Published online before print June 3, 2004, doi: 10.1161/01.ATV.0000134299.89599.53
© 2004 American Heart Association, Inc.
Vascular Biology |
EGF-Like Domain of Tenascin-C Is Proapoptotic for Cultured Smooth Muscle Cells
From the Atherosclerosis Research Center (K.W., C.L., P.K.S., K-J.W., B.G.S.), Division of Cardiology, and Burns and Allen Research Institute, Cedars-Sinai Medical Center, and UCLA School of Medicine, Los Angeles, Calif; and the Department of Pathology (S.M.S.), University of Washington, Seattle.
Correspondence to Dr Behrooz G. Sharifi, Cedars-Sinai Medical Center, Davis Building, 1016 8700 Beverly Boulevard, Los Angeles, CA 90048. E-mail Sharifi{at}cshs.org
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Abstract |
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Objective— Based on our previous observations on the expression of Tenascin-C (Tn-C) in human atherosclerotic plaques and its colocalization with macrophages, we explored whether Tn-C undergoes fragmentation and the potential pathobiological significance of this fragmentation.
Methods and Results— Using cultured human smooth muscle cells (SMCs), we found that Tn-C upregulates expression of matrix metalloproteinases (MMPs). Western blot analysis revealed that Tn-C substrate is fragmented and most of the cleavage products have fibronectin-like and epidermal growth factor-like (EGF-like) domains of Tn-C. One fragment that contains an EGF-like domain was found in some human atherosclerotic plaques. Cell culture studies revealed that the recombinant EGF-like domain inhibits growth, induces apoptosis of SMCs in a dose-dependent, time-dependent, and caspase-dependent manner, and activates caspase-3 before SMC detachment. Conversely, the caspase inhibitor z-YVAD.cmk, serum, and protease inhibitors blocked cell apoptosis conferred by the EGF-like domain. In addition, these inhibitors blocked EGF-like domain-induced caspase-3 activation. In contrast to this EGF-like domain, intact Tn-C, its fibronectin-like, and its fibrinogen-like domains were inactive.
Conclusions— Together with our previous observations, our data suggest that Tn-C upregulates MMP expression that cleaves Tn-C into fragments containing the EGF-like domain. This domain has proapoptotic activity for SMCs.
Tenascin-C was found to undergo fragmentation in cultured smooth muscle cells and in human atherosclerotic plaques generating a fragment that contains the epidermal growth factor-like domain of tenascin-C. Recombinant epidermal growth factor-like domain of tenascin-C induces apoptosis of cultured smooth muscle cells, an activity that was blocked by chymotrypsin inhibitor and MMP inhibitor.
Key Words: Tenascin-C • metalloproteinases • atherosclerosis • apoptosis • EGF-like domain
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Introduction |
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The reasons for loss of smooth muscle cell (SMC) as atherosclerotic plaques progress are not known. One possible explanation is that the loss of cellularity as lesions evolve is a result of changes in the extracellular matrix. Indeed, cell death resulting from loss of cell adhesion to the extracellular matrix has been called "anoikis."1 Activated inflammatory cells and vascular cells produce various proteases including matrix metalloproteinases (MMPs),2,3 which disrupt the matrix structure that supports endothelial cells and SMCs and, thereby, enhance the apoptotic propensity of cells.4–11 We have previously shown that serum protease inhibitors are required for spreading of SMCs in a fibrin gel because of their ability to inhibit fibronectin proteolysis.12 Our earlier study showed that Tn-C upregulates expression of MMPs in cultured macrophages, and it is strongly expressed in advanced human atherosclerotic plaques, particularly around the lipid core and plaque shoulders,13 regions with high levels of MMP activity, and SMC apoptosis.14,15 Little is known about the role of Tn-C in human atherosclerotic plaques. We now report that Tn-C undergoes fragmentation in cultured SMCs, and in human atherosclerotic plaques and one of the fragments contains the EGF-like domain of Tn-C. Using a recombinant EGF-like domain, we found that this domain promotes apoptosis of cultured SMCs and that chymotrypsin inhibitor blocks this apoptosis.
