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(Circulation Research. 1997;81:17-23.)
© 1997 American Heart Association, Inc.

Articles

Diverse Effects of Heparin on Mitogen-Activated Protein Kinase–Dependent Signal Transduction in Vascular Smooth Muscle Cells

Günter Daum, Ulf Hedin, Yunxia Wang, Trevina Wang, , Alexander W. Clowes

From the Department of Surgery, University of Washington, Seattle.

Correspondence to Dr Günter Daum, Department of Surgery, Box 356410, University of Washington, Seattle, WA 98195.

	Abstract

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Abstract Proliferation of vascular smooth muscle cells (SMCs) is implicated in pathological events, including atherosclerosis and intimal hyperplasia following angioplasty. The glycosaminoglycan heparin is a growth inhibitor of SMCs in vitro and in vivo. The underlying mechanism, however, is still poorly understood. In the present study, we report that heparin inhibited the activation of the mitogen-activated protein kinase (MAPK) in baboon SMCs by serum but not by platelet-derived growth factor (PDGF). When fibroblast growth factor was used, heparin had a stimulatory effect on MAPK. The only MAPK-activating kinase found in SMCs was MAPK kinase (MAPKK)-1, although MAPKK-2 was present in comparable amounts. Activation of MAPKK-1 and DNA synthesis were affected by heparin in a similar fashion. Heparin does not appear to exert its effects through members of the protein kinase C family, which are downregulated by phorbol esters, because it was still capable of inhibiting MAPK/MAPKK-1 stimulation by FCS in phorbol ester–pretreated cells. The present findings support the conclusions that the effects of heparin depend on the nature of the mitogen and that heparin inhibits SMC proliferation by preventing activation of MAPKK-1.

Key Words: atherosclerosis • restenosis • proliferation • heparin • mitogen-activated protein kinase

	Introduction

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Excessive growth of SMCs contributes to the pathogenesis of atherosclerosis and plays an important role in the development of a neointima and luminal narrowing in diseased vessels treated by angioplasty or surgery (reviewed in References 1 and 2^{1 2} ). The glycosaminoglycan heparin inhibits growth and migration of cultured SMCs (reviewed in References 3 and 4^{3 4} ) and prevents the formation of neointima following vascular injury in rats⁵ but fails to do so in primates.⁶ To explain these differences in vivo, the precise knowledge of the molecular mechanisms of how heparin mediates its antiproliferative effects is necessary. These mechanisms have not been defined, although many effects of heparin have been described, including altered expression of matrix molecules,^{7 8} inhibition of proteases,^{9 10 11} the repression of the early genes c-fos and c-myc,¹² a decrease of activator protein 1 binding to DNA,^{13 14} and the inhibition of MAPK activation by serum.¹⁵ Our interest has focused on the mechanism of how heparin impairs MAPK activation, because this kinase seems to be important for transducing mitogenic signals, since its activity is stimulated by numerous mitogens. A direct link between MAPK and proliferation was demonstrated in fibroblasts by expression of a dominant-negative mutant and antisense cDNA.¹⁶ Both resulted in a decrease of the growth rate. In the last few years, it has become clear that MAPKs are a family of protein kinases consisting of at least three groups (reviewed in Reference 17¹⁷ ): the "classical" MAPKs, also referred to as extracellular signal-regulated kinases, which are involved in many cellular signaling events, including proliferation and differentiation; the JUN kinases or stress-activated protein kinases, which are part of inflammatory and stress-induced signaling pathways; and p38, a homologue to the yeast HOG kinase, which is involved in the cellular response to osmotic stress. All MAPKs are activated by specific kinases (MAPKKs) that phosphorylate a threonine and tyrosine residue in the highly conserved TXY motif. The signaling module that is involved in cell proliferation consists of two isozymes for each kinase, MAPK-1 (p44^MAPK) and -2 (p42^MAPK) and MAPKK-1 and -2, respectively (reviewed in Reference 18¹⁸ ). Activation of MAPKK-1 and -2 requires phosphorylation on two adjunct serine residues.^{19 20} This reaction can be catalyzed by various MAPKKKs. Signaling of most mitogens seems to involve the MAPKKK Raf-1. Activation of Raf-1 is dependent on its association with the GTP-bound form of the small G-protein Ras that translocates Raf-1 to the membrane,^{21 22} where it is fully activated by a not yet completely understood mechanism (reviewed in Reference 23²³ ). Ras is linked to different receptor systems, including tyrosine kinase receptors and seven-transmembrane-domain receptors that are coupled to heterotrimeric G proteins.^{24 25}

On the basis of the recent observation that heparin prevented MAPK activation in rat SMCs,¹⁵ we addressed two questions in the present report: (1) In baboon SMCs, does heparin, compared with serum, interfere with signaling of PDGF-BB and bFGF, since both molecules are thought to play an important role in injury-induced SMC growth? (2) What molecules mediate the inhibitory effect of heparin on MAPK activation?

