Thrombin Stimulates Syndecan-1 Promotor Activity and Expression of a Form of Syndecan-1 That Binds Antithrombin III in Vascular Smooth Muscle Cells
Abstract Vascular smooth muscle (VSM) cells express transmembrane proteoglycans of the syndecan gene family. We reported previously that the expression of syndecans by VSM cells is regulated by mitogens such as serum, platelet-derived growth factor, and basic fibroblast growth factor and that syndecan expression is induced after balloon injury in vivo. We now show that thrombin is a potent inducer of syndecan-1 expression in VSM cells. Transient transfection experiments with a rat syndecan-1 promoter construct demonstrated that thrombin stimulates transcription of the syndecan-1 gene. Syndecan expression in response to thrombin was not inhibited by downregulation of protein kinase C. Thrombin-induced syndecan-1 expression was dependent on tyrosine kinase activity. Calcium was necessary for syndecan-1 expression, but increasing the intracellular calcium levels was not sufficient to induce syndecan-1 expression. Analysis of antithrombin III (AT III) binding activity revealed that thrombin caused an increase in the synthesis of syndecan-1 molecules that exhibited high-affinity AT III binding. These results suggest that VSM cells could play an important role in controlling local thrombus formation subsequent to vascular injury, via a feedback mechanism that involves thrombin-induced stimulation of an inhibitor of thrombin activity.
- Received March 17, 1997.
- Accepted August 7, 1997.
Syndecans are a family of transmembrane cell surface proteoglycans that are expressed in highly regulated cell and development specific patterns. Syndecans have been implicated in a number of important processes, including cell-cell and cell-extracellular matrix adhesion, growth factor binding, and hemostasis, by virtue of their ability to bind extracellular ligands via their covalently attached heparan sulfate chains.1,2
The vascular wall is rich in proteoglycans.3,4 Syndecans are expressed in all three layers of the vascular wall.5,6 We have shown previously that vascular smooth muscle (VSM) cells express the mRNAs for all four mammalian syndecans and that the expression of individual syndecan types in VSM cells is differentially regulated by mitogens.7 In general, these studies have shown that VSM cells express syndecan-2 constitutively but that expression of syndecan-1 and syndecan-4 is induced by exposure to mitogens such as serum, PDGF, or bFGF. Expression of syndecan-1 and syndecan-4 is also induced in vivo following balloon catheter injury of rat carotid arteries.8
One consequence of injury to a tissue is the formation of thrombin. Thrombin is a multifunctional serine protease that can activate signaling pathways in a variety of cells, including VSM cells, by binding to plasma membrane receptors that are coupled to heterotrimeric G proteins.9 Thrombin stimulation has been shown to cause a variety of cellular responses, including an increase in intracellular calcium, a stimulation of protein synthesis, and induction of expression of c-fos.10 In addition to being a ligand for signaling receptors, thrombin is also a critical component of the blood coagulation cascade. Generation of proteolytically active thrombin is a rate-limiting step in thrombus formation. For this reason, organisms have evolved complex mechanisms for regulating thrombin activity and localizing coagulation to appropriate sites. One of the mechanisms for inhibiting thrombin activity is the formation of a complex between thrombin and antithrombin III (AT III) in a manner that is strictly dependent on the binding of heparin or structurally similar molecules to AT III.11 The high-affinity binding of heparin and related compounds to AT III is dependent on a specific pattern of modification of the polysaccharide structure that occurs only rarely even in heparin. It has been shown that heparan sulfate chains on membrane-associated proteoglycans can be modified structurally in such a way as to be able to carry out high-affinity binding to AT III but that this modification is a relatively rare event. For example, previous work has demonstrated that 2% to 5% of syndecan molecules synthesize by endothelial cells exhibit high-affinity binding to AT III.12 Thus, endothelial cell membrane HSPGs have the potential to be able to bind and activate AT III and, as a consequence, inhibit coagulation in the local environment.
In this study, we have examined the effects of thrombin treatment on syndecan expression by VSM cells. These results presented here demonstrate that thrombin induces a significant increase in the expression of syndecan-1, including an increase in the amount that binds with high-affinity to AT III. These results suggest that VSM cells play an important role in regulating thrombus formation after vascular injury and provide evidence for a feedback mechanism that involves thrombin-induced stimulation of an inhibitor of thrombin activity.
