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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:1608-1614.)
© 1999 American Heart Association, Inc.

Vascular Biology

Protein Kinase C Mediates Basic Fibroblast Growth Factor–Induced Proliferation Through Mitogen-Activated Protein Kinase in Coronary Smooth Muscle Cells

Adriane Skaletz-Rorowski; Johannes Waltenberger; Joachim G. Müller; Ewa Pawlus; Kai Pinkernell; Günter Breithardt

From the Institute for Arteriosclerosis Research (A.S.-R., J.G.M., E.P., K.P., G.B.), University of Münster , Münster, Germany; the Department of Internal Medicine II (Cardiology [J.W.]), Ulm University Medical Centre, Ulm,Germany; and the Department of Cardiology and Angiology (J.G.M., E.P., G.B.), University of Münster, Münster, Germany.

Correspondence to Dr Adriane Skaletz-Rorowski, Institute for Arteriosclerosis Research, University of Münster, Domagkstr.3, D-48149 Münster, Germany. E-mail skaletz{at}uni-muenster.de

	Abstract

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Abstract—Proliferation of coronary smooth muscle cells (cSMCs) contributes to the pathogenesis of arteriosclerosis and restenosis after angioplasty, and basic fibroblast growth factor (bFGF) is a powerful mitogen for cSMCs. In this study, we investigated the involvement of mitogen-activated protein kinase (MAPK), protein kinase C (PKC), and the transcription factor c-myc in bFGF-stimulated mitogenesis, as well as the functional relationship between these factors. cSMC stimulation with bFGF resulted in phosphorylation of p42 MAPK, as well as the phosphorylation and increased expression of c-myc. The MAPK kinase (MEK) inhibitor PD98059 blocked bFGF-stimulated MAPK phosphorylation and resulted in both a decrease of c-myc expression and inhibition of bFGF-stimulated DNA synthesis in cSMCs. bFGF also increased PKC activity in cSMCs in a time-dependent manner. The inhibition of PKC by chelerythrine or its downregulation by phorbol 12-myristate 13-acetate (PMA) inhibited bFGF-induced DNA synthesis and blocked the phosphorylation of MAPK and c-myc expression in response to bFGF. This indicates an involvement of phorbol ester–sensitive PKC isoforms in MAPK activation and mitogenic signaling by bFGF. Western blot analysis revealed the presence of the phorbol ester–sensitive isoforms PKC , , and as well as the PKC isoforms , , µ, and in cSMCs. In this study, we show that the MAPK cascade is required for bFGF-induced proliferation and that phorbol ester–sensitive PKC isoforms contribute to the bFGF-induced cSMC mitogenesis in cSMCs.

Key Words: basic fibroblast growth factor • coronary artery smooth muscle cells • mitogen-activated protein kinase • protein kinase C • c-myc • arteriosclerosis

	Introduction

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Proliferation of coronary smooth muscle cells (cSMCs) with subsequent formation of intimal thickening is an important event in the development of arteriosclerosis and restenosis after PTCA¹ and stent implantation.² Basic fibroblast growth factor (bFGF) synthesized by vascular SMC^{3 4} is a powerful mitogen for SMC replication during atherogenesis⁵ and in response to vessel wall injury.⁶ Previous studies have shown that (1) bFGF, released from arterial SMC after injury, is a potent mitogen,⁷ (2) SMC replication appears to be correlated to arterial damage,⁸ (3) high bFGF expression and cell replication correlate in injured rat arteries,⁹ and (4) neointimal proliferation is significantly inhibited by anti-bFGF antibodies.⁸ Our previous studies have shown that bFGF expressed by vascular SMC is retained primarily in the intracellular compartment but is delivered to the pericellular compartment during proliferation, reaching maximum values in the phase of exponential growth.^{3 4} The formation of an active complex composed of bFGF, its specific tyrosine kinase receptor (FGF-R1), and the coreceptor heparan sulfate proteoglycan (HSPG) is required for the initiation of the bFGF-induced mitogenic pathway.¹⁰

Receptor tyrosine kinases activate a number of intracellular signaling pathways including phosphoinositide 3-kinase (PI3-kinase), 70 kDa S6 kinase, phospholipase C, and mitogen-activated protein kinase (MAPK).¹¹ However, the functional role of intracellular downstream signaling elements in bFGF-induced proliferation of cSMCs is not fully understood.

