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Cardiovascular Research 2005 67(1):142-150; doi:10.1016/j.cardiores.2005.03.009
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Copyright © 2005, European Society of Cardiology

PKC {delta}-induced activation of MAPK pathway is required for bFGF-stimulated proliferation of coronary smooth muscle cells

Adriane Skaletz-Rorowski*, Heike Eschert, Jutta Leng, Birgit Stallmeyer, Jürgen R. Sindermann, Ewa Pulawski1 and Günter Breithardt

Leibniz-Institute for Arteriosclerosis Research, University of Münster, Domagkstr. 3, 48149 Münster, Germany and Department of Cardiology and Angiology, University of Münster, Albert-Schweitzer Str. 33, 48129 Münster, Germany

* Corresponding author. Tel.: +49 251 83 55326; fax: +49 251 83 52980. Email address: skaletz{at}uni-muenster.de

Received 25 November 2004; revised 17 February 2005; accepted 10 March 2005


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Basic fibroblast growth factor (bFGF)-stimulated proliferation of coronary smooth muscle cells (cSMC) contributes to the pathogenesis of arteriosclerosis and restenosis. However, the molecular mechanisms involved are not fully understood. We have shown previously that protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) are required for the bFGF-stimulated mitogenic process in bovine cSMC. In this study, we determined the PKC isoform(s) involved and investigated their functional role in the bFGF-stimulated signaling and cell cycle progression in human and bovine cSMC.

Methods and Results: Downregulation of PKC by phorbol 12-myristate 13-acetate (PMA) inhibited bFGF-induced DNA synthesis, the activation of MAPK, and the expression of c-myc, demonstrating the involvement of PMA-sensitive PKC isoforms in growth factor-induced proliferation and the MAPK pathway. The PMA-sensitive classical PKC isoforms {alpha}, β, {gamma}and novel PKC isoforms {delta} and {varepsilon} were found in human cSMC. Whereas blocking of the classical PKC isoforms had no influence, the suppression of PKC {delta} by genetic and pharmacological approaches inhibited the bFGF-stimulated c-Raf1–MEK–MAPK–c-myc signaling and DNA synthesis in cSMC. In contrast to PKC {varepsilon}, our results showed that bFGF activated PKC {delta} by phosphorylation in a time-dependent manner. In addition, inhibition of PKC {delta} induced a hypophosphorylation of the retinoblastoma protein and suppression of the cyclins D1 and A, demonstrating the importance of PKC {delta} for bFGF-induced cell cycle progression through the G1 phase in cSMC.

Conclusions: Our results show that PKC {delta} is required for the bFGF-stimulated c-Raf1–MEK–MAPK–c-myc signaling pathway involved in the proliferation of cSMC. Therefore, it may be an interesting therapeutic target for preventing proliferative vascular disorders.

KEYWORDS Growth factors; MAP kinase; Signal transduction; Smooth muscle cells; Protein kinase C


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Growth factor-induced smooth muscle cell (SMC) proliferation in the intimal area of vascular vessels plays an important role in the development of arteriosclerotic lesions [1,2]. During the initial stages SMC migrate into the intimal layer of the arterial wall where the cells reenter the cell cycle in response to mitogens. Cell cycle re-entry is imposed by activation of molecules consisting of cyclin regulatory subunits and cyclin-dependent kinases (CDK). G1 phase progression requires cyclin D1-dependent CDK complexes [3] which induce the hyperphosphorylation of the retinoblastoma protein (pRb). This induces the release of the transcription factor E2F with a consecutive activation of genes required for further cell cycle progression, including cyclin A [4].

Basic fibroblast growth factor (bFGF), synthesized by vascular SMC [5], is a powerful growth factor for SMC cycle progression [6] during arteriogenesis [7] and in response to vessel wall injury [8]. A prior study showed that the tyrosine kinase receptor–mitogen-activated protein kinase (MAPK) pathway is required for bFGF-induced proliferation [9]. Moreover, our previous data indicated that phorbol 12-myristate 13-acetate (PMA)-sensitive protein kinase C (PKC) isoforms contribute to this proliferative process in bovine cSMC [10]. The PKC isoforms play different roles in cell function [11] and are classified on the basis of their PMA-sensitivity and Ca2+-dependence into three subfamilies: the PMA-sensitive and Ca2+-dependent classical PKC, the PMA-sensitive but Ca2+-independent novel PKC and the PMA- and Ca2+-independent atypical PKC [12]. The purpose of the present study was to determine which specific PMA-sensitive PKC isoform(s) is involved in bFGF-induced proliferation and how the specific PKC isoform(s) and the bFGF-induced signal transduction pathway are merged in cSMC.