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Methods |
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Please see online methods section at http://atvb.ahajournals.org for further details. Tn-C was purified from conditioned media of BHK cells overexpressing the large isoform of Tn-C, as described.16 The recombinant proteins corresponding to the full-length fibrinogen-like and unspliced fibronectin-like domains of Tn-C were expressed and purified from the bacteria BL-21 Escherichia coli as described.17 GM 6001 metalloproteinase inhibitor was obtained from Chemicon. Other reagents were purchased from Sigma. All reagents were tested for endotoxin activity using E-TOXATE kit (Sigma) and were found to be endotoxin-free.
Cultured SMCs
Rat vascular SMCs were cultured as we described previously.18 Cultured human SMCs were kindly provided by Dr Navab (University of California Los Angeles). For more details, please see http://www.ahajournals.org.
Zymography
Zymography was performed essentially as described previously.13 For more details, please see http://www.ahajournals.org.
Cloning of Rat EGF-like
Total RNA derived from rat SMC was used as a template for the reverse-transcription polymerase chain reaction, which was performed as described16 using primers containing a KEX2 site. The polymerase chain reaction product was placed on agarose gel, isolated, cut with XhoI/EcoRI, and subcloned into pPIC9 yeast vector, essentially as recommended by Invitrogen. After transformation, cells were screened for Mut+ and Muts transformants. For more details, see please see http://www.ahajournals.org.
Purification and Characterization of the Recombinant EGF
The ammonium sulfate pellet was resuspended in 10% acetonitrile and loaded onto a C8 reverse-phase column that was eluted with a gradient of 10% to 60% acetonitrile containing 0.1% trifluoroacetic acid. For more details, please see http://www.ahajournals.org.
Antibody Techniques
The anti–Tn-C monoclonal antibody was obtained from Sigma (clone BC-24). The antibody recognizes an epitope within the N-terminal EGF sequence of the molecule. The antibodies to the fibronectin-like domain of Tn-C were a gift from Dr Erickson (Duke University, Durham, NC). Tn-C degradation was measured by Western blot as we previously described.19 For more details, please see http://www.ahajournals.org.
Cell Apoptosis
Cell apoptosis was measured by several methods. Please see http://atvb.ahajournals.org.
All the experiments have been performed in triplicate and repeated 3 times with different preparations of Tn-C or recombinant EGF-like domain to ensure reproducibility. The results are representative of either 3 experiments or average±SEM of a triplicate determinations.
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Results |
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We have previously reported that Tn-C upregulates MMPs synthesis by macrophages.13 Because SMCs are major components of atherosclerotic plaques, we asked whether Tn-C affects expression of MMPs in these cells. To explore this possibility, SMCs were plated onto Tn-C–coated dishes in defined media for 24 hours. Collagen-coated dishes were used as a control. SMCs cultured on collagen type I constitutively secreted only 1 band, 70-kDa, with gelatinolytic activity. Cells cultured on Tn-C substrate expressed 2 bands, 70-kDa and 65-kDa, with gelatinolytic activity (Figure 1). The 70-kDa enzyme activity was increased >4-fold as compared with collagen (Figure 1), and the 65-kDa activity was detected primarily on the Tn-C substrate. Similar results were obtained with rat and porcine SMCs (data not shown), indicating that the effect of Tn-C is conserved. Kinetic studies revealed that the 65-kDa protease activity was detectable 8 to 9 hours after plating onto Tn-C substrate and reached saturation levels after 24 hours. Past studies have shown that Tn-C is sensitive to degradation by MMPs.20,21 Because Tn-C upregulates MMP expression, we reasoned that this upregulation may, in turn, lead to degradation of Tn-C. To explore this possibility, human SMCs were plated onto Tn-C–coated dishes in 10% fetal bovine serum or defined serum-free, and then extracted 10 hours or 24 hours after plating. The extracted Tn-C was analyzed by Western blot using antibodies specific to the fibronectin-like or EGF-like domains of Tn-C. The 2 antibodies showed that Tn-C remained intact in the presence of serum (Figure 2A through 2C, lane