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Cardiovascular Research 1999 41(3):641-653; doi:10.1016/S0008-6363(98)00261-2
© 1999 by European Society of Cardiology
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Copyright © 1999, European Society of Cardiology

Platelet-derived growth factor induces cellular growth in cultured chick ventricular myocytes1

Tatsuya Shimizua,*, Koh-ichiro Kinugawaa, Atsushi Yaoa, Yasuyuki Sugishitaa, Kazuro Sugishitaa, Kazumasa Haradaa, Hiroshi Matsuia, Osami Kohmotob, Takashi Serizawab and Toshiyuki Takahashia

aThe Second Department of Internal Medicine, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
bThe Second Department of Internal Medicine, Saitama Medical School, 38 Morohongo, Moroyama-cho, Iruma-gun, Saitama 350-0451, Japan

* Corresponding author. Tel.: +81-3-3815-5411 ext. 3076; Fax: +81-3-3814-0021; E-mail: ruriko25@mxb.meshnet.or.jp

Received 3 March 1998; accepted 11 August 1998


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objectives: Platelet-derived growth factor (PDGF) stimulates growth in various types of cells, but little is known about its effect on cardiac myocytes. Therefore, we examined whether PDGF had a direct effect on cardiac myocytes and investigated their intracellular signaling pathways. Methods: A primary culture of chick embryonic (Hamburger and Hamilton stage 36) ventricular myocytes was prepared. Cellular growth was estimated by 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide assay and 5-bromo-2'-deoxyuridine incorporation assay. The number of PDGF binding sites was measured by binding assay. Induction of c-fos mRNA was analyzed by Northern blot analysis. The binding activity of activator protein (AP)-1 was examined by electrophoretic mobility shift assay. The activation of mitogen-activated protein kinase (MAPK) and signal transducers and activators of transcription (STATs) was analyzed by Western blot analysis, immunoprecipitation, and immunocytochemistry. Furthermore, intracellular Ca2+ concentration ([Ca2+]i) was measured with indo-1 and L-type Ca2+ channel current (ICa) was recorded with the patch clamp technique. Results: PDGF-AB and -BB, but not PDGF-AA, increased viable cell number (5 ng/ml of PDGF-AA, -AB, -BB: 101±4%, 115*±4%, 122*±4%, respectively, n=4, *P<0.05) and DNA synthesis (104±11%, 202*±18%, 295*±25%, respectively, n=4, *P<0.05). Scatchard analysis demonstrated that the maximal number of PDGF-AA, -AB, -BB binding sites was 5±1, 63±12, 126±24 fmol/106 cells, respectively. PDGF-BB provoked induction of c-fos mRNA and increases in binding activity to the AP-1 site. PDGF-BB also induced tyrosine phosphorylation and nuclear translocation of MAPK. The c-fos induction, the increased AP-1 binding activity and the acceleration of DNA synthesis were all attenuated by genistein (100 µM) or MAPK kinase inhibitor (10 or 50 µM PD98059). Interestingly, protein kinase C inhibitor (250 nM calphostin C) attenuated the increases of AP-1 binding activity to some extent, but did not inhibit the c-fos induction at all. The phosphorylation states of STATs were not significantly affected by PDGF-BB. PDGF-BB did not alter [Ca2+]i or ICa. Conclusions: We conclude that PDGF can exert direct effects on embryonic cardiac myocytes and induce their growth. MAPK cascade may play an important role in the PDGF-induced embryonic myocardial growth.

KEYWORDS Culture/isolation; Growth factors; Myocytes; Receptors; Signal transduction


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Platelet-derived growth factor (PDGF) stimulates various types of cells, including smooth muscle cells, fibroblasts, endothelial cells and glia cells, and is implicated in the processes of development, differentiation, wound healing, atherosclerosis and tumorigenesis [1]. However, little is known about its direct effects on cardiac myocytes. Some studies revealed that PDGF stimulated DNA synthesis in cardiac myocytes [2, 3]. Recently, it was demonstrated that PDGF-B gene-deficient mice had perinatal abnormalities, including the dilatation of the heart and the thin walled ventricle [4]. These studies suggest that PDGF may play an important role in embryonic myocardial growth.