A crucial role in signal transduction, cell growth, and differentiation is played by protein kinase C (PKC).¹² The family of structurally related PKC isoforms consists of products of different genes that have been classified on the basis of their Ca²⁺ and phorbol ester sensitivities into 3 subfamilies: classical PKC (, ß, ), novel PKC (, , , ), and atypical PKC (, , ).¹³ In addition, recent evidence warrants the designation of a fourth group of PKC which is based on the finding that the catalytic domain of PKCµ (classified originally as a member of the novel PKC family) is more closely related to Ca²⁺/calmodulin-dependent protein kinases and contains signal and transmembrane moieties that are absent in other PKC family members.¹⁴ Although PKC activity plays a role in bFGF-induced cell growth, its effect depends on the cell type studied. Whereas the mitogenic effect of bFGF was found to be PKC independent in fibroblasts,¹⁵ studies in endothelial cells have suggested that the activation of PKC is involved in the mitogenic pathway of bFGF.¹⁶

The MAPK signaling cascade is activated by bFGF as well, shown in studies with SMC from porcine thoracic aorta.¹⁷ However, the relationship between PKC and MAPK signaling in bFGF-induced proliferation of cSMCs is unknown. Furthermore it is unclear whether the transcription factor c-myc, an important factor in proliferation,¹⁸ participates in bFGF-induced proliferation of cSMCs.

The purpose of the present study was to determine whether bFGF leads to activation of MAPK, c-myc, and PKC in cSMCs after stimulation, whether these signaling elements exhibit a functional relationship, and whether they are required for bFGF-induced proliferation in cSMCs.

We demonstrate that bFGF causes phosphorylation of MAPK, induces the phosphorylation and a marked increase of c-myc, as well as activation of PKC. Furthermore, we show that (1) bFGF-induced expression of c-myc requires the activation of MAPK, (2) c-myc expression and MAPK phosphorylation are mediated in part by a phorbol ester–dependent PKC isoform(s), and (3) MAPK and phorbol ester–sensitive isoforms of PKC are required for bFGF-induced proliferation of cSMCs.

	Methods

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Materials
[³H]Thymidine (1.10 Tbq/mmol · L^-1) was obtained from Amersham Buchler. Cell culture media were purchased from Seromed; fFCS and soybean trypsin inhibitor (SBTI) were supplied by Boehringer Mannheim. Chemicals for the SDS-PAGE/immunoblotting, such as 5-Bromo-4-chloro-3-indolyl phosphate (BCIP) and Nitro Blue Tetrazolium (NBT) were products of AppliChem. All other chemicals were of analytical grade or the best grade available and were purchased from Boehringer Mannheim, Merck, or Serva. Anti-phosphotyrosine, anti-p42 MAPK, and anti-PKC antibodies were obtained from Transduction Laboratories. All other antibodies were products of Sigma.

Cell Culture
Primary cultures of cSMCs were isolated from explants of bovine coronary arteries and cultured in medium with 10% FCS at 37°C in a humidified, 5% CO₂/95% air atmosphere. Cultures of the third to sixth passage were used in the experiments.