S). In contrast, in defined media, Tn-C was fragmented 10 hours after plating and further fragmented at 24 hours (Figure 2A through 2C, lanes 10 and 24). The patterns of Tn-C fragmentation detected by the 2 antibodies were similar with 1 exception: the anti-EGF-like monoclonal antibody (Figure 2B and 2C), but not anti-fibronectin domain antibody (Figure 2A), detected an additional band corresponding to 85-kDa, suggesting that this band contains EGF-like domain. To further determine whether the 85-kDa fragment possesses an EGF-like domain, we performed an antibody pull-down assay by incubating the anti-EGF-like domain monoclonal antibody with a 0.1-µmol/L solution of recombinant EGF-like or fibronectin-like domain of Tn-C before addition to the blot. Pre-incubation with the recombinant fibronectin-like domain had no effect on the bands recognized by anti-EGF-like domain antibody, whereas pre-incubation with the recombinant EGF-like domain blocked the ability of antibody to detect the 85-kDa band (data not shown). We concluded that the 85-kDa band is likely a fragment of Tn-C that contains its EGF-like domain. To determine whether degradation of Tn-C is mediated by MMPs, SMCs were cultured in defined media onto Tn-C–coated dishes in the presence of 25 µmol/L GM6001, a MMP inhibitor that blocks the activities of MMP 1 to 3, MMP-8, and MMP-9. SDS-PAGE analysis of cell extracts showed that the MMP inhibitor blocked degradation of Tn-C (Figure 2D; lanes 10 and 24), suggesting that Tn-C degradation is mediated by MMPs produced by cultured cells. It has been reported that proteoglycans undergo fragmentation in unstable human endarterectomy specimens,22 suggesting that matrix proteins may be fragmented in the plaque. We previously reported that Tn-C expression colocalizes with macrophages in human atherosclerotic plaques;13 therefore, we hypothesized that this colocalization could lead to Tn-C fragmentation. To explore this idea, Tn-C integrity was determined in human endarterectomy specimens by Western blot. Antifibronectin-like domain antibodies detected 220-kDa and 280-kDa bands in all 16 endarterectomy specimens, which most likely correspond to the small and large isoforms of Tn- C, respectively (data not shown). However, anti-EGF-like domain monoclonal antibody not only detected the 220-kDa and 280-kDa bands in all 16 specimens but also reacted with 85-kDa and 65-kDa proteins in 4 specimens (Figure 3), suggesting that under some circumstances, Tn-C is fragmented in human atherosclerotic plaques and that the fragments most likely contain an EGF-like domain. To determine the biological consequences of Tn-C fragmentation, we cultured SMCs onto Tn-C–coated dishes in defined media for 24 hours. Cells plated onto fibronectin-coated dishes in defined media were used as control. Consistent with our previous report,17 we noted that SMCs adhered to Tn-C substrate; however, cells remained round and did not spread within 1 hour after plating, in contrast to cells cultured onto fibronectin-coated dishes (not shown). Examination of cells 24 hours after plating showed that most cells were detached from the Tn-C–coated dishes and those that remain attached assumed morphology characteristic of apoptotic cells (Figure IA, bottom, available online at http://atvb.ahajournals.org). In contrast, cells cultured onto fibronectin-coated dishes did not exhibit apoptotic morphology (Figure IA, top), suggesting that apoptosis of cells is related to Tn-C substratum. Because we noted that under these culture conditions Tn-C is degraded and that MMP inhibitor blocked fragmentation, we asked whether cell apoptosis is related to Tn-C degradation. To answer this question, we cultured cells onto Tn-C–coated dished in defined media in the presence and absence of GM6001 MMP inhibitor and cell apoptosis was measured by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining. In the absence of inhibitor, cells showed condensed red nuclei (Figure IB, bottom); however, in the presence of inhibitor, cells were fully spread and showed expanded gray nuclei, indicating that they are viable (Figure IB, top). These data suggest that SMC apoptosis is related to degradation of Tn-C, most likely by MMPs. To determine which domain of Tn-C mediates the apoptotic activity of Tn-C, we preformed a protein pull-down assay by incubating the conditioned media collected from culture cells that were plated onto Tn-C–coated dishes in defined media for 24 hours. The