	Materials and Methods

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Materials
Antibodies against MAPKK-1, MAPKK-2 (both monoclonal), and all PKC isoforms were from Transduction Laboratories. Antiserum 7884 raised against p42/p44 MAPK was a generous gift from Rony Seger.²⁶ Alkaline phosphatase– coupled anti-IgG was purchased from Promega; GF109203X, from Calbiochem; Gö6976, from Alexis; and [³H]thymidine and [-³²P]ATP, from Du Pont-New England Nuclear. Tissue culture solutions, including FCS, were from GIBCO-BRL. MBP and catalytically inactive histidine-tagged MAPK-2 (K52R mutant) were prepared with minor modifications as described previously.^{27 28} PDGF-BB was a generous gift from Zymogenetics. bFGF was purchased from R&D Systems. Prestained molecular weight markers for SDS-PAGE, protein A agarose, PMA, staurosporine, chondroitin sulfate, and heparin were from Sigma Chemical Co. All other chemicals were obtained from standard suppliers.

Cell Culture
Baboon aortic SMCs were prepared as described previously.²⁹ Cells of 5 to 20 passages were grown in DMEM supplemented with 10% FCS, 200 U/mL penicillin, and 200 µg/mL streptomycin. Cells were starved for 2 to 3 days before the experiment in DMEM/Ham's nutrient mix F-12 supplemented with 6 µg/mL insulin, 5 µg/mL transferrin, 1 mg/mL ovalbumin, 200 U/mL penicillin, and 0.2 mg/mL streptomycin. When heparin was included, it was administered simultaneously with the mitogen.

Buffers and Solutions
Buffer A contained 50 mmol/L HEPES-NaOH (pH 7.5) and 5 mmol/L 2-mercaptoethanol; buffer G, 10 mmol/L Tris-HCl (pH 7.8), 15 mmol/L 2-glycerophosphate, 1 mmol/L EDTA, 1 mmol/L EGTA, 5% glycerol, 0.1% 2-mercaptoethanol, 0.1 mmol/L Na₃VO₄, and 0.05% Triton X-100; buffer HEB, 25 mmol/L HEPES (pH 7.5), 10% glycerol, 5 mmol/L EDTA, 5 mmol/L EGTA, 150 mmol/L NaCl, 100 mmol/L sodium pyrophosphate, 50 mmol/L NaF, 1 mmol/L sodium vanadate, 1 mmol/L benzamidine, 0.1% 2-mercaptoethanol, 1% Triton X-100, 1 µmol/L pepstatin A, 2 µg/mL leupeptin, and 20 kallikrein inhibitor units/mL aprotinin; buffer K, 50 mmol/L HEPES (pH 7.5), 10 mmol/L MgCl₂, 0.1% Triton X-100, and 0.1% 2-mercaptoethanol; kinase buffer (MAPK), 20 mmol/L HEPES-NaOH, 20 mmol/L MgCl₂, and 2 mmol/L dithiothreitol; and TTBS, 25 mmol/L Tris-HCl, 500 mmol/L NaCl, and 0.1% Triton X-100.

MAPK Assay
Subconfluent cells were stimulated as indicated in the text, washed twice with ice-cold PBS, and harvested in buffer HEB. To 1 vol of lysate, 0.5 vol of 4x Laemmli buffer and 0.15 vol of 20% SDS were added. MAPK activity was measured using an in-gel phosphorylation assay according to the methods described in References 30 and 31^{30 31} . Briefly, 10 to 50 µg protein was subjected to SDS-PAGE³² after the 10% gels were polymerized in the presence of 0.25 mg/mL MBP. After electrophoresis, the gels were incubated for 1 hour at room temperature in 50 mmol/L HEPES-NaOH (pH 7.5) and 20% 2-propanol, followed by buffer A, with one change of each solution. Protein was denatured by immersing the gels in buffer A, containing 6 mol/L urea, twice for 15 minutes each time and renatured overnight at 4°C in buffer A containing 0.05% Tween-20 with two buffer changes. After a 30-minute incubation at room temperature in kinase buffer, phosphorylation was performed by soaking the gels for 45 minutes in kinase buffer containing 20 µmol/L [-³²P]ATP (1000 to 2000 cpm/pmol). Nonbound radioactivity was removed by six 30-minute washes with 5% TCA and 1% sodium pyrophosphate. Before drying, the gels were stained with Coomassie brilliant blue G to control for equal loading of protein. Incorporated radioactivity was analyzed by phosphorimaging (facility at the Department of Pharmacology, University of Washington, Seattle). The sum of activities of MAPK-1 and MAPK-2 are referred to as MAPK activities. These are presented relative to control values. Protein concentrations were determined using the Bradford reagent.³³