DNA Synthesis Assay
To assay for DNA synthesis, 25 000 cSMCs were seeded into Petri dishes (diameter, 35 mm) in medium supplemented with 10% FCS and cultured for 4 days. After a serum-free incubation for 48 hours, cSMCs were pretreated with different concentrations of the PKC inhibitor chelerythrine for 48 hours or with the MAPK kinase (MEK) inhibitor PD98059 for 30 minutes before addition of bFGF (2 ng/mL). DNA synthesis was measured as the amount of [³H]thymidine incorporation (during a 12-hour pulse label) into cSMCs as described previously.¹⁹

Immunoblotting
Cells were lysed with boiling 2x concentrated electrophoresis sample buffer (1x=62.5 mmol/L Tris-HCl [pH 6.8], 3% SDS, 10% glycerol, 5% ß-mercaptoethanol, 1% bromophenol blue) and scraped off with a rubber policeman. After brief sonication, the sample was boiled and centrifuged for 5 minutes. After separation on a 7.5 to 12.5% SDS polyacrylamide gel, the proteins were transferred to a nitrocellulose membrane (Schleicher & Schuell, Inc) and stained with Ponceau S to verify equivalent amounts of protein. After blocking with 3% BSA, incubation with a specific antibody and an alkaline phosphatase–conjugated secondary antibody, the protein was detected with BCIP/NBT or CDP-Star (Boehringer).

MAPK Phosphorylation Analyzed by Western Blot
The experiments were carried out as previously described²⁰ with slight modifications. In brief, serum-starved cSMCs were stimulated with bFGF (2 ng/mL) at 37°C for indicated times. Lysates were used for immunoprecipitation with the anti-p42 monoclonal MAPK antibody. Samples were analyzed by Western blot with use of the polyclonal anti-phosphotyrosine antibody PY20 or the specific MAPK antibody for specific control as well as for quantification of protein content.

Downregulation and Inhibition of PKC
PKC was downregulated by treating cSMCs with 0.1 to 1 µmol/L phorbol 12-myristate 13-acetate (PMA) in culture medium for 72 hours in the presence or absence of 10% FCS. The catalytic PKC domain was blocked by incubating cSMCs with 1 to 2.5 µmol/L chelerythrine in culture medium with or without 10% FCS for 48 hours. Nondownregulated or noninhibited control cells were maintained in the medium for an equal period of time.

PKC Assay
In vitro PKC activity was determined by using a nonradioactive method based on an enzyme immunoassay supplied by Upstate Biotechnology. For the assay, cells grown in 35-mm (diameter) culture dishes were harvested into a 50-µL/dish of extraction buffer containing Triton X-100, and the extracts were clarified by centrifugation in a microfuge. (The data were normalized to protein concentration of the eluate.) The assay utilized a synthetic peptide and a mouse monoclonal antibody (2B9) that recognizes the phosphorylated form of the peptide. The PKC present in the samples catalyzed the phosphorylation of the peptide coated on the microwell plate. The biotinylated antibody (2BG) then bound the phosphorylated peptide and was subsequently detected with streptavidin conjugated to peroxidase.

Other Methods and Statistics
Cell counting and protein determination were performed according to standard methods. Results are expressed as mean±SEM of the specified number of experiments carried out on different cultures of cSMCs in duplicate or triplicate. Statistical significance was assessed using the Student's t test for paired comparisons, and P<0.05 was considered significant.

	Results

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Phosphorylation and Expression of c-myc Is Mediated Through MEK-MAPK Pathway in bFGF-Induced Proliferation in cSMCs
Our previous results have shown that treatment of subconfluent SMC with bFGF caused an induction of DNA synthesis in SMC.³ Because activation of MAPK²¹ is a rather early mitogenic signaling event in a variety of cell types, we investigated the ability of bFGF to stimulate MAPK in cSMCs. Quiescent cSMCs were stimulated with bFGF (2 ng/mL) for 5, 60, 90, and 120 minutes. After immunoprecipitation with a monoclonal MAPK antibody, the tyrosine phosphorylation status of MAPK was analyzed by Western blot using the anti-phosphotyrosine antibody PY20. As demonstrated in Figure 1A, bFGF caused a rapid phosphorylation of the p42 MAPK, the predominant MAPK isoform in these cells (data not shown). This remained phosphorylated for 120 minutes. The activation of MAPK was confirmed by a highly selective activity assay for MAPK (data not shown).