media were collected, incubated with antibodies to intact Tn-C, fibrinogen-like, fibronectin-like, or EGF-like domains of Tn-C, and then added to SMCs cultured onto fibronectin-coated dishes. Pre-incubation of media with intact Tn-C (Figure IC, lane Tn-Cg), anti-fibronectin-like (Figure IC, lane Fn-L) or antifibrinogen-like domain (Figure IC, lane Fbg-L) antibodies did not influence the proapoptotic activity of the conditioned media. In contrast, pre-incubation of media with anti-EGF-like antibodies (Figure IC, lane EGF-like) neutralized the proapoptotic activity of the conditioned media, suggesting that the apoptotic activity is a soluble molecule related to the EGF-like domain of Tn-C. Although there are extensive data about the activity of intact Tn-C and its fibronectin-like and fibrinogen-like domains, little is known about the activity of its EGF-like domain. The full-length EGF-like domain of Tn-C consists of 447 amino acids, with 15.5 repeats of a 31 amino acid EGF-like domain. The EGF-like repeats share 6 cysteine residues, which are linked together by a special configuration of disulfide bonds.23 The presence of 87 cysteine residues and nonsequential arrangement of the disulfide bonds excludes the use of an E. coli system for the expression of the EGF-like domain because of lack of disulfide isomerase activity. Therefore, we used yeast to express this domain of Tn-C. To determine the activity of recombinant EGF-like domains, a 0.1-µmol/L solution of intact long Tn-C isoform, recombinant fibronectin-like, fibrinogen-like, or EGF-like domain was added to fibronectin-coated cultured SMCs in the absence or presence of 1 nM PDGF-BB in serum-free media. As expected, in the absence of PDGF-BB, there was no DNA synthesis. However, DNA synthesis was stimulated in the presence of PDGF-BB and Tn-C domains, with the exception of the EGF-like domain (Figure IIA, available online at http://atvb.ahajournals.org). The cultured SMCs in the presence of recombinant EGF-like domain exhibited apoptotic morphology, suggesting that inhibition of DNA synthesis is related to cell apoptosis. To further investigate this idea, cells were analyzed for annexin V externalization. Cells were cultured onto fibronectin-coated dishes in defined media in the presence of recombinant EGF-like domain for 24 hours. Cells treated with the actinomycin D under similar condition were used as a positive control. Fluorescence-activated-cell sorter analysis revealed that 78±7% of cells treated with EGF-like underwent apoptosis (Figure IIB). Under similar conditions, 65±8% of cells treated with actinomycin D underwent apoptosis (Figure IIB). Untreated cells remained viable. Because activation of caspases is central to cell apoptosis, SMCs were treated with the recombinant EGF-like domain in the presence of increasing concentrations of z-VAD.fmk, a widely used caspase inhibitor, and cell viability was measured by MTT assay. Unexpectedly, this inhibitor was toxic to the cells; however, other caspase inhibitors, z-DEVD.fmk or z-YVAD.cmk, were not toxic. The most effective caspase inhibitor was z-YVAD.cmk, which at 100 µmol/L reduced EGF-like domain-induced cell death by 50% (Figure IIIA, available online at http://atvb.ahajournals.org). Increasing concentrations of the inhibitor to 200 to 400 µmol/L completely blocked cell death (data not shown). To further confirm the involvement of caspases, we measured activation of caspase-3 by Western blot in both attached and detached SMCs. The activation of caspase-3, as measured by intensity of the 11-kDa caspase fragment, was weakly detectable 10 hours after treatment that significantly increased at 24 hours in the attached SMCs (Figure IIIB). In detached cells, activation of caspase-3 was observed at 24 to 48 hours of treatment with EGF-like domain, which was suppressed by z-YVAD.cmk in a concentration- dependent manner. Addition of 100 µmol/L of the inhibitor reduced activation of caspase-3, whereas 300 µmol/L completely suppressed it (Figure IIIB). In addition, PARP, a caspase-3 substrate, was also cleaved in a time-dependent manner (data not shown). Collectively, we conclude that the addition of recombinant EGF-like domain to cultured SMCs activates caspase-3 before cell detachment. Because we previously reported that SMC viability is dependent on its interaction with extracellular matrix proteins and that serum factors prevent cell death by inhibiting matrix degradation,19 we asked whether the apoptotic activity of the recombinant EGF-like domain is a protease-dependent event. Serum blocked the proapoptotic activity of the recombinant EGF-like domain (data not shown). To identify the nature of this inhibition, SMCs were treated with the recombinant EGF-like domain in defined media containing various protease inhibitors. We noted that a 0.32-µg/mL solution of