Here, we demonstrate that the novel PKC isoform {delta} is required for the bFGF-induced cell cycle progression through the G1 phase by activating the c-Raf1–MEK–MAPK–c-myc signaling pathway in human and bovine cSMC.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Materials
[3H]thymidine (1.10 Tbq/mM) was obtained from Amersham Buchler (Braunschweig, Germany) and chemicals for SDS-PAGE were products of AppliChem (Darmstadt, Germany). Antibodies against pRb were from PharMingen (San Diego, CA, USA), antibodies against p42 MAPK, phospho-MAPK, phospho-MEK, MEK and phospho-PKC {delta} (Thr 505) were obtained from NEB (Beverly, MA, USA), anti-PKC {alpha}, β, {varepsilon}, {gamma}, {theta}, {eta}, {zeta} and c-Raf1 antibodies were supplied by Transduction Laboratories (Lexington, KY, USA) and anti-phospho-PKC {varepsilon} (Ser 719) was from Upstate Biotechnology (Lake Placid, NY, USA). Anti-cyclin A, E and D1, anti-c-myc and PKC {delta} antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The PKC inhibitors rottlerin and Gö6976 were from Calbiochem-Novobiochem (Schwalbach, Germany) and the human recombinant bFGF from PromoCell (Heidelberg, Germany).

2.2. Cell culture and viability
Human cSMC (PromoCell) were cultured in Smooth Muscle Cell Growth Medium-2 (SmGM-2, PromoCell) at 37 °C in a humidified, 5%CO2/95% air atmosphere. cSMC from passages two to five were used. To analyze DNA synthesis, protein levels and the phosphorylation status of proteins under serum starved conditions human cSMC were incubated in a low-serum DMEM (AppliChem) supplemented with 0.125% FCS to prevent cells from undergoing cell death. Subsequently, the cells were treated with or without inhibitors for 24 h, followed by bFGF (15 ng/ml) stimulation. For transfection experiments bovine cSMC were used, because our results have shown that in contrast to human cSMC, bovine cSMC presented a higher transfection efficiency. Bovine cSMC were cultured in DMEM supplemented with 10% FCS. Serum-starved cells were established by a serum-free incubation for 48 h. Cell viability of human and bovine cSMC was measured via trypan blue exclusion and a LIVE/DEAD fluorescence-based viability/cytotoxicity kit (Molecular Probes, Eugene, OR, USA).

2.3. Downregulation of PKC and specific inhibition of PKC isoforms
PMA-sensitive PKC isoforms were downregulated by treating serum-starved cSMC with 0.1 µM PMA for 72 h as described previously [10]. The protein downregulation was verified by Western blot. Classical PKC isoforms were inhibited (24 h) by Gö6976 (10–500 nM) and the novel PKC isoform {delta} was blocked (24 h) by rottlerin (0.05–0.5 µM).

2.4. Western blot analysis
Cell lysates and SDS-PAGE were processed as described previously [10]. After blocking with 3% non-fat dry milk, incubation with a specific antibody and horseradish peroxidase-conjugated secondary antibody, the immunoreactivity was visualized using the ECL chemiluminescence detection system (Amersham, Arlington Heights, IL, USA). Samples analyzed by anti-phospho-p42 MAPK, anti-phospho MEK and anti-phospho PKC {delta} antibodies were controlled for the quantification of protein content by p42-MAPK, MEK and PKC {delta} antibodies. Blots were treated with an anti-alpha-tubulin antibody (Sigma, Taufkirchen, Germany) as a control for protein loading.

2.5. Northern blot analysis
Total RNA of human cSMC was prepared as described previously [13]. Digoxigenin labelled (Roche Diagnostics, Mannheim, Germany) RNA run-off transcripts were generated from human PKC {alpha}, β1, {delta}, {varepsilon} and {gamma} gene sequence (ATCC, Manassas, VA, USA).

2.6. DNA synthesis assay
Seeded into dishes (35 mm diameter) were 50.000 human cSMC in SmGM-2 and cultured for 4 days (25.000 bovine cSMC were seeded into DMEM supplemented with 10% FCS). After an incubation of 48 h low serum-starved human cSMC and serum-free incubated bovine cSMC were treated with different concentrations of PKC inhibitors (24 h) before bFGF was added. DNA synthesis was measured as described previously [10].