View larger version (26K): [in this window] [in a new window]   Figure 1. Effect of bFGF on the phosphorylation of MAPK, and effect of PD98059 on bFGF-induced DNA synthesis in cSMCs. A, cSMCs were plated at a density of 25 000 cells in culture dishes (35-mm diameter) in a medium containing 10% FCS and cultured for 4 days. After serum-free incubation (48 hours) quiescent cSMCs (control) were stimulated for 5, 60, 90, and 120 minutes with bFGF (2 ng/mL). The cell lysates were immunoprecipitated with an anti-MAPK antibody that recognizes the p42 isoform of MAPK and size-fractionated by SDS-PAGE. The gel was blotted and filters were probed with the anti-phosphotyrosine antibody PY20. Molecular weight marker is indicated on the right. Similar results were obtained in 4 independent additional experiments. B, Quiescent cSMCs (control) were pretreated with indicated concentrations of PD98059 30 minutes before stimulation with bFGF (2 ng/mL) for 24 hours. DNA synthesis was measured as the amount of [3H]thymidine incorporation into cSMCs. Values represent means of 4 experiments performed in duplicate. *P<0.05 compared with bFGF.

To test whether bFGF-induced DNA synthesis is dependent on MAPK activation in cSMCs we used PD98059, a selective inhibitor of the MEK protein kinase that activates MAPKs by phosphorylation on both threonine and tyrosine residues. PD98059 blocks the activation of MEK by Raf-1 without affecting other known serine/threonine and tyrosine kinases.²² With Western blot analysis, pretreatment of cSMCs with PD98059 (35 to 70 µmol/L) for 30 minutes suppressed the bFGF-induced phosphorylation of p42 MAPK (data not shown). In addition, PD98059 inhibited bFGF-stimulated DNA synthesis in a dose-dependent manner, with 30% inhibition observed at a concentration of 35 µmol/L and 63% at 70 µmol/L (Figure 1B).

MAPK has been shown to induce the transcriptional activity of early-response genes.¹⁸ Therefore we examined the early-response gene c-myc which encodes a nuclear transcription factor that is both necessary and sufficient to trigger entry into the S phase of the cell cycle.²³ As shown in Figure 2A, treatment of quiescent cSMC cultures with bFGF for 60 minutes caused phosphorylation of c-myc and an increase of the c-myc protein level which was sustained for up to 150 minutes. As judged by scanning densitometry (Figure 2B), bFGF stimulation enhanced c-myc expression (P<0.05) in cSMCs by 3.3x over the unstimulated cells.

View larger version (29K): [in this window] [in a new window]   Figure 2. Effects of bFGF and PD98059 on the phosphorylation and expression of c-myc in cSMCs. A, Quiescent cSMCs were untreated (control) or incubated with bFGF (2 ng/mL) for indicated times. Equivalent amounts of protein were subjected to SDS-PAGE, transferred to a nitrocellulose membrane, and incubated with a monoclonal anti–c-myc antibody. The immunoreactive protein was visualized by use of an alkaline phosphatase detection system. Similar results were obtained in 3 additional independent experiments. B, Serum-starved cSMCs were treated with PD98059 (35 µmol/L) for 30 minutes and stimulated with bFGF (2 ng/mL) for 60 minutes. The Western blot was done as described in Figure 2A. The c-myc protein levels were quantified by scanning densitometry. The figure shows the levels of c-myc protein as percentage of the maximal level of expression. Values are means of 3 experiments done in duplicate. *P<0.05 compared with bFGF.

To investigate the relationship between MAPK and c-myc in bFGF signaling in cSMCs, we incubated the cells with 35 µmol/L PD 98059. As shown in Figure 2B, 35 µmol/L PD 98059 inhibited the ability of bFGF to stimulate c-myc protein expression in cSMCs by 45%.

bFGF-Induced Proliferation Is Mediated in Part Through Phorbol Ester–Sensitive PKC Isoforms
In initial experiments, we examined whether bFGF could induce PKC activity in cSMCs. Serum-starved cSMCs were stimulated with bFGF (10 ng/mL) for up to 2 hours, and the total PKC activity in cSMCs was measured. After 5 minutes of stimulation, the PKC activity was already elevated and increased substantially after 30 minutes of stimulation (Figure 3). The PKC activity reached a maximum of 40% increase above baseline after 1 hour of bFGF stimulation and returned to basal levels after 2 hours.