Cell Culture and Reagents
VSM cells were obtained from medial strips dissected from aortas of male Sprague-Dawley rats and cultured in Dulbecco’s Modified Eagle’s (DME) medium containing 10% fetal calf serum (FCS) as described previously.5 Cells were grown to 70% to 80% confluence and then made quiescent in DME medium containing 0.4% FCS for 48 hours. Cells were then switched to fresh medium containing 0.4% FCS or 0.4% FCS plus bovine thrombin (15 to 30 nmol/L) (Sigma Chemical Company) as indicated in the Results section. Cycloheximide (Sigma Chemical Company) was used to inhibit protein synthesis at a concentration of 10 μg/mL. In some experiments, PDGF-AB (Upstate Biotechnology, Inc) was used to stimulate syndecan expression at a concentration of 10 ng/mL. Media and FCS were purchased from Life Technologies, Inc. PMA (phorbol 12-myristate 13-acetate), Herbimycin A (HA) ionomycin, and BAPTA-AM [1, 2-bis-(2-aminophenoxy)ethane-N1 N1 N1 N1- tetraacetic acid tetra-(acetoxymethyl) ester] were purchased from Sigma, and stock solutions were prepared in DMSO at a concentration of 0.05%.
Northern Blot Analysis
Total RNA was extracted from the cultured cells using Ultraspec RNAzol (Cinna Biotech) as described previously.7 Twenty milligrams of total RNA from each sample was separated by electrophoresis in agarose/formaldehyde gels, transferred to nylon membranes, and hybridized to random primed 32P-labeled cDNA probes, as described previously. The preparation of cDNAs for rat syndecan-1,5 syndecan-2,7 and syndecan-48 has been described previously. c-fos cDNA was obtained from American Type Culture Collection. Quantitation of the hybridization signals was done by scanning exposed x-ray films with a laser densitometer or by phosphorimager analysis (Molecular Dynamics). Signals were normalized to the quantity of 18S rRNA in the samples as described previously.7
Isolation of Rat Syndecan-1 Promoter
The sequence of the mouse syndecan-1 promoter was reported previously.13, 14 The mouse gene sequence was used to design oligonucleotides that were used to isolate a 667-bp upstream fragment from the rat syndecan-1 gene that contained the transcription start sites, TATA and CAAT boxes, and potential transcription factor–binding sites. Rat liver DNA was used as template. The sense and antisense primers were GCT CTT CCA GAC AGT GCT CA, and CTG CGT TAG GCT CTG TCT CC. PCR conditions were 94°C for 1 minute, 57°C for 2 minutes, and 72°C for 4 minutes for 40 cycles. The resulting 667-bp PCR product was gel purified and sequenced and was subcloned into the chloramphenicol acetyl transferase (CAT) reporter vector (Promega) to produce plasmid p syn-1-CAT.
Transient Transfection of VSM Cells
VSM cells at 70% to 80% confluency were transfected with 6 μg of p syn-1-CAT plasmid DNA and 20 μL of lipofectamine (Gibco-BRL) per 60-mm dish for 3 hours. Cells were cotransfected with pPGL3 reporter vector (Promega) containing luciferase coding sequences under transcriptional control of the SV40 enhancer and promoter to normalize for transfection efficiency. After overnight incubation in 10% FCS, cells were made quiescent in 0.4% FCS for 48 hours. The cells were then stimulated with thrombin or serum. After 8 hours, the cells were harvested and resuspended in 100 μL of 0.25 mol/L Tris-HCl pH 8.0 per 60-mm dish. The cell extracts were lysed by three freeze-thaw cycles, and CAT activity was assayed in the presence of [14C]chloramphenicol and n-butyl CoA according to the manufacturer’s protocol. Radioactivity in the organic phase was counted by liquid scintillation spectroscopy and corrected for the background value obtained by using a control sample with no cells. Transfection efficiencies were determined by assaying luciferase gene activity in the same extracts.