2.7. Transfection with antisense oligodeoxynucleotides
Phosphorothioate-modified ODNs were purchased from Eurogentec (Seraing, Belgium). The antisense sequences used were 5'-GGC-GAT-GCG-CAG-GAA-CGG-3' for PKC {delta}, 5'-GAA-CAC-TAC-CAT-GGT-CGG-3' for PKC {varepsilon} and 5'-AAC-GTC-AGC-CAT-GGT-CCC-3' for PKC {alpha}. The antisense sequences are based near the ATG start codon. The sequences of the sense ODNs were synthesized as follows: 5'-CCG-TTC-CTG-CGC-ATC-GCC-3' for PKC {delta}, 5'-CCG-ACC-ATG-GTA-GTG-TTC-3' for PKC {varepsilon} and 5'-GGG-ACC-ATG-GCT-GAC-GTT-3' for PKC {alpha}. ODNs were modified by fluorescein in order to prove transfection efficiency. Each ODN was screened for complementary gene sequences in the Genbank and EMBL databases using the DNA search program BLASTN. None of the ODNs were found to have complementary hybridization sequence to genes other than their intended targets.

As human cSMC were characterized by a very low transfection efficiency, bovine cSMC were used for this set of experiments. In contrast to our previous work which failed to detect PKC {delta} protein expression in bovine cSMC [10], the use of the anti-PKC {delta} antibody (C-20) from Santa Cruz Biotechnology (Santa Cruz, CA, USA) confirmed the expression of the PKC {delta} protein by Western blot in bovine cells.

The amount of 1 x 105 bovine cSMC was seeded in DMEM supplemented with 10% FCS and cultured for 24 h. cSMC were transfected in serum-free medium with a premixed ODN(400 nM)/DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate) solution (Roche Diagnostics, Mannheim, Germany) for 6 h at 37 °C. After an incubation (18 h) in fresh DMEM, serum-starved cells were stimulated with 6 ng/ml bovine bFGF (Roche Diagnostics) and prepared for Western blot analysis as described [10].

2.8. Vectors and the transient transfection assay
pSRD vectors expressing wild-type PKC {delta} (wtPKC-{delta}) and dominant negative PKC {delta} (dnPKC-{delta}) were generously provided by Prof. Shigeo Ohno (Yokohama City University School of Medicine, Yokohama, Japan) and have been described previously [14–16]. Bovine cSMC were seeded at a density of 1 x 105 cells/dish (60 mm diameter) in DMEM supplemented with 10% FCS and grown for 24 h. We introduced plasmid DNA (18 µg) into the cells by using Transfast (1 µg DNA:6 µl Transfast) following the manufacturer's instructions (Promega Corp., Madison, Wi, USA). Transfection was conducted in 1.5 ml serum-free medium for 1 h and subsequently in 10% FCS medium for an additional 23 h. Cells were washed twice with HANKS (AppliChem) and serum-starved for 48 h before the addition of bFGF (6 ng/ml). [3H]thymidine DNA incorporation was evaluated as described. Values are the means ± S.D. of three experiments performed in triplicate.

2.9. Other methods and statistics
Cell counting and protein determination were performed according to standard methods. Results are expressed as mean ± S.D. of the specified number of experiments carried out on different cSMC cultures in duplicate or triplicate. Statistical significance between 2 groups was assessed using the unpaired Student's t-test and ANOVA among more than 2 groups. A value of P<0.05 was considered to indicate significant difference.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. PMA-sensitive PKC isoforms are required for bFGF-induced mitogenic pathway in human cSMC
Our previous data showed that downregulation of PMA-sensitive PKC isoforms which belong to the classical and novel PKC subfamilies significantly inhibited the bFGF-induced proliferation in bovine cSMC [10]. Our present data demonstrated that downregulation of classical and novel PKC isoforms by PMA (Fig. 1A, insert) significantly inhibited DNA synthesis after bFGF stimulation in human cSMC (Fig. 1A). Furthermore, as demonstrated by Western blot PMA-sensitive PKC isoforms were involved in the bFGF-induced MAPK pathway (Fig. 1B) and in the upregulation of the MAPK-dependent transcription factor c-myc (Fig. 1C). To determine candidates for the PKC isoforms involved in human cSMC proliferation, we studied the mRNA levels of the PMA-sensitive PKC isoforms at two different stages during the cell growth. As summarized in Fig. 1D, human cSMC expressed the mRNA of the classical PKC {alpha}, β, {gamma} and of the novel PKC isoforms {delta} and {varepsilon}. Western blot analysis verified the data of the Northern blot analysis and indicated that the novel PKC isoforms {theta} and {eta} could not be detected in human cSMC (data not shown).