Western Blotting
Proteins were transferred to nitrocellulose,³⁴ and the blots were submerged in 1% BSA in TTBS, followed by an incubation for 2 hours at room temperature in 0.1% BSA in TTBS containing the appropriate primary antibody in concentrations recommended by the manufacturer. MAPK antiserum was diluted 1:5000. The proteins were detected by anti-IgG–coupled alkaline phosphatase using a standard protocol provided by the manufacturer.

Chromatography of Cell Extracts on Mono Q
Cells were starved for 2 days and stimulated with 10% FCS or 10 ng/mL PDGF-BB for 20 minutes. After two washes with ice-cold PBS, cells were harvested in 1 mL buffer G supplemented with 1% Triton X-100, 1 mmol/L benzamidine, 1 µmol/L pepstatin A, 2 µg/mL leupeptin, and 20 kallikrein inhibitor units/mL aprotinin and kept on ice. The homogenate was spun for 15 minutes at the highest speed in a microfuge. The supernatant containing 0.5 to 1.0 mg protein was subjected to chromatography on Mono Q (Pharmacia). Protein was eluted by a 40-mL gradient from 0 to 250 mmol/L NaCl in buffer G with a flow rate of 0.5 mL/min. Fractions of 1 mL were collected.

DNA Synthesis
Cells were starved in 24-well plates for 3 days and stimulated as indicated in the text in the presence of 1 µCi/mL [³H]thymidine for 24 hours. Cells were rinsed twice in ice-cold PBS and incubated overnight in 1 mL 10% TCA at 4°C. After the TCA was aspirated, 0.4 mL of 0.1 mol/L NaOH was added, and the plates were shaken at room temperature for 1 hour before the radioactivity of 0.35 mL of the sample was measured by liquid scintillation counting. The remainder was used for protein determination.³³

MAPKK Assay
Cells were harvested as described above for MAPK assays. The lysate was kept on ice for 20 minutes, vigorously mixed, and spun for 5 minutes at maximum speed in a microfuge. One microgram of antibody and 10 µL of protein A–Sepharose slurry (1 mg/mL protein A) were added to the supernatant containing 0.1 to 0.5 mg protein. The sample was stirred overnight at 4°C or for 4 hours at room temperature. The beads were washed in buffer HEB, followed by two washes in TTBS containing 0.1% 2-mercaptoethanol and one wash in buffer K. The kinase reaction on the beads was performed in buffer K containing 1 µg K52R-MAPK-2 per assay and 50 µmol/L [-³²P]ATP (5000 cpm/pmol). The assay was incubated for 30 minutes at room temperature, and the reaction was terminated by adding 10 µL of 4x Laemmli buffer. The samples were subjected to SDS-PAGE, and the extent of MAPK phosphorylation was determined as described above for MAPK in-gel assays. Assays were performed in duplicate.