Purification of Proteoglycans
VSM cells were incubated for 18 to 24 hours in medium containing 35SO4 to label the proteoglycans15 and then for an additional 48 hours in medium containing 35SO4 in the absence or presence of 30 nM of thrombin. The conditioned culture medium was collected. The cells were rinsed with phosphate-buffered saline (PBS, 50 mmol/L sodium phosphate, 150 mmol/L NaCl, pH 7.5) and extracted by scraping in 2 mL/100 mm plate of 50 mmol/L Tris-HCl, pH 7.5, 0.5% Nonidet P40 (United States Biochemical Corporation, Cleveland, Ohio), 0.5% deoxycholate, 0.1% sodium dodecyl sulfate. PMSF (final concentration, 1 μmol/L) was added to inhibit protein degradation. DEAE-Sephacel, equilibrated with PBS, 0.5% NP-40, was added to both the medium and the cell extract samples (2-mL sample). Proteins were bound to the matrix overnight with gentle rocking at 4°C. After overnight binding, the samples were centrifuged for 5 minutes at 200g to pellet the DEAE-Sephacel. The supernatant, containing the unbound fraction, was removed. The pellets were washed twice for two hours at 4°C with gentle rocking with PBS, 0.5% NP-40 followed by one wash with PBS, 0.5% NP-40 plus 0.15 mol/L NaCl. The tightly bound proteins (including proteoglycans) were eluted by incubating in PBS, 0.5% NP-40 plus 1 mol/L NaCl. All fractions were dialyzed overnight against 6 L of 50- mmol/L Tris-HCl, pH 7.4, 1.0 mmol/L EDTA at 4°C.
Antithrombin III–Binding Assay
The method that was used was a slight modification of a previously described assay.12 Antithrombin III agarose beads (Sigma Chemical Company) were equilibrated with column buffer (50 mmol/L Tris-HCl, 1 mmol/L EDTA, 30 mmol/L octylglucoside, pH 7.5) overnight at 4°C with gentle rocking. The purified proteoglycan fractions were bound to the antithrombin III agarose overnight at 4°C. The loading was equalized by applying equal quantities of 35SO4-labeled proteoglycans from control and thrombin-treated media and extract samples. The unbound proteins were removed from the beads by centrifugation for 5 minutes. The beads were washed twice with column buffer containing 4 μmol/L of dextran sulfate and 0.15 mol/L of NaCl. A step gradient of 0.15 mol/L, 0.50 mol/L, and 1.0 mol/L NaCl was used to elute the bound proteins. Each step elution was carried out for 2 hours with gentle agitation at 4°C using 2.5 mL of buffer. All samples were dialyzed overnight against 6 L of 50 mmol/L Tris-HCl, pH 7.5, at 4°C before further analysis.
DEAE-Sephacel and AT III-agarose-purified samples were analyzed by immunoblotting. Samples were electrophoresed through 3.9% to 15% polyacrylamide gradient gels, transferred to Immobilon, and incubated with anti-syndecan-1 antibody as described previously. Bound antibodies were detected by Enhanced Chemiluminescence (ECL) (Amersham Life Science, Buckinghamshire, England) according to the manufacturer’s protocol.
VSM cells were grown in eight-well glass chamber slides (Nunc, Naperville, Illinois) at an initial density of 250 000 cells/well. The cells were cultured in DME-10% FCS to 60% to 70% confluency. On reaching this density, the cells were made quiescent by incubation for 48 hours in DME-0.4% FCS. The cultures were subsequently stimulated with 30 nmol/L of thrombin for 48 hours. Control wells were treated with fresh DME-0.4% FCS without thrombin. Indirect immunofluorescence was carried out as follows: The culture medium was removed, and the cells were rinsed with PBS. The cells were fixed with methanol at −20°C for 15 minutes. After fixation, the cells were rinsed with PBS three times for 15 minutes. Affinity-purified anti-syndecan-1 antibodies5 diluted in 50 mmol/L Tris-HCl, 100 mmol/L NaCl, pH 7.4, 5.0% nonfat dry milk (Blotto) were added, and the cells were incubated for 1 hour at room temperature. After two Blotto rinses, the fluorescein-conjugated secondary antibodies were added, and the cultures were incubated for 30 minutes at room temperature in the dark. Slides were subsequently rinsed and mounted with a glass coverslip.