Figure 1
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  		Fig. 1 Involvement of PMA-sensitive PKC isoforms in DNA synthesis, MAPK–c-myc pathway and proliferation in human cSMC. PMA pre-treatment downregulated the expression of classical and novel PKC isoforms (A, insert), inhibited the bFGF-induced DNA synthesis (A), the bFGF-induced phosphorylation of MAPK (B) and the c-myc protein expression (C). For all experiments serum-starved (control) and PMA-treated cSMC were stimulated with bFGF (15 ng/ml) for indicated times. (A) Western blot analysis shown in insert demonstrates that PMA (0.1 µM) pre-treatment downregulated the expression of the classical PKC isoform {gamma} and of the novel PKC isoform {delta}, but not of the atypical PKC isoform {zeta}. DNA synthesis was measured as described in Methods. *P<0.05 vs. control; #P<0.05 vs. bFGF. (B) Upper panel: for Western blot analysis membranes were probed with an anti-phospho-MAPK antibody that recognized the 42-kDa isoform of MAPK and crossreacts with the 44-kDa MAPK isoform. Samples analyzed by anti-phospho-p42 MAPK antibodies were normalized to protein content of p42-MAPK, respectively. Blots were treated with an anti-alpha-tubulin antibody as a control for protein loading. Similar results were obtained in 3 additional experiments. Lower panel: quantification of the bFGF-induced phosphorylation status of MAPK in PMA-treated cells; *P<0.001 vs. control, #P<0.01 vs. bFGF (15 min). (C) Upper panel: effect of PMA on c-myc protein expression in bFGF-stimulated cSMC. Lower panel: quantification of the c-myc protein expression; *P<0.01 vs. control, #P<0.01 vs. bFGF. (D) Densitometry of Northern blot analysis shows the growth status-dependent expression of PMA-sensitive PKC isoforms in human cSMC. The mRNA expression was calculated as ratio of PKC isoform and GAPDH signals. Among the detected PMA-sensitive PKC isoforms the novel isoform {delta} was most abundantly expressed during the proliferating growth status. Northern blot analysis was repeated 3 times. Confluent growth status did not significantly affect cell viability compared with proliferating cSMC (proliferating growth status: 3 days; confluent growth status: 7 days after seeding).

 
3.2. Classical PKC isoforms are not involved in the bFGF-induced proliferation
To examine whether the proliferative effect was mediated via the classical PKC isoform(s) we measured the content of bFGF-induced DNA synthesis after exposure of human cSMC to Gö6976, a specific inhibitor of classical PKC isoforms. As shown in Fig. 2A, treatment with Gö6976 at various concentrations had no influence on bFGF-stimulated DNA synthesis in human cSMC demonstrating that classical PKC isoforms are not involved in the bFGF-induced proliferation. Further results obtained by Western blot analysis showed that the inhibition of the classical PKC had no effect on the bFGF-stimulated activation of the MAPK pathway in human cSMC (Fig. 2B). In accordance with these results, treatment of bovine cSMC with Gö6976 (10–500 nM) had no effect on the cSMC proliferation and the bFGF-induced MAPK pathway (data not shown).


Figure 2
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  		Fig. 2 Inhibition of classical PKC isoforms and the influence on the bFGF-induced DNA synthesis and MAPK phosphorylation in human cSMC. Gö6976 treatment had no effect on bFGF-stimulated DNA synthesis (A) and bFGF-induced MAPK phosphorylation (B). Serum-starved cSMC (control) were pre-treated with Gö6976 (24 h) at indicated concentrations and subsequently stimulated with bFGF (15 ng/ml) for 24 h (DNA synthesis) or indicated times (Western blot). [3H]thymidine incorporation studies represent means of 3 experiments performed in triplicate. *P<0.05 vs. control.