View larger version (38K): [in this window] [in a new window]   Figure 3. PKC activity induced by bFGF in cSMCs. Cells were stimulated with bFGF (10ng/mL) and processed for PKC activity measurements as described in Methods. Similar results were obtained in 4 independent additional experiments. Results are expressed as the mean±SEM of the percent increase in PKC activity in bFGF-stimulated cells compared with cells incubated with carrier (PBS).

To examine whether inhibition of PKC could affect bFGF-induced proliferation in cSMCs, the cells were treated with 2 specific PKC antagonists that have different mechanisms of inhibition. First, the cells were incubated with the PKC inhibitor chelerythrine, which binds to the catalytic domain of PKC and appears to have no effect on other protein kinases.²⁴ A 48-hour incubation of cSMCs with chelerythrine (2.5 µmol/L) produced a 65% inhibition of bFGF-induced DNA synthesis (Figure 4). Lower concentrations of chelerythrine reduced the bFGF-induced DNA synthesis in a dose-dependent manner, with an 35% inhibition by 1 µmol/L (Figure 4).

View larger version (36K): [in this window] [in a new window]   Figure 4. Effect of PKC inhibition by chelerythrine on bFGF-induced DNA synthesis in cSMCs. After serum-free incubation (48 hours) quiescent cSMCs (control) were pretreated with indicated concentrations of chelerythrine for 48 hours before addition of bFGF (2 ng/mL). DNA synthesis was measured as the amount of [3H]thymidine incorporation into cSMCs. Values represent means of 4 experiments performed in duplicate. *P<0.05 compared with bFGF.

Next we determined the influence of phorbol ester–sensitive PKC isoforms on bFGF-induced DNA synthesis and cell number. Prolonged incubation (72 hours) of cSMCs with 1 µmol/L PMA induced a downregulation of PKC isoforms that were sensitive to phorbol ester (data not shown). PMA-treated cSMCs proliferated significantly more slowly than the corresponding control cells when stimulated with 10 ng/mL bFGF (Figure 5A). Furthermore, in parallel experiments and consistent with the notion above, we could show that PKC downregulation by PMA (72 hours) significantly inhibited [³H]thymidine incorporation after bFGF stimulation (Figure 5B).

View larger version (81K): [in this window] [in a new window]   Figure 5. Effect of PMA pretreatment on the mitogenic effect of bFGF in cSMCs. A, cSMCs were incubated in 10% serum (control) or in serum with PMA (1 µmol/L; downregulation) before stimulation with bFGF. Results indicate means of 3 experiments performed in triplicate. B, After serum deprivation for 48 hours, cSMCs were treated for 72 hours with PMA (0.1 µmol/L). Cells were then stimulated with bFGF (2 ng/mL) for additional 24 hours. DNA synthesis was measured as the amount of [3H]thymidine incorporation into cSMCs. Values represent means of 4 experiments performed in duplicate. *P<0.05 compared with bFGF.

To test which phorbol ester–sensitive PKC isoforms are present in cSMCs, we immunoblotted for the phorbol ester–sensitive PKC isoforms , ß, , , , , and the PKC isoforms , , µ, . In Western blot analysis (Figure 6) PKC showed an expression of a single band at 82 kDa, PKC at 90 kDa, and PKC at 74 kDa. No immunoreactivity for PKC ß, , and was observed. In addition, PKC (80 kDa), (74 kDa), µ (115 kDa), and (72 kDa) were strongly expressed. Thus, the Western blot results show that cSMCs express at least 3 phorbol ester–sensitive PKC isoforms that are candidates to mediated bFGF-stimulated cSMC mitogenesis.