As for PDGF-induced signaling pathways, many studies have revealed a network of various signaling molecules [5, 6]. Once PDGF is bound to its receptor, the receptor is autophosphorylated on its tyrosine residues. The autophosphorylated receptors serve docking sites for several molecules that have Src homology region 2 (SH2), including growth factor receptor-bound protein 2 (Grb2). Grb2 activates Raf and mitogen-activated protein kinase (MAPK). PDGF also activates phospholipase C {gamma} and consequently induces phosphatidylinositol turnover and the activation of protein kinase C (PKC). Recently, increasing evidence has indicated that PDGF activates signal transducers and activators of transcription (STATs) [7]as do a wide variety of cytokines and growth factors [8, 9]. Tyrosine-phosphorylated STATs are translocated into the nucleus, and then transactivate the genes including c-fos. These data suggest that PDGF activates various signaling pathways in many types of cells, but there has been no report about the PDGF-induced signaling pathways in cardiac myocytes.

Growth-related intracellular molecular mechanism has been extensively examined after hypertrophic responses in cardiac myocytes. Mechanical stretch [10–12], norepinephrine [13, 14], angiotensin II [15–17], and endothelin-1 [18–20]provoke myocardial hypertrophy. PKC activation, intracellular Ca2+ increase, and MAPK activation have been considered to play central roles in these types of stimuli causing cardiac hypertrophy. The crosstalks of the several signaling pathways in cardiac hypertrophic response have also been reported.

Therefore, the objectives of this study were: (1) to examine whether PDGF could cause growth-promoting effects on cultured chick embryonic ventricular myocytes; (2) to investigate which intracellular signals contributed to chick embryonic myocardial growth.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
The investigation conforms with the Guide for the Care and Uses of Laboratory Animals published by the US National Institutes of Health (NIH publication No.85-23, revised 1985).

2.1 Primary culture of chick embryo ventricular myocytes
Spontaneously contracting chick ventricular cells were prepared according to the previously-described method [21]. Briefly, ventricles from chick embryos at Humberger and Hamilton stage 36 [22]were digested at 37°C in Hank’s solution containing 0.025% trypsin (Gibco BRL). After digestion of four cycles for 8 min each, the resulting cell suspension was incubated in the culture medium consisting of 6% fetal calf serum (Summit), 40% Medium 199 (Gibco BRL), 0.1% penicillin–streptomycin solution (Gibco BRL), 2.7 mM glucose, and 54% balanced salt solution containing 116 mM NaCl, 1.0 mM NaH2PO4, 0.8 mM MgSO4, 1.18 mM KCl, 0.87 mM CaCl2 and 26.2 mM NaHCO3. The cell suspension was appropriately diluted and plated in 60-mm dishes (2·106 cells/dishes) for RNA, protein, and nuclear protein extraction, or in the 100-mm dishes containing 25-mm circular cover slips for immunocytochemistry (1·106 cells/dishes), intracellular Ca2+ measurement (3·106 cells/dishes), and L-type Ca2+ channel current measurement (0.5·106 cells/dishes). Cells were also plated in 24-well plates for 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (0.1·106 cells/well), binding assay (0.3·106 cells/well) and plated in 96-well plates for 5-bromo-2'-deoxyuridine (BrdU) incorporation assay (0.5·105 cells/well). After incubation for 3 days, the culture medium was changed to the serum-free medium consisting of 20% Medium 199, 20% Ham’s F-12 (Gibco BRL), 54% balanced salt solution, 0.6 mM KCl, and 2.7 mM glucose. All experiments were performed after incubation in the serum-free media for at least 24 h.