 
3.3. The novel PKC isoform {delta} is required for the bFGF-induced proliferation in cSMC
As our data suggested that classical PKC isoforms were not involved in bFGF-stimulated proliferative effects, we studied the role of the novel PKC isoforms {delta} and {varepsilon} in bFGF-induced mitogenic processes. First, we investigated if bFGF induces the activation of these isoforms by phosphorylation. Western blot analysis showed that bFGF induced PKC {delta} phosphorylation in a time-dependent manner at the Thr 505 residue, which is important for determining the PKC activity (Fig. 3A, upper panel). The phosphorylated form of PKC {delta} was already detected after 15 min, reached a maximum after 1 h and returned to basal level after 7 h in human cSMC (Fig. 3A, lower panel). In contrast to PKC {delta}, no bFGF-induced effect on the phosphorylation status of PKC {varepsilon} at Ser 719, the residue involved in the activation of the other novel PKC isoform expressed in human cSMC, was detected (Fig. 3A).


Figure 3
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  		Fig. 3 The novel PKC isoform {delta} is involved in bFGF-induced proliferation. (A) bFGF induced PKC {delta} phosphorylation in human cSMC. Serum-starved cSMC were untreated (control) or incubated with bFGF (15 ng/ml) for indicated times. Upper panel: endogenous PKC {delta} protein and phosphorylation at Thr 505 as well as the phosphorylation of the PKC isoform {varepsilon} at Ser 719 was detected by Western blot. Similar results were obtained in four additional experiments. Lower panel: quantification of the phosphorylation status of PKC {delta} and {varepsilon}. Samples were normalized to the PKC protein content; *P<0.05 vs. control. (B) Effect of rottlerin on bFGF-stimulated DNA synthesis in human cSMC. Serum-starved cSMC were untreated (control) or treated with rottlerin (24 h) at indicated concentrations and stimulated with bFGF (15 ng/ml) for 24 h. Values represent means of three independent experiments performed in triplicate; *P<0.05 vs. control; #P<0.05 vs. bFGF. (C) DNA synthesis in dnPKC-{delta}, wtPKC-{delta} and pSRD transfected bovine cSMC. Transfected cells were serum-starved (48 h) before bovine bFGF (6 ng/ml) was added for 24 h. [3H]thymidine DNA incorporation was evaluated as described. Values are means of five independent experiments performed in triplicate. The insert demonstrates the expression of the PKC {delta} protein in transfected bovine cSMC by Western blot. {dagger}{dagger}P<0.001 vs. unstimulated cSMC; {dagger}P<0.05 vs. unstimulated cSMC; *P<0.05 vs. bFGF-stimulated pSRD-transfected cells; #P<0.05 vs. bFGF-stimulated wtPKC-{delta}-transfected cells.

 
Second, in order to prove the involvement of PKC {delta} in bFGF-induced proliferation we examined the effect of the PKC {delta} specific inhibitor rottlerin on the bFGF-induced DNA synthesis in cSMC. Rottlerin significantly inhibited the ability of bFGF to stimulate DNA synthesis in human (Fig. 3B) and bovine cSMC (data not shown) in a dose-dependent manner. Treatment with 1 µM rottlerin did not further decrease bFGF-induced DNA synthesis (data not shown).

Finally, we examined the effect of a dominant negative mutant of PKC {delta} (dnPKC-{delta}) on the bFGF-stimulated proliferation. Bovine cSMC were used for the transfection experiments, because these cells were characterized by a higher transfection efficiency than human cSMC. In accordance to human cSMC, the protein expression of PKC {delta} in bovine cSMC was confirmed by Western blot analysis with an anti-human PKC {delta} antibody which crossreacted with bovine PKC {delta} protein (Fig. 3C, insert). The empty vector (pSRD) was transfected as a control. As shown in Fig. 3C, transfection of dnPKC-{delta} significantly decreased the bFGF-induced DNA synthesis in cSMC. In contrast, transfection of the wild type PKC {delta} DNA (wtPKC-{delta}) increased the bFGF-induced DNA synthesis in cSMC compared to bFGF-stimulated pSRD-transfected cells (Fig. 3C).