Thrombin Induces Syndecan-1 and Syndecan-4 Expression in VSM Cells
Rat aortic VSM cells were made quiescent by incubation in medium containing 0.4% FCS for 48 hours. The cells were treated with thrombin, and total RNA was extracted as described in the Materials and Methods section. Expression of syndecan mRNAs was analyzed by Northern blot analysis. As shown in Fig 1⇓, thrombin caused a large increase in syndecan-1 mRNA levels. In contrast, thrombin resulted in a decrease in syndecan-2 mRNA levels and caused a modest increase in syndecan-4 mRNA levels (see below). The effect of thrombin appeared not to result from its proteolytic activity, since treatment with plasmin, another serine protease, had no effect on syndecan mRNA expression (Fig 1⇓).
Increased syndecan-1 mRNA levels correlated with increased proteoglycan expression in the cells. Proteoglycans were extracted from the cells and analyzed by immunoblotting with affinity purified anti-syndecan-1 antibodies. As shown in Fig 2⇓, thrombin-treated cells contained more syndecan-1 than did quiescent untreated cells. In contrast, there was no apparent increase in total proteoglycan synthesis, as determined by 35SO4 incorporation into high-molecular-weight polypeptides. The increase in the steady-state level of syndecan-1 was also demonstrated by immunofluorescent staining of control and thrombin treated VSM cells (Fig 2B⇓).
Thrombin Treatment Increases the Synthesis of Anticoagulant Syndecan-1 by VSM Cells
We examined the AT III binding activity of syndecan-1 molecules synthesized by control and thrombin treated VSM cells. Proteins released into the culture medium and present in extracts of the cells were subjected to affinity chromatography on a matrix of immobilized AT III. Nonbinding and weakly binding proteins were eluted from the column by washing with buffer containing dextran sulfate. Proteins that remained bound in the presence of dextran sulfate but were eluted by buffer containing 0.5 mol/L of NaCl were considered to exhibit high-affinity AT III binding. Syndecan-1 molecules were visualized by immunoblot analysis. As shown in Fig 3⇓, a minority population of syndecan-1 molecules synthesized by VSM cell bound with high affinity to AT III. After thrombin treatment, however, the culture medium and cell extract of thrombin-treated cells contained significantly more syndecan-1 molecules that exhibited high-affinity binding to AT III. In contrast, the proportion of total 35SO4-labeled proteoglycans that bound with high affinity to AT III was not increased significantly by thrombin treatment of VSM cells. Thus, thrombin treatment causes a selective increase in expression of syndecan-1 molecules that exhibit high-affinity AT III–binding activity.
Thrombin-Stimulated Production of Syndecan-1 mRNA Expression Requires New Protein Expression
Previously, we reported that serum and bFGF stimulated syndecan-1 and syndecan-4 expression in VSM cells and that the syndecan-4 response but not the syndecan-1 response exhibited characteristics of an early response gene.8 As shown in Fig 4⇓, induction of syndecan-1 mRNA by thrombin was completely inhibited by treatment of the cells with cycloheximide. In contrast, syndecan-4 mRNA expression induced by thrombin was increased by the inhibition of protein synthesis. The effect of thrombin and cycloheximide on the expression of c-fos, an immediate early gene product, is also shown in Fig 4⇓. Similar to syndecan-4 mRNA expression, c-fos mRNA levels were increased by cycloheximide treatment. However, the temporal patterns of c-fos and syndecan-4 mRNA expression were distinct, the increase in syndecan-4 mRNA occurring only after a lag period of 1 to 2 hours. The effects of thrombin on syndecan-1 and syndecan-4 mRNA expression are thus qualitatively similar to effects of serum and mitogens described previously.
Thrombin Increases Syndecan-1 Gene Transcription
To determine whether the increase in syndecan-1 mRNA levels in response to thrombin treatment resulted from an increase in the rate of transcription of the gene, we isolated a 0.7-kb segment of the rat syndecan-1 gene upstream region and generated a CAT reporter construct containing this fragment (see the “Methods” section). This portion of the mouse syndecan-1 gene was shown to contain the core promoter element as well as numerous potential cis-acting regulatory sites.13, 14 The rat sequence was found to be 89% identical to the mouse sequence within this region (Fig 5⇓). The effect of thrombin treatment on promoter activity was determined by analysis of CAT activity in VSM cells that were transiently transfected with the syndecan-1 reporter construct. As shown in Fig 6⇓, thrombin treatment resulted in an approximately 2.5-fold increase in syndecan-1 promoter activity. Treatment with 10% FCS, which also induces syndecan-1 mRNA expression, caused a nearly 4-fold increase. Transcription from a construct that lacked the syndecan-1 upstream sequence was minimal. These results demonstrate that the thrombin-dependent increase in syndecan-1 mRNA expression is caused by increased transcription of the syndecan-1 gene.