2.2 Immunocytochemistry
Immunostaining of the cultured cells was performed as described previously [23]. Cells on coverslips were fixed in 4% paraformaldehyde or 100% acetone. The fixed cells were exposed to first antibodies [1:200 diluted monoclonal anti-sarcomeric {alpha}-actin antibody (Sigma), monoclonal anti-smooth muscle {alpha}-actin antibody (Sigma), or 1:500 diluted phospho-specific MAPK antibody (New England Biolabs] in 3% bovine serum albumin (Sigma)–phosphate-buffered saline (PBS) for 1 h at room temperature. After a PBS wash, 1:50 diluted fluorescein isothiocyanate (FITC)-labeled second antibodies (Wako) in 3% bovine serum albumin–PBS were applied for 30 min at room temperature. The coverslips were then mounted in Mowiol 4-88 (Calbiochem) containing 2.5% 1,4-diazabicyclo[2.2.2]octane, and illuminated by 490-nm wavelength excitation light. The emitted fluorescent light passing through a dichroic mirror and filter (525 nm) was collected by an objective lens (Fluor x40, Nikon Diaphot, Japan). The fluorescence image was obtained by means of an image intensified charge coupled device (CCD) camera (Hamamatsu Photonics, Japan). The image collected by the CCD camera was fed to a computed analyzing system (Argus-50, Hamamatsu Photonics). Nuclei of the cultured cells were also stained simultaneously with ethidium bromide (1 µg/ml). Ethidium bromide was applied with FITC-labeled second antibodies. The emitted light (606 nm) by 526-nm wavelength excitation light was collected as described above.

2.3 3-(4,5-Dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide assay
The MTT assay was performed to examine viable cell number as described previously [23]. Cells were cultured in 24-well plates. After exposure to PDGF (0.1~100 ng/ml, Sigma) for 72 h, the media were supplemented with 200 µg/ml MTT (Sigma) for 4 h. The medium was removed, and the cells were harvested in 1 ml of 0.04 M HCl–isopropanol. The extent of MTT reduced to formazan within the cells was quantified by measurement of the ratio of absorbance at 570 and 630 nm and used as an index of the viable cell number. The results were expressed as a percentage of the 570/630-nm absorbance ratio of control cells.

2.4 5-Bromo-2'-deoxyuridine incorporation assay
BrdU incorporation assay was performed to examine DNA synthesis with BrdU labeling and detection kit III (Boehringer Mannheim). Cells were cultured in 96-well plates. After exposure to PDGF for 12 h, the media were supplemented with 10 µM BrdU and incubated for 3 h at 37°C. After fixation with 0.5 M HCl–70% ethanol, cellular DNA was partially digested by being incubated in nuclease solution for 30 min at 37°C. Next, peroxidase-labeled antibodies against BrdU (200 mU/ml) were added to the cells, which were then incubated for 30 min at 37°C. After PBS washing, peroxidase substrates were added to each well. The peroxidase enzyme catalyses the cleavage of the substrate, producing a colored reaction product. The absorbance of the samples at 405 nm with a reference wavelength at 490 nm were measured by a microplate reader (MicroReaderTM4, Hyperion). The results were expressed as percentages of the 405/490-nm absorbance ratio of control cells.

2.5 Binding assay
Binding assay was performed with 125I-labeled PDGF-AA, -AB, and -BB. 125I-labeled PDGF-BB was purchased from Dupont NEN, which was iodinated by Bolton–Hunter method, possessing a specific activity of 30 000~54 000 cpm/ng. 125I-labeled PDGF-AA and 125I-labeled PDGF-AB were labeled with 125I (Dupont NEN) according to the chloramine-T method [24]to specific activities of 23 000~37 000 and 55 000~75 000 cpm/ng, respectively. Cells were cultured in 24-well plates. After a PBS wash, the cells were incubated at 4°C for 3 h with 2% bovine serum albumin–PBS containing 125I-labeled PDGF (1~5 ng/ml) and various concentrations (0~500 ng/ml) of the corresponding unlabeled PDGF. The cells were then washed with ice-cold PBS three times, and harvested with 1 M NaOH. The total 125I incorporated into the cells was determined in a gamma scintillation counter. Nonspecific binding was defined as the binding in the presence of unlabeled PDGF of 100-fold molar excess. The dissociation constant (Kd) and maximal number of binding sites (Bmax) were obtained by Scatchard analysis [25].