	Results

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Heparin Inhibits the Activation of MAPK by FCS but Not by PDGF-BB or bFGF
In order to test whether MAPK activation by FCS is inhibited in baboon SMCs as it is in rat SMCs,¹⁵ kinase activities were determined in the absence and presence of heparin by using an in-gel assay with MBP, as described in "Materials and Methods." Heparin attenuated MAPK activation by FCS in baboon SMCs. Fig 1A illustrates a time-course experiment, and Fig 1B shows the resulting graph after quantifying kinase activities by phosphorimaging. MAPK exhibited a considerable basal activity in starved SMCs, and its activation reached a maximum 30 minutes after stimulation with a 3.7-fold increase (SD=1.2, n=10) of kinase activity. In the presence of heparin, a lower activation of MAPK was observed at all time points (see also Fig 2). Since both PDGF-BB and bFGF are thought to play an important role in intimal hyperplasia following vessel injury (reviewed in Reference 2² ), we measured the effect of heparin on cells stimulated with these growth factors compared with FCS in regard to MAPK activation. Maximum MAPK activities were measured at 30 minutes with PDGF-BB and at 15 minutes with bFGF. Compared with FCS, PDGF-BB was slightly less effective in activating MAPK, increasing its activity 2.8-fold (SD=1.0, n=3). bFGF, however, in the absence of heparin, was a very weak activator of MAPK; it elicited only a 1.4-fold (SD=0.5, n=3) stimulation over starved cells. As shown in Fig 2, heparin inhibited FCS-induced MAPK activity by 40% but had little effect when PDGF-BB was the mitogen (10% inhibition). Moreover, in the presence of bFGF, heparin stimulated MAPK activity 2-fold. Thus, the effect of the glycosaminoglycan on MAPK activation completely depends on the nature of the mitogen.

View larger version (26K): [in this window] [in a new window]   Figure 1. Heparin inhibits activation of MAPK by FCS. Starved SMCs were stimulated with 10% FCS with or without 0.1 mg/mL heparin. Kinase activities were determined in in-gel assays from cell extracts prepared at the time points indicated. A, Autoradiograph showing a typical time-course experiment (number of independent experiments exceeds 10). B, Graph based on data obtained by phosphorimaging of the gel shown in panel A. Activities in the absence () and the presence () of heparin are given in fold stimulation.

View larger version (15K): [in this window] [in a new window]   Figure 2. Effect of heparin on MAPK activation by FCS, PDGF-BB, and bFGF. Starved SMCs were stimulated for 30 minutes with 10% FCS and 10 ng/mL PDGF-BB and for 15 minutes with 10 ng/mL bFGF in the absence and presence of 0.1 mg/mL heparin. Cells were extracted, and MAPK activity was determined using an in-gel assay. Activities in the presence of heparin (mean±SD for 10 [FCS] and 3 [PDGF-BB and bFGF] experiments) are reported as a percentage of the control value (without heparin) indicated by the dotted line.

Interestingly, independent of the mitogen used, 80% to 90% of total MAPK activity could be attributed to p42 MAPK on the basis of the in-gel assay. To confirm that this was not due to a failure of p44 MAPK to renature during the in-gel assay, we performed a column shift assay to determine the amount of activated MAPK protein. Because of the introduction of two negatively charged phosphate groups in the TEY motif of subdomain VIII, active MAPK, compared with inactive kinase, elutes at higher salt concentrations from anion exchange columns.³⁵ Extracts of SMCs stimulated with FCS or PDGF-BB were fractionated on a Mono Q column. Both mitogens gave identical results. The fractions were analyzed for MAPK activity in an in-gel assay (Fig 3A) and for MAPK protein by Western blotting (Fig 3B). Scanning of the blot allowed quantification of relative protein amounts per fraction. We determined that the p42/p44 MAPK protein ratio was 2.5:1. After stimulation, 40% of p42 eluted at higher salt concentrations (Nos. 71 to 73 in Fig 3; note in panel A that No. 70 consists of mostly inactive kinase), whereas this was the case for only 20% of the p44 isoform (Nos. 74 to 76). We further calculated that p44 contributed only 15% to the total "shifted" MAPK. Thus, by two independent methods, activity and retardation in chromatography, we found that p44 is the dominant active isoform of MAPK in SMCs. This is due to both a higher concentration and a higher degree of activation of p42 over p44 MAPK.

View larger version (30K): [in this window] [in a new window]   Figure 3. p42 is the dominant mitogen-activated MAPK isoform in baboon SMCs. Extracts of PDGF-BB–stimulated baboon SMCs were fractionated on a Mono Q column as described in "Materials and Methods." In the experiment shown, gradient fractions were 41 to 80. Each fraction was analyzed for MBP kinase activity by an in-gel assay (A) and MAPK protein by Western blotting (B).

Effect of Heparin on DNA Synthesis Induced by FCS, PDGF-BB, and bFGF
To test whether the effect of heparin on the activity of MAPK is reflected in the proliferative response of the cells, we measured DNA synthesis in the absence and presence of the glycosaminoglycan. As shown in Fig 4, FCS, PDGF-BB, and bFGF increased DNA synthesis 8-fold, 6-fold, and 2-fold, respectively, over control values. When heparin was added, an average 45% inhibition was observed when FCS was used as mitogen. Heparin failed, however, to significantly inhibit DNA synthesis by PDGF-BB, and it stimulated thymidine incorporation by bFGF 2-fold. These observations suggest a strong correlation between mitogenesis and the activity of the MAPK.