Thrombin Signaling Pathways and Syndecan-1 Expression
Thrombin has been shown to activate a number of signaling pathways in various cells. We carried out experiments to identify the pathways by which thrombin activates syndecan-1 expression in VSM cells. To determine whether the effect of thrombin on syndecan-1 expression is dependent on activation of tyrosine kinase activity, VSM cells were treated with an inhibitor of tyrosine kinases. Cells pretreated with HA for 24 hours were stimulated with thrombin, and syndecan expression was measured by Northern blot analysis. As shown in Fig 7⇓, HA inhibited the thrombin-stimulated increase in syndecan-1 mRNA in a dose-dependent manner. Thus, these results demonstrate an essential role for tyrosine kinase activity in thrombin-dependent syndecan-1 expression.
Thrombin has also been reported to exert some of its effects on cells by modulating levels of intracellular calcium. To determine whether changes in intracellular calcium levels were involved in thrombin-stimulated syndecan-1 expression, VSM cells were treated with thrombin in the presence of ionomycin, a calcium ionophore, or BAPTA-AM, a calcium chelator. As shown in Fig 8⇓, ionomycin treatment did not induce syndecan-1 mRNA expression in the absence of thrombin. Ionomycin produced a moderate inhibition of syndecan-1 mRNA induction by thrombin. Treatment of cells with BAPTA-AM caused both a depression of basal expression of syndecan-1 and a complete inhibition of the thrombin-dependent increase in syndecan-1 mRNA. These results demonstrate that calcium is required for syndecan-1 mRNA expression but that changes in intracellular calcium concentrations alone are not sufficient to induce syndecan expression.
Activation of protein kinase C has been shown to be a point of convergence for integrating signals from receptor tyrosine kinases and G-protein linked receptors. To determine whether protein kinase C was involved in thrombin-dependent expression of syndecan-1, VSM cells were treated with PMA for 24 hours and then stimulated with thrombin. Syndecan-1 mRNA levels were determined by Northern blot analysis. Prolonged treatment of cells with PMA causes a downregulation of protein kinase C activity. As shown in Fig 9⇓, PMA treatment did not block the ability of thrombin to stimulate syndecan-1 expression. There was, in contrast, a modest enhancement of syndecan-1 expression in response to thrombin. This result suggests that protein kinase C might have an inhibitory effect on thrombin-induced syndecan-1 mRNA expression. We have shown previously that syndecan-1 expression is induced in VSM cells by PDGF and serum. As shown in Fig 9⇓, serum-induced syndecan-1 mRNA expression was inhibited by PMA, whereas PDGF induced expression was not affected.
VSM cells are the main cellular component of the normal arterial wall and play a major role in several vascular diseases. Under normal conditions, these cells are quiescent and form a distinct muscular layer. Injury to the blood vessel wall results in the exposure of the VSM cells to several mitogens at the injury site. Here we have demonstrated that one of these mitogens, thrombin, induces the expression of the cell surface heparan sulfate proteoglycan syndecan-1 in VSM cells.
Syndecan expression has been shown to be cell-type and tissue-type specific. This is the first report to demonstrate that thrombin increases syndecan-1 expression in VSM cells. The effect of thrombin appeared to be at the level of transcription but required the synthesis of new proteins. The increase in transcription may be due to activation of a thrombin-induced transcription factor(s) or activation of existing factors in response to mitogens released by thrombin-treated cells. Recently, a thrombin response element was identified in the PDGF-B chain promoter,16 as well as a thrombin-induced nuclear factor (TINF) that binds to this site. Interestingly, TINF appears to bind to the same site on the DNA as SP1, although the two transcription factors are not related. The rat syndecan-1 promoter sequence has four consensus thrombin response elements within the 667-bp fragment that confers thrombin-induces transcription in VSM cells. We are in the process of identifying the specific regions that are responsible for thrombin-induced syndecan-1 expression in VSM cells. Our studies also revealed that serum treatment induces syndecan-1 transcription in quiescent VSM cells. A similar response was reported previously in murine mammary gland cells and fibroblasts.17
Syndecan-4 expression is also induced by thrombin, but, in contrast to syndecan-1, this appears to be a direct effect of thrombin stimulation and does not require the synthesis of new proteins. In this respect, syndecan-4 can be considered to be a primary response gene, whereas syndecan-1 can be classified as a secondary response gene product.18 This is similar to what we observed in an earlier study of bFGF and serum-treated VSM cells.8 Therefore, this is likely to be a property of the mechanisms of regulation transcription of these syndecan genes rather than a characteristic of thrombin-induced gene expression.