-macroglobulin significantly reduced and a 0.6-µg/mL solution completely blocked EGF-L–induced cell apoptosis (Figure 4). Chymostatin, a chymotrypsin inhibitor, at a concentration of 3 µmol/L blocked the apoptotic activity of EGF-like domain (Figure 4). In contrast, trypsin inhibitor, plasmin inhibitor, and thrombin inhibitor had no effect (data not shown). These data suggest that the proapoptotic activity of EGF-L domain is mediated by a protease. To further understand the role of EGF-L domain in SMC apoptosis, we measured caspase-3 activation in the presence of protease inhibitors. We found that 1 µmol/L chymostatin significantly reduced and 4 µmol/L completely suppressed caspase-3 activation induced by the EGF-like domain (Figure 5). In contrast, hirudin, a thrombin inhibitor (1 U/mL) and plasmin inhibitor (15 µg/mL) had no effect (Figure 5). To further examine whether the inhibitory activity of chymostatin is specific to apoptosis caused by EGF-like domain, SMC death was induced by staurosporine and actinomycin D in the absence and presence of 4 µmol/L chymostatin. Although the proapoptotic reagents activated caspase-3, chymostatin did not block the activation (Figure 5). From these data, we conclude that protease inhibitors blocked the EGF-like domain activity through inhibition of caspase-3 activation.
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Discussion |
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It is believed that destruction of matrix protein by matrix-degrading enzymes leads to apoptosis of adherent cells caused by deprivation of cell attachment. In this report, we described another mechanism by which matrix-degrading enzymes may induce cell apoptosis. We found that culturing of SMCs onto Tn-C substrate, in the absence of serum, leads to fragmentation of Tn-C. Tn-C fragmentation was also detected in some human atherosclerotic plaques. Based on the motilities of Tn-C fragments on SDS-PAGE, Western blot data, and pull-down assays, it appears that proteolysis of Tn-C leads to generation of fragments that contain the EGF-L domain. Proteolysis of Tn-C in cultured SMCs and in atherosclerotic plaques was not surprising because others have noted that decorin, biglycan, versican, apoB, apo(a), and apoE undergo proteolysis in human endarterectomy specimens.22 Although the significance of this fragmentation is unknown, past studies have shown that MMP-2 cleaves decorin in a region that is responsible for transforming growth factor-ß binding, thus increasing availability of this growth factor in an active form.24 Because decorin interaction with collagen is critical to fibrillogenesis, proteolysis of decorin may affect tissue integrity and maintenance. Thus, it appears that cells are exposed to matrix fragments, in addition to intact protein. Therefore, it is important to assess the fragmentation of matrix molecules and activity of their fragments.
We found that Tn-C upregulates MMP expression and that MMP inhibitor blocked Tn-C fragmentation, suggesting an autocrine loop may be involved in the degradation of Tn-C in both SMCs and macrophages. Upregulation of MMPs appears to be a common activity of Tn-C, because others have noted that a mixed substrate of Tn-C and fibronectin upregulates collagenase, stromelysin, and gelatinase gene expression in rabbit fibroblast.25 Because Tn-C is sensitive to degradation by MMP-1, MMP-2, MMP-3, and MMP-7,20,21 it is likely that Tn-C undergoes degradation in inflamed tissues and cells are exposed to Tn-C fragments in addition to the intact protein. These fragments may exhibit an activity that is not found in the intact molecule. For example, cleavage of the laminin-5 
2 chain by MMP-2 exposes a cryptic site within laminin, inducing migration of malignant breast epithelial cells, an activity not found in the intact protein.26 Similarly, we found that the EGF-L domain induces SMC apoptosis, an activity not exhibited by the intact molecule. Taken together, these data suggest that local proteinase concentration and matrix degradation determine cell behavior in multiple ways.
