimeric G-proteins can be linked to integrin activation through a mechanism other than association of IAP.
In contrast to its effects on chemotaxis, TSP-1-induced DNA synthesis was unaffected by both RGD and LM609, but dose-dependently inhibited by the function blocking ß1 antibody. Indeed TSP-1-induced DNA synthesis was specifically inhibited by a function-blocking antibody to the integrin 3ß1, but not by antibodies to
2ß1 or
5ß1. Furthermore TSP-1-induced DNA synthesis was unaffected by either B6H12 or PTX.
Similarly TSP-1-stimulated activation of Erk2 was completely inhibited by both the function-blocking ß1 integrin antibody and heparin, but was not significantly affected by the RGD peptide. This further differentiates the receptor-binding and concomitant signalling mechanisms involved in initiating this functional response from those involved in the induction of TSP-1-stimulated cell chemotaxis, and suggests that non-RGD interactions with 3ß1 integrins are critically important for TSP-1-induced DNA synthesis in HVSMC. This is consistent with data from Jurkat T cells, which demonstrated that TSP-1-induced tyrosine phosphorylation of MAPK was mediated by a ß1 integrin-receptor (Wilson et al., 1999
). Similarly TSP-1-binding to
3ß1 has been reported in breast carcinoma cells (Krutzsch et al., 1999
), and ligation of the
3ß1 integrin has been shown to modulate TSP-1-induced endothelial cell proliferation (Chandrasekaran et al., 2000
). Intriguingly a recognition sequence for
3ß1 in the N-terminal, heparin-binding domain of TSP-1 has been identified (Krutzsch et al., 1999
). This is not only RGD-independent but correlates well with the ability of heparin to inhibit both TSP-1-induced Erk2 activation shown here, and its previously demonstrated ability to inhibit TSP-1-induced DNA synthesis in VSMC [(Majack et al., 1986
) M.K.P., K.L.G., A.D.H. et al., unpublished].
In the context of integrin signalling mechanisms these data exhibit interesting differences to previous reports. Individual integrins can recognise several ECM proteins, and individual matrix molecules can bind to several integrins (Wary et al., 1996); the signalling pathway activated is dependent on the class of integrin. Intracellular signals elicited by ß1 and
v integrins resemble those induced by receptor tyrosine kinases, and while most integrins activate FAK, only a subset are linked to the Erk pathway (Wary et al., 1996
; Wary et al., 1998
). The differential effects of LM609 and CD29 on TSP-1-induced cellular responses, coupled with the differential effects of RGD on specific signalling molecules however, suggest that while TSP-1 activation of a ß1 integrin stimulates Erk it does not result in significant FAK activation. Similarly the binding of TSP-1 to
vß3 results in FAK activation but does not lead to Erk activation.
The ability of certain ligands to induce discrete, functional consequences within cells, is a concept that has gained increasing attention, with a number of recent publications demonstrating its importance in specific circumstances. Indeed earlier work from this laboratory has shown that different PDGF-BB concentrations differentially induce chemotaxis and DNA synthesis in HVSMC (Clunn et al., 1997) via the PDGFß receptor. Indeed TSP-1 has been shown to exhibit chemotactic and anti-proliferative activities in melanoma cells which may be ascribed to differential receptor signalling (Guo et al., 1998
). The differential effect of TSP-1 on proliferation in these two cell types may be due to the range of integrins expressed, or perhaps to the specific signals, not investigated in the melanoma cells, generated in what are essentially cells with opposing functions. HVSMC by their nature proliferate only slowly in culture, and generally not at all in vivo, whereas melanoma cells are rapidly proliferating. This may lead to TSP-1-induced stimulation resulting in a negative feedback mechanism, or in increased apoptosis of the melanoma cells.
This report demonstrates that TSP-1 induces distinct and differential cellular signals, in a single cell type, through discrete and separable receptor-binding mechanisms. This offers a mechanism by which extensive and subtle control of cellular responses to TSP-1 can be achieved through modulation of integrin expression and/or affinity. TSP-1 is emerging as a powerful modulator of vascular function through its ability to induce both smooth muscle cell movement and growth. While these functions are inherently necessary for vascular repair, the ability to differentiate between these processes may have particular relevance in terms of human vascular disease.
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Acknowledgments |
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