View larger version (32K): [in this window] [in a new window]   Figure 4. Effect of heparin on DNA synthesis. Incorporation of [3H]thymidine was determined after 24-hour stimulation of starved SMCs by 10% FCS, 10 ng/mL PDGF-BB, and 10 ng/mL bFGF in the absence and presence of 0.1 mg/mL heparin. Numbers are given in percent incorporation compared with 10% FCS (mean±SD for two [bFGF] and four [FCS and PDGF-BB] experiments with three or four plates per experiment).

The Effect of Heparin Is Mediated by MAPKK-1
We addressed the question whether the effect of heparin on MAPK is mediated directly, by inhibition of the kinase, or indirectly, by affecting the immediate MAPK activator MAPKK. Since there are two MAPKK isoforms, we first investigated which isoform plays a role in activating MAPK in baboon SMCs. After stimulation of SMCs with FCS, cells were extracted, and MAPKK-1 and -2 were immunoprecipitated using monoclonal antibodies. When kinase activities were determined, only MAPKK-1 was found to be active (Fig 5A), although both isoforms were present, as confirmed by Western blotting (Fig 5B). Experiments with PDGF-BB as a mitogen showed identical results (data not shown). Others have demonstrated the ability of the same anti–MAPKK-2 antibody to immunoprecipitate active kinase.^{36 37} Thus, our data suggest that only MAPKK-1, but not MAPKK-2, is part of the mitogenic signaling cascade in baboon SMCs.

View larger version (27K): [in this window] [in a new window]   Figure 5. MAPKK-1 is the activator of MAPK in baboon SMCs. MAPKK-1 and -2 were immunoprecipitated, and kinase activity was determined with a histidine-tagged K52R mutant of p42MAPK as substrate. The arrows indicate the position of MAPK (A). The presence of MAPKK protein in the immunoprecipitates is demonstrated by Western blotting (B). The arrows indicate the position of MAPKK. The protein band above MAPKK is due to mouse IgG detected by the secondary antibody. IP indicates immunoprecipitation.

Heparin had the same effects on MAPKK-1 as on MAPK stimulation (Fig 6). Whereas it blocked FCS-mediated MAPKK-1 stimulation (70% inhibition), little difference was observed with PDGF-BB (17% inhibition), and when bFGF was used, heparin increased MAPKK-1 activity 3.3-fold. These findings support the view that the heparin effect on MAPK is mediated by targeting the MAPK activator MAPKK-1 or further upstream signaling elements. Fig 7 shows the dose dependence of the heparin effect in the presence of FCS. Heparin (10 µg/mL) inhibited MAPKK-1 activity by 50%. The same concentration of chondroitin sulfate had no effect (data not shown), indicating that the inhibition of MAPKK-1 by heparin is specific and does not only depend on the negative charges of the sulfate groups.

View larger version (15K): [in this window] [in a new window]   Figure 6. Effect of heparin on MAPKK-1 activation by FCS, PDGF-BB, and bFGF. Starved SMCs were stimulated for 30 minutes with 10% FCS and 10 ng/mL PDGF-BB and for 15 minutes with 10 ng/mL bFGF in the absence and presence of 0.1 mg/mL heparin. Cells were extracted, and MAPKK-1 activity was determined. Activities in the presence of heparin (mean±SD for 10 [FCS] and 3 [PDGF-BB and bFGF] experiments) are reported as a percentage of the control value (without heparin) indicated by the dotted line.

View larger version (16K): [in this window] [in a new window]   Figure 7. The inhibition of MAPKK-1 by heparin is dose dependent. Starved SMCs were incubated with 10% FCS in the presence of the indicated concentrations of heparin. After 30 minutes, cells were harvested, and extracts were analyzed for MAPKK-1 activity. The activity in the absence of heparin was set at 100%. The experiment was repeated with equal results.