3.4. PKC {delta} mediates bFGF-induced proliferation through the c-Raf1–MEK–MAPK–c-myc pathway in cSMC
Further to analyze the role of the novel PKC isoforms {delta} and {varepsilon} in the bFGF-induced mitogenic signaling pathway, we applied antisense PKC {delta} and PKC {varepsilon} ODN to specifically suppress the expression of these isoforms in cSMC. As described in Methods these experiments were performed with bovine cSMC because these cells were characterized by a higher transfection efficiency than human cSMC. As shown in Fig. 4, downregulation of the PKC {delta} expression by antisense PKC {delta} ODN led to a clear inhibition of the bFGF-induced phosphorylation of MAPK, but had no effect on the MAPK protein expression. In contrast to PKC {delta} antisense ODN, antisense ODN against PKC {varepsilon} did not affect the bFGF-induced phosphorylation of MAPK (Fig. 4). Furthermore, treatment with sense PKC {delta} and sense PKC {varepsilon} ODN had no effect on the MAPK phosphorylation status (Fig. 4).


Figure 4
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  		Fig. 4 The novel PKC isoform {delta} is required for bFGF-induced phosphorylation of MAPK in bovine cSMC. Antisense PKC {delta} ODN specifically inhibited bFGF-induced MAPK phosphorylation. Serum-starved cSMC were untreated (control), treated with DOTAP (*) or with premixed ODN/DOTAP solutions and stimulated with bFGF (6 ng/ml) for indicated times. Upper panel: phosphorylation status of MAPK and endogenous MAPK protein expression were detected by Western blot. Similar results were obtained in three independent experiments. Lower panel: quantification of the MAPK phosphorylation status in antisense or sense PKC {delta} ODN treated cells. *P<0.05 vs. unstimulated cSMC; #P<0.05 vs. bFGF-stimulated cells.

 
Since our data indicated that PKC {delta} activates MAPK in cSMC, we subsequently analyzed whether PKC {delta} was able to stimulate the c-Raf1–MEK–MAPK–c-myc pathway in cSMC. Therefore we studied the effect of the PKC {delta} inhibitor rottlerin on the MAPK phosphorylation and on the expression and phosphorylation of c-Raf1 and MEK, which are both activators of MAPK. As shown in Fig. 5A (upper and lower panel), treatment with rottlerin significantly inhibited the bFGF-induced MAPK phosphorylation in human cSMC in a dose-dependent manner.


Figure 5
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  		Fig. 5 Inhibition of PKC {delta} prevented bFGF-induced activation of the c-Raf1–MEK–MAPK–c-myc pathway and the induction of cell cycle regulatory proteins in human cSMC. Serum-starved human cSMC were untreated (control) or treated with rottlerin at indicated concentrations prior to stimulation with bFGF (15 ng/ml) for 30 min (A) or indicated times (B, C). (A) Upper panel: Western blot analysis demonstrates the bFGF-induced phosphorylation status and expression of MAPK in rottlerin treated cSMC. Lower panel: quantification of the phosphorylation status of MAPK; *P<0.001 vs. control, #P<0.01 vs. bFGF, ##P<0.001 vs. bFGF. (B) Upper panel: the effect of rottlerin (0.5 µM) on c-Raf1, c-myc and MEK as well as the phosphorylated (Ser217/221) form of MEK in bFGF-stimulated cSMC. The phosphorylated (~74 kDa) and the unphosphorylated (~70 kDa) forms of c-Raf1 are indicated by lines. Experiments were repeated four times. Lower panel: quantification of the bFGF-induced phosphorylation status of c-Raf1, MEK and of the c-myc expression under rottlerin treatment; *P<0.001 vs. control, {dagger}P<0.001 vs. control, {ddagger}P<0.01 vs. control, **P<0.05 vs. bFGF (15 min), {dagger}{dagger}P<0.01 vs. bFGF (15min), {ddagger}{ddagger}P<0.01 vs. bFGF. (C) Upper panel: the effect of rottlerin on the bFGF-stimulated pRb phosphorylation and the expression of cyclins D1, E and A. In comparison with the mitogenic signaling pathway, the influence of bFGF on cell cycle regulatory proteins is a rather late event. Therefore, we investigated these effects at a time period of 1–36 h. Hypophosphorylated (pRb) and hyperphosphorylated (ppRb) forms of pRb are indicated by lines. Experiments were performed in quadruplicate. Lower panel: quantification of ppRb, cyclin D1, E and A expression under rottlerin treatment. {dagger}P<0.001 vs. control, {dagger}{dagger}P<0.001 vs. bFGF, *P<0.001 vs. control, **P<0.01 vs. bFGF, {ddagger}P<0.001 vs. control, {ddagger}{ddagger}P<0.001 vs. bFGF.