View larger version (27K): [in this window] [in a new window]   Figure 6. Western blot analysis of PKC isoforms in cSMCs. Equivalent amounts of protein lysate from cSMCs were subjected to SDS-PAGE, transferred to a nitrocellulose membrane, and incubated with antibodies specific for each of the PKC isoforms. The immunoreactive protein was visualized by use of an alkaline phosphatase detection system. Western blot was repeated 4 times. *Phorbol ester–sensitive PKC isoform.

Finally, we tested whether PKC might be regulating proliferation by affecting bFGF-induced phosphorylation of MAPK and induction of c-myc in cSMCs. To downregulate the PKC-mediated pathway, quiescent cSMCs were preincubated with 0.1 µmol/L PMA (72 hours) and stimulated with bFGF (2 ng/mL) for 1 hour. As shown in Figure 7A, treatment with bFGF resulted in the tyrosine phosphorylation of MAPK; in contrast, PMA-pretreated cells exhibited significantly attenuated MAPK phosphorylation in response to bFGF stimulation.

View larger version (13K): [in this window] [in a new window]   Figure 7. Effects of PKC inhibitors on the bFGF-induced MAPK phosphorylation and c-myc expression in cSMCs. A, Quiescent cSMCs were treated with (0.1 µmol/L) or without (control) PMA for 72 hours. The cells were then stimulated for 60 minutes with bFGF (2 ng/mL) and processed as described for Figure 1A. Molecular weight marker is indicated on the right. Similar results were obtained in 4 additional independent experiments. B, Quiescent cells were treated with (2.5 µmol/L) or without (control) chelerythrine for 48 hours and then incubated with bFGF (2 ng/mL) for 60 minutes. The membrane was probed with a monoclonal anti–c-myc antibody. Similar results were obtained in 2 additional independent experiments.

Furthermore, we determined the involvement of PKC in the bFGF-mediated induction of c-myc in cSMCs. Experiments shown in Figure 7B, demonstrate that the bFGF-induced expression of c-myc protein is inhibited by a chelerythrine pretreatment. This result was confirmed by bFGF stimulation of PMA-pretreated cSMCs (data not shown).

	Discussion

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The data of this study demonstrate that the growth-promoting effects of bFGF correlate with phosphorylation of MAPK, as well as the phosphorylation of and a marked increase in c-myc, a downstream target of MAPK signals. Furthermore, we show that PMA-sensitive PKC isoforms mediate the bFGF-induced DNA synthesis in part by activating the MAPK cascade in cSMCs.

To investigate the involvement of MAPK in the stimulation of DNA synthesis by bFGF in cSMCs we used the synthetic compound PD98059, which selectively blocks the phosphorylation and activity of MEK by Raf-1 and, as a consequence, blocks the phosphorylation and activity of p42 and p44 MAPK isoforms.^{22 25} PD98059 is a valuable tool to inhibit the cellular activity of MAPK in cSMCs and, in fact, PD98059 inhibits bFGF-induced DNA synthesis in a dose-dependent manner. This demonstrates the importance of the Raf-1-MEK-MAPK cascade in bFGF-induced proliferation in cSMCs.

Furthermore, we showed that PD98059 significantly inhibited the ability of bFGF to stimulate the c-myc protein expression in cSMCs, demonstrating the involvement of the MAPK cascade in the bFGF-induced expression of the transcription factor c-myc in cSMCs. These findings are in accordance with the data of Seth et al²⁶ who have shown that the signaling role of MAPK within the nucleus is characterized by phosphorylation at Ser⁶² of the c-myc protein. Phosphorylation at Ser⁶² stimulated the activity of the NH₂-terminal transactivation domain of c-myc,²⁶ which is known to be involved in the regulation of gene expression.²⁷

However, the most important finding in the present study is that PMA-sensitive PKC isoforms mediate the bFGF-induced DNA synthesis in part by activating the MAPK cascade. General observations argue in favor of this hypothesis: first, bFGF stimulated a long-lasting increase of the PKC activity which is required to induce proliferation in endothelial cells,²⁸ and second, treatment of the cells with the specific PKC inhibitor chelerythrine inhibited, dose-dependently, the growth-promoting effects of bFGF.