Role of PKC in Heparin Inhibition of MAPKK-1
Previously, it was suggested that heparin exerts its effects, including the inhibition of c-fos mRNA, by affecting a PKC-dependent pathway.³⁸ Subsequently, PKC was identified as the target for heparin inhibition of proliferation in rat SMCs.³⁹ We tested the possibility that the inhibitory effect of heparin on FCS-induced MAPK/MAPKK-1 activity was also mediated by PKC. By immunoblotting of SMC extracts using various PKC antibodies, we found that PKC, PKC, PKC, PKC, PKC, PKC, and PKC were expressed. PKCß, PKC, and PKCµ were not detected. At this point, we cannot rule out the possibility that these isoforms are also present but were not detected by our technique. Pretreatment of SMCs with PMA resulted in a depletion of the diacylglycerol-dependent PKC, PKC, PKC, and PKC, whereas the expression of PKC, PKC, and PKC remained unchanged. Data are shown for PKC in Fig 8A. If any of the PMA-sensitive PKC isoforms are mediating the inhibitory effect of heparin on MAPK/MAPKK-1 activation by FCS, one would expect that heparin has no effect in SMCs that are pretreated with PMA. We found that MAPK and MAPKK-1 were less active in PMA-pretreated cells but that heparin was still capable of further decreasing their activity (Fig 8B). The same observation was made in the presence of 3 µmol/L Gö6976, which is a PKC inhibitor with restricted specificity toward the calcium-dependent isoforms, such as PKC⁴⁰ (data not shown). We conclude from these data that the activation of the MAPK/MAPKK-1 signaling module by FCS is at least partially dependent on PKC and that heparin does not inhibit that process by affecting the PMA-sensitive PKC isoforms , , , and .

View larger version (16K): [in this window] [in a new window]   Figure 8. Heparin inhibits MAPK/MAPKK-1 activation by FCS in PKC-depleted SMCs. SMCs were starved for 2 days in the absence or presence of 1 µmol/L PMA for the last 24 hours. A, Extracts (20 µg per lane) were analyzed by Western blotting for the presence of PKC. MW indicates molecular weight. B, Activities of MAPK and MAPKK-1 were determined in PMA-pretreated SMCs after 30 minutes of stimulation with 10% FCS in the absence (solid bars) or presence (open bars) of 0.1 mg/mL heparin. Activities (mean±SD for two or three experiments) are presented as percentages of control values (stimulation for 30 minutes with 10% FCS in the absence of heparin and PMA).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The inhibitory effect of heparin on signal transduction depends on the nature of the mitogen. On the basis of the recent finding that heparin inhibits MAPK activation by FCS in rat SMCs,¹⁵ we compared the effects of the glycosaminoglycan on baboon SMCs stimulated by FCS, PDGF-BB, and bFGF because the latter two growth factors are thought to play an important role in SMC function in vivo (reviewed in Reference 2² ). In order to distinguish between the two known isoforms of MAPK, p42 and p44, that are activated by mitogens, we took advantage of an in-gel assay in which kinase activities are measured after the enzymes have been separated by SDS-PAGE. We found that p42 MAPK contributes 80% to 90% of the MAPK activity. These results were confirmed by shift assays on Mono Q that are based on a retardation of elution of activated kinases due to the presence of the two phosphate groups in the TEY motif.

Heparin was capable of blocking FCS-mediated MAPK activation, but it had little effect when PDGF-BB was used and even increased the activation of MAPK by bFGF. The same pattern was observed when we determined the influence of heparin on DNA synthesis. To exclude the possibility that the heparin effects described are due to a specific property of baboon SMCs, we also measured DNA synthesis in Fischer rat SMCs under identical conditions and obtained the same results (authors' unpublished data, 1996). The activating effect of heparin on signaling by bFGF is in accordance with recent reports demonstrating that heparin promotes the oligomerization of FGF molecules and thereby increases binding of these complexes to the receptor and, consequently, receptor activation and signaling.⁴¹ Heparin also failed to inhibit MAPK activation by epidermal growth factor in rat SMCs.¹⁵ Thus, in accordance with our findings, it appears that heparin does not interfere with mitogenic signaling by tyrosine kinase receptors. In summary, our observations in vitro and in vivo in the rat and the baboon suggest the conclusion that the efficacy of heparin as an inhibitor of intimal hyperplasia and SMC growth depends mainly on the mix of mitogens and cytokines present and much less on the species from which the SMCs are derived. Future work in our laboratory will be directed to the identification of the mitogen in FCS that is inhibited by heparin and to the determination of which mitogens are involved in the process of neointimal formation in rats and baboons.