 
Retardation of the c-Raf1 electrophoretic mobility ("band shift") which correlated with increased phosphorylation and activation of c-Raf1 [17] was present in bFGF-treated cells as compared to the unstimulated control (Fig. 5B). PKC {delta} inhibition decreased the c-Raf1 retardation and the c-Raf1 protein expression level (Fig. 5B, upper panel). In addition, rottlerin treatment resulted in the suppression of the bFGF-induced time-dependent phosphorylation of MEK (Fig. 5B, upper panel). As shown in Fig. 5B (lower panel), the inhibitory effect of rottlerin on c-Raf1 and MEK phosphorylation status is significant.

Finally, we determined the influence of PKC {delta} on the bFGF-induced and MAPK-mediated expression of the transcription factor c-myc. As demonstrated in Fig. 5B (upper and lower panel), treatment with rottlerin significantly suppressed the bFGF-induced expression of c-myc in human cSMC. In accordance with human cSMC rottlerin treatment of bovine cSMC inhibited the c-Raf1–MEK–MAPK–c-myc pathway, too (data not shown).

Since c-myc is necessary and sufficient to trigger entry into the S phase of the cell cycle by regulating the expression of G1 phase cyclin D1/cdk complexes [18,19], we examined the effect of PKC {delta} on bFGF-induced expression of cell cycle G1 phase proteins. As shown in Fig. 5C (upper panel), after 24 h stimulation with bFGF, a mobility shift of pRb was observed indicating an increase in pRb phosphorylation which is required for cell cycle progression through the G1 phase. This shift was significantly suppressed in PKC {delta}-inhibited human cells (Fig. 5C, lower panel) and bovine cSMC (data not shown). Moreover, we examined the effect of bFGF on the expression of the G1 phase cyclins D1, E and A (Fig. 5C, upper panel). While bFGF had no effect on the cyclin E expression, it induced the expression of cyclin D1 and A which was significantly inhibited by rottlerin treatment (Fig. 5C, lower panel).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
In the present study we demonstrate that the novel PKC isoform {delta} is linked to the bFGF-induced cell cycle progression through the G1 phase via the c-Raf1–MEK–MAPK–c-myc signaling pathway in cSMC.

4.1. PKC isoform {delta}
In accordance with our previous data carried out on bovine cSMC [10], this study showed that downregulation of PMA-sensitive PKC isoforms significantly decreased the ability of bFGF to activate mitogenic processes in human cSMC. Furthermore, to the best of our knowledge this study represents the first description of the PMA-sensitive PKC isoform pattern expressed in human cSMC. However, the most important finding in the present study is that in human and bovine cSMC the PMA-sensitive PKC isoform {delta} mediates the bFGF-induced proliferation by activating the MAPK pathway. As the inhibition of the classical PKC isoforms failed to prevent the bFGF-induced cell proliferation, we delineated the specific role of the novel PKC isoforms {delta} and {varepsilon} expressed in cSMC. Using different pharmacological and genetic approaches we analyzed the role of the PKC isoforms {delta} and {varepsilon} in the bFGF-induced mitogenic process in detail. We verified that bFGF activated PKC {delta} in a time-dependent manner by phosphorylation at Thr 505. This amino acid is localized at the activation loop and is important for the PKC activity [20]. Recent data of Kitamura et al. demonstrated that the activation of PKC {delta} correlates with the phosphorylation of Thr 505 [21]. Furthermore, it was shown that Thr 505 phosphorylation is involved in growth factor-stimulated PKC {delta} activation [22]. In contrast, using antibodies against the phosphorylated Ser 719 residue of PKC {varepsilon} which is involved in mitogen-stimulated activation of PKC {varepsilon} [23], no bFGF-stimulated effect on the phosphorylation status of PKC {varepsilon} was observed in the present study.

4.2. PKC isoform {delta} and the MAPK pathway
The importance of PKC {delta} for the bFGF-induced mitogenic signaling was revealed by a dominant negative mutant of PKC {delta} which entailed a reduced DNA synthesis in bovine cSMC after bFGF stimulation. Moreover, transfection of bovine cSMC with specific antisense ODN against PKC {delta} inhibited the bFGF-induced phosphorylation of MAPK, whereas an antisense ODN against PKC {varepsilon} had no effect. For all transfection experiments bovine cSMC were used, because these cells were characterized by a higher transfection efficiency than human cSMC. In contrast to our previous study which failed to detect PKC {delta} in bovine cSMC [10], the use of a new anti-PKC {delta} antibody (characterized by a cross reaction with the bovine species) allowed the detection of PKC {delta} protein expression in bovine cSMC as demonstrated in Fig. 3C (insert). Thus, comparable to human cSMC the bovine cSMC express PKC {delta} and are useful tools to define the role of this isoform in mitogenic signaling and proliferation.