We have previously reported that pericellular proteolysis of matrix proteins induces SMCs apoptosis.12 However, the EGF-L–induced apoptosis described in this article is different from anoikis, in which denying cell adhesion leads to cell apoptosis. We previously reported that SMCs adhere to Tn-C and determined that the fibrinogen-like domain of Tn-C accounts for this adhesive activity.17 However, we noted that in the absence of serum, this adhesion did not lead to cell spreading; rather, adherent cells remained round and underwent apoptosis. We have now discovered that apoptosis can be blocked by the addition of serum and protease inhibitors (such as -macroglobulin or chymostatin), suggesting that a protease may be involved in this process. We further identified that a degradation product of Tn-C that contains its EGF-L domain has proapoptotic activity. Thus, our data suggest that degradation of Tn-C and generation of EGF-L fragments are linked to SMCs apoptosis, an event that is independent of interference with cell adhesion.
Although our data cannot rule out other mechanisms promoting SMC death, it is important to realize that very little is known of the factors that control the loss of these cells from the human plaque. The only specific hypothesis has been that apoptosis in atherosclerotic plaques is caused by the expression of Fas and its ligand, FasL.27 However, experimental literature does not provide strong support for this hypothesis. For example, in vivo studies have demonstrated that intimal lesions grow more rapidly in FasL-transduced arteries, which is primarily caused by the accumulation of intimal SMCs with no significant apoptosis.28 Cell culture studies have also shown no correlation between Fas/FasL expression and SMC apoptosis. Cultured human SMCs express relatively high levels of Fas/FasL and remain viable in the presence of soluble FasL or agonist anti-Fas antibody.29 SMCs are insensitive to apoptosis induced by Fas ligand27 and must be pretreated with interferon- (INF-) to become sensitive to apoptosis by anti-Fas antibody. It has also been proposed that IFN- pretreatment upregulates Fas, leading to the induction of apoptosis.27 However, neither tumor necrosis factor- nor IFN- upregulates Fas in human SMCs.29 In addition, media collected from activated T-cell clones isolated from atherosclerotic plaques had no proapoptotic activity despite strong expression of proinflammatory cytokines such as IFN-.30,31 Taken together, these data suggest that the increased expression of Fas/FasL in atherosclerotic plaques or cultured cells may not be related to SMC apoptosis.
In summary, we provide evidence that Tn-C upregulates MMP expression in cultured SMCs, which leads to Tn-C fragmentation and cell apoptosis. These findings are potentially important because advanced plaques contain SMCs, macrophages, T cells, and mast cells, which are cell types that are capable of expressing MMPs and inducing apoptosis of SMCs through fragmentation of Tn-C. Because proteolysis is controlled at a multitude of levels, this pathway of apoptosis would increase the complexity of cell death in the vasculature.
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Acknowledgments |
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Supported by National Institutes of Health grant HL50566, Established Investigator Award 0040201N from the American Heart Association, and the Eisner Foundation.
Received March 4, 2004; accepted May 12, 2004.
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 A. Della Corte, C. Quarto, C. Bancone, C. Castaldo, F. Di Meglio, D. Nurzynska, L. S. De Santo, M. De Feo, M. Scardone, S. Montagnani, et al. Spatiotemporal patterns of smooth muscle cell changes in ascending aortic dilatation with bicuspid and tricuspid aortic valve stenosis: Focus on cell matrix signaling J. Thorac. Cardiovasc. Surg., January 1, 2008; 135(1): 8 - 18. [Abstract] [Full Text] [PDF]
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