In the remainder of the present study, additional insight into how heparin inhibits proliferation of cultured SMCs was provided. To identify the MAPK-activating kinases in SMCs, MAPKK-1 and -2 were assayed in immunoprecipitates from extracts of stimulated cells. Only MAPKK-1, but never MAPKK-2, was found to be activated, although both kinases were present in comparable amounts in the immunoprecipitates. Because the same anti–MAPKK-2 antibodies have been used by others to precipitate the active enzyme,^{36 37} we assume that MAPKK-1 is solely responsible for activating MAPK after mitogenic stimulation. Why there is a selective involvement of one isoform of MAPKK is not yet clear. It is possible that there is a scaffold protein like STE5 in yeast⁴² that allows only one isoform to assemble with its activating kinase. Indeed, in NIH 3T3 fibroblasts, only MAPKK-1, but not MAPKK-2, was identified in signaling particles in complex with Ras and Raf after stimulation of the cells.⁴³

In the presence of heparin, stimulation of MAPKK-1 was affected in a manner similar to that of MAPK, suggesting that heparin targets not MAPK itself but its activator or further upstream signaling elements. It is noteworthy that the inhibitory effect of heparin on FCS-stimulated cells was stronger on MAPKK levels than on MAPK levels. One explanation is that a small fraction of the MAPKK-1 pool is already sufficient to fully activate MAPK. This conclusion is supported by the observation that the heparin concentration of 10 µg/mL that blocks 50% of MAPKK-1 activity does not have any significant effect on MAPK activity nor on DNA synthesis (data not shown). Our attempts to determine the effect of heparin on the MAPKK-1–activating kinase Raf-1 were inconclusive because we measured only a very low activation (1.5-fold) by FCS that was not significantly altered by heparin. In addition, we have recently reported that in SMCs kinases other than Raf-1 might be responsible for MAPKK activation⁴⁴ upon stimulation with G protein–coupled mitogens. In order to find the target of heparin in SMC signal transduction, we will first try to identify the responsible MAPKK-1 kinase stimulated by FCS and the receptor tyrosine kinase ligands PDGF-BB and bFGF.

Since it has been suggested previously that heparin affects PKC-dependent pathways³⁸ and since PKC was recently identified as a target for heparin in inhibiting proliferation,³⁹ we attempted to define a role for this isoform in heparin inhibition of the MAPK/MAPKK-1 signaling module. We obtained somewhat conflicting results in that heparin was still capable of blocking the stimulation of both kinases after the cells were depleted of PKC. Besides PKC, we detected PKC, PKC, PKC, PKC, PKC, and PKC, of which PKC, PKC, and PKC were also absent in SMCs pretreated with PMA. These data suggest that heparin does not mediate its inhibitory effect on MAPK/MAPKK-1 through PKC, PKC, PKC, and PKC. This conclusion was confirmed by experiments using the PKC inhibitor that is specific toward the calcium-dependent PKC isoforms, such as PKC,⁴⁰ in that heparin still blocked FCS-induced MAPK/MAPKK-1 activation in the presence of Gö6976. It remains to be determined whether heparin affects isoforms other than the PMA-sensitive PKC isoforms to inhibit MAPK/MAPKK-1. When we replaced Gö6976 with PKC inhibitors with broad specificity, including GF109203X⁴⁵ and staurosporine,⁴⁶ a significant effect of heparin could not be determined, because the activation of MAPK/MAPKK-1 was greatly impaired even in the absence of heparin (data not shown). Future work in our laboratory will be directed to determine the effect of PKCs by using an antisense oligonucleotide approach that allows the depletion of single PKC isoforms in SMCs.

	Selected Abbreviations and Acronyms

 

bFGF = basic FGF FGF = fibroblast growth factor MAPK = mitogen-activated protein kinase MAPKK = MAPK kinase MAPKKK = MAPKK kinase MBP = myelin basic protein PDGF = platelet-derived growth factor PKC = protein kinase C PMA = phorbol 12-myristate 13-acetate SMC = smooth muscle cell TCA = trichloroacetic acid

	Acknowledgments

 
This study was supported by National Institutes of Health grants HL-18645 and HL-30946. We thank Melanie Cobb for the plasmid encoding the histidine-tagged K52R-MAPK-2 mutant, Rony Seger for p42/p44 MAPK antiserum 7884, and Zymogenetics for PDGF-BB.

Received January 29, 1997; accepted April 18, 1997.

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Top Abstract Introduction Materials and Methods Results Discussion References

 

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