In addition to our genetic approaches performed on bovine cSMC, treatment of human and bovine cSMC with the PKC {delta} inhibitor rottlerin suppressed the bFGF-induced activation of MAPK, c-Raf1 and MEK. Since the phosphorylation of the MAPK activators c-Raf1 and MEK was inhibited by rottlerin, we conclude that the PKC isoform {delta} is involved in early steps of the bFGF-induced cell proliferation.

In addition to PKC {delta} dominant negative mutant and antisense experiments, we used rottlerin as a selective inhibitor of PKC {delta} activation (IC50=3–6 µM) [24]. Notwithstanding rottlerin was reported to inhibit the phosphorylation processes in unspecific manner in rat parotid acinar cells [25]. However, this unspecific effect is controversially discussed depending on the cell type used. Thus, Frank et al. showed that rottlerin specifically blocked processes necessary for PKC {delta} activation in vascular SMC [26]. These data are supported by our results showing that rottlerin inhibited the bFGF-induced signaling processes in human cSMC in a dose- and time-dependent manner. Taken together, our data obtained by pharmacological and genetic approaches clearly supported the important role of PKC {delta} in the bFGF-induced activation of the c-Raf1–MEK–MAPK–pathway and cell proliferation in cSMC.

Comparable to our data Ueda et al. demonstrated that PKC {delta} activates the MEK–MAPK pathway in a c-Raf1-dependent manner in TPA- and EGF-stimulated COS1 cells [16].

Although the results of our study clearly demonstrate that PKC {delta} activates the bFGF-induced MAPK–c-myc pathway in a c-Raf1-dependent manner, a slight activation of the MAPK pathway (~34%) was also observed in PKC {delta} inhibited cells. We suggest that besides the PKC {delta}-dependent activation a PKC {delta}-independent stimulation of the MAPK pathway is involved in bFGF-induced proliferation of cSMC. A PKC-independent activation of the c-Raf1-MEK-MAPK pathway was already discussed for choriocapillary endothelial cells [27].

4.3. PKC isoform {delta} and the cell cycle progression
In the present study we demonstrated a connection between PKC {delta} and the MAPK–c-myc pathway. Previous data showed that the transcription factor c-myc linked the MAPK pathway to the G1–S phase cell cycle progression [28]. Correspondingly, we found that the inhibition of PKC {delta} suppressed the bFGF-induced effects on G1–S phase cell cycle machinery such as the upregulated expression of cyclins D1 and A as well as the pRb hyperphosphorylation. In accordance with our results obtained by human and bovine cSMC experiments it has been reported that PKC {delta} plays an important role in PDGF-induced G1–S cell cycle progression in NIH3T3-derived cell lines [29]. In contrast, in experiments with rat SMC cell line A7r5 which stable overexpress PKC {delta}, this PKC isoform inhibited the cell proliferation [30]. In this context, using transiently overexpressing PKC {delta} cells Kitamura and co-workers showed that PKC {delta} played a role in the late G1 phase through stimulation of the DNA synthesis [21]. Furthermore, their data demonstrated that cell lines stable overexpressing PKC {delta} were characterized by an inhibition of the G2–M transition. The authors suggest that a possible reason for this antiprolifertive effect may be a PKC {delta}-independent process. This process can be caused by a general influence of the stable transfection method on the expression of proteins and mRNAs involved in growth regulation. Taken together, the data of Kitamura and co-workers imply the general importance of PKC {delta} to promote cell cycle progression. This is in agreement with our data showing PKC {delta} as an initial part of the bFGF-induced c-Raf1–MEK–MAPK–c-myc pathway and ultimately as an activator of the G1 phase cell cycle progression in cSMC.


   Acknowledgements
 
A.S.-R. was supported by the Lise-Meitner Fellowship of the "Ministerium für Schule und Weiterbildung, Wissenschaft und Forschung des Landes Nordrhein-Westfalen", by the German–Israeli Foundation-Young Scientists Program and in part by "Innovative Medizinische Forschung der Westfälischen Wilhelms-Universität Münster".


   Notes
 
1 Present address: Department of Cardiology, Heart and Diabetes Center Bad Oeynhausen, Germany. Back

Time for primary review 18 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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