Our findings related to thrombin-induced syndecan expression are similar to what has been observed for thrombin-induced mitogenesis in VSM cells. VSM cells express G protein–linked thrombin receptors19 that can initiate cellular signaling events such as phosphoinositide hydrolysis.20 On the other hand, it has been shown that thrombin-induced mitogenesis in VSM cells can be inhibited by inhibition of tyrosine kinase activity. In our experiments, a similar inhibition of thrombin-stimulated syndecan-1 expression by a tyrosine kinase inhibitor was found in VSM cells. A possible explanation for this effect is that thrombin stimulates the release of growth factors such as PDGF21 and bFGF22 from the cells. The receptors for these growth factors have intrinsic tyrosine kinase activities that are essential for their activity. Nonreceptor kinases may also be involved. In thrombin-treated platelets, a nonreceptor tyrosine kinase, p72 syk, was found to be translocated to the cytoskeleton and activated during platelet activation.23 In VSM cells, basal syndecan-1 mRNA and protein expression is minimal, so it cannot be detected by Western blot, either in the cell or in the medium. However, we are able to show the presence of the proteoglycan expression in the medium of the cultured cells that are treated with thrombin. One possibility would be the protease effect of thrombin on the cells that can release the cell surface proteins. To confirm that this is not the case, we used a cell line (Schwann cells) that does not express syndecan-1. However, Schwann cells that are stably transfected with syndecan-1 express syndecan-1 protein at the cell surface.24 When we treated these cells with thrombin in the same experimental conditions that we used for VSM cells, we did not detect any change in the cell surface expression, a finding that also confirms that thrombin-induced syndecan-1 expression in VSM cells is not due to its protease effect (data not shown).
Thrombin has also been shown to induce release of calcium from intracellular stores by a mechanism that is independent of tyrosine phosphorylation.25 Thrombin-induced syndecan-1 expression required the presence of Ca+2 ions. However Ca+2 ionophores alone did not induce syndecan-1 expression. Similar results were reported for thrombin-induced c-fos expression in VSM cells.26
Syndecans have been shown to bind a number of extracellular ligands. Thus, the functional consequence of thrombin-induced syndecan-1 expression may be complex. Our results demonstrate that syndecan-1 expressed by VSM cells has anticoagulant activity based on high-affinity binding to AT III. This is similar to what was shown previously for endothelial cell syndecans.12 Previous studies with heparin showed that the presence of a unique carbohydrate structure that includes 2-O, 3-O, and 6-O sulfates was required for AT III binding to heparin4,27,28 and that 3-O-sulfated glucosamine was essential for AT III binding. This is the first report of VSM cell syndecan-1 as an anticoagulant molecule.
We can conclude from our studies that syndecans isolated from VSM cells have AT III–binding activity. Thus, the most critical component of the coagulation cascade, thrombin, regulates the expression of an anticoagulant proteoglycan. These results suggest that a function of VSM cell syndecan-1 is to serve as a membrane-based regulator of local coagulation in the vascular wall. This may be especially important after vascular injury, in which the endothelium is absent. Thus, these data provide evidence for a negative feedback loop wherein thrombin, a procoagulant, stimulates the expression of proteins at the surface of VSM cells, which generate anticoagulant activity through high-affinity binding of AT III.
This work was supported by NIH Grant HL-48740 (to D.J.C.) and a Grant-In-Aid from the American Heart Association, PA Affiliate, (to G.C.S.). We thank Linda Dearman for help in manuscript preparation.
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