To characterize the PKC isoforms involved, PKC was downregulated by prolonged PMA incubation, which led to a significant reduction in the quantity of phorbol ester–sensitive PKC isoforms (Yamamura et al²⁹ and Skaletz-Rorowski et al, unpublished results). Our data demonstrate that PKC downregulation reduced bFGF-induced DNA synthesis and cell number and indicate that phorbol ester–sensitive isoforms of PKC are involved in this mitogenic process.

We next identified the pattern of expression of PKC isoforms in cSMCs. The phorbol ester–sensitive PKC isoforms , , and are possible candidates for bFGF-induced proliferation in cSMCs. The involvement of PMA-sensitive PKC isoforms in bFGF-induced proliferation is supported by the data of Haller et al³⁰ who have observed that bFGF induced a rapid association of the PMA-sensitive isoforms and with nuclear structures to induce endothelial cell growth. Furthermore, Hrzenjak and Shain³¹ have shown that bFGF promoted activation of PKC and in rat prostate cancer cells and suggested that the effector-mediated cell proliferation is achieved by processes involving both of these PKC isoforms. In addition, the effects of PMA-sensitive PKC isoforms and on cellular growth have been examined by overexpressing PKC and in selected cell lines. Overexpression of PKC had little effect on fibroblast growth, whereas overexpression of PKC increased the growth rate significantly.²⁴ Besides PKC and , the phorbol ester–sensitive PKC isoform that was detected in cSMCs in this study may be a candidate for bFGF-mediated proliferation, too. A possible role of PKC in the growth factor–mediated proliferation is supported by recent data demonstrating a relationship between the inhibition of proliferation by suramin and a suramin-dependent suppression of the activity of the PKC isoform .³²

The effect of PKC on proliferation in our study is, in part, mediated by activation of MAPK: chelerythrine or PMA treatment reduces the ability of bFGF to phosphorylate MAPK and to phosphorylate and increase c-myc expression. This is in compliance with previous studies which have shown that, after PKC downregulation, both angiotensin II- and platelet-derived growth factor (PDGF)–stimulated MAPK activation were substantially reduced, demonstrating the PKC involvement in the growth factor–stimulated MAPK pathway in aortic SMC.³³

The relationship between PMA-sensitive PKC isoforms and MAPK cascade in mitogenic processes is supported by some reports.^{34 35} Thus, Raf-1 can be activated by a PKC –mediated direct phosphorylation in NIH3T3 fibroblasts.³⁴ Recent results of Perletti et al³⁵ suggested that PKC –induced proliferation of rat colonic epithelial cells is associated with increased Raf-1 activation. Furthermore, the specific role of PKC in the growth factor–induced proliferation is supported by the data of Malarkey et al,³⁶ who have shown that PKC is involved in the angiotensin II–induced activation of MAPK. In addition, using recombinant baculoviruses expressing PKC and Raf polypeptides, Sozeri et al³⁷ have shown that, besides the conventional PKC isoforms and ß, the PKC isoform is able to activate Raf on coexpression in insect cells.

Taken together, our data contribute to clarifying the mechanism of the bFGF-induced PKC-dependent proliferation of cSMCs and suggest a signal transduction cascade which has the possible sequence: PMA-sensitive PKC isoformsRaf-1MEKMAPKc-myc. Further investigation is required to fully understand the exact mechanisms of bFGF-induced PKC activation and to identify the PKC isoform(s) involved in activation of MAPK and c-myc leading to bFGF-stimulated cSMC proliferation.

	Acknowledgments

 
The authors thank Marion Ziebarth, Annette Thormann, Barbara Glaß, and Marianne Opalka for skillful technical assistance; and Dr Bodo Levkau for helpful discussions. The work presented in this manuscript has been awarded the Prevention Price of the Deutsche Herzhilfe e.V.

Received March 6, 1998; accepted December 10, 1998.

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