2.6 RNA isolation and northern blot analysis
Total RNA was isolated from the cells in 60-mm dishes with the acid guanidinium phenol–chloroform method as described previously [26]. Aliquots (20 µg) of total RNA were size-fractionated by electrophoresis in 1% agarose–3% formaldehyde gels containing 0.5 µg/ml ethidium bromide and transferred to nylon membranes (HybondTM-N; Amersham Life Science) in 10x standard sodium citrate (SSC; 1x=0.15 M sodium chloride and 0.015 M sodium citrate). After ultraviolet crosslinking for 2 min, membranes were prehybridized at 42°C overnight in the solution mix consisting of 40% formamide, 5xSSC, 5xDenhardt’s solution, 0.5% sodium dodecyl sulfate (SDS), 20 mM sodium phosphate buffer (pH 7.2), 100 µg/ml salmon sperm DNA. The blots were then hybridized in the freshly-prepared same solution mix at 52°C overnight with 0.26 kb SacI–PstI fragment of the chicken v-fos [27]labeled with ready-to-go DNA labeling kit (Pharmacia) and [{alpha}-32P]dCTP (3000 Ci/mmol, Dupont NEN). The filters were washed stringently with the final wash in 0.5x SSC–0.1% SDS at 55°C for 30 min. The blots were then exposed to X-ray films (New RX, Fuji Film, Japan) with intensifying screens at –80°C for 24–72 h. Hybridized signals for c-fos mRNA were quantified with laser densitometry (LKB 2202 Ultra Laser Densitometer, Pharmacia) and densitometric scores were normalized by those of PDGF-BB-induced signals at 30 min, which were set as 100%.

2.7 Nuclear protein extraction
Nuclear protein was extracted according to the previously described method [28]. Briefly, after each intervention, the cells in 60 mm dishes were homogenated in Tris-buffered saline (TBS, pH 7.4). The centrifuged pellets were resuspended in ice-cold buffer A (10 mM Hepes, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonyl fluoride (PMSF)) by gentle pipeting. The cells were allowed to swell on ice for 15 min, and mixed with 0.625% Nonidet NP-40 (Fluka) by vortexing. The homogenates were centrifuged for 30 s. After removal of the supernatants, the pellets containing nuclear protein were resuspended in ice-cold buffer B (20 mM Hepes, pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF), and were vigorously rocked at 4°C for 5 min on a shaking platform. After centrifugation, the supernatants were saved as nuclear extracts.

2.8 Electrophoretic mobility shift assay
Double-stranded DNA fragments containing activator protein (AP) -1 consensus region (provided by GelShiftTM Assay Kit, Stratagene, [5'-CTAGTGATGAGTCAGCCGGATC-3']) were labeled with {gamma}-[32P] ATP (6000 Ci/mM; Dupont NEN) and T4 polynucleotide kinase. Nuclear extract (5 µl=~5 µg protein) was mixed with 15 µl of the incubation buffer (25 mM Hepes, 5 mM EDTA, 0.5 mM DTT, 5 mM PMSF, 50 mM KCl, 10% glycerol, 100 µg/ml poly (dI-dC)), and the labeled probe (~5·104 counts). In the presence or absence of competitors, these products were incubated for 30 min at room temperature, followed by mixing with 0.1% bromphenol blue. For competition experiments for AP-1 binding, a mutant oligonucleotide (Sawady, [5'-CTAGTGATGAGTTGGCCGGATC-3']) was also used. The DNA–protein complexes were electrophoresed at 4°C in 5% non-denaturing polyacrylamide gel. The gels were exposed to X-ray films for 12–48 h. The AP-1 binding activities were quantified with laser densitometry and densitometric scores were normalized by those of control samples, which were arbitrarily set as 100%.

2.9 Western blot analysis
For immunoblotting, the cells in 60 mm dishes were harvested in the MAP lysis buffer containing 25 mM Tris–HCl (pH 7.4), 25 mM NaCl, 10 mM NaF, 10 mM Na2P2O7, 0.5 mM EGTA, 1 mM Na3VO4, 10 nM okadaic acid, 1 mM PMSF, 1.6 µM leupeptin (Sigma), 1.5 nM aprotinin (Sigma), and 38 mM p-nitrophenyl phosphate (Sigma). The cell-free lysates were prepared from the cell lysates after freeze-and-thaw process. Aliquots of the cell-free lysates were mixed with the loading buffer (62.5 mM Tris–HCl, pH 6.8, 2% SDS, 5% mercaptoethanol, 10% glycerol and 0.15% bromphenol blue) and 100 mM DTT. The mixtures were electrophoresed in 10% SDS-polyacrylamide gel. After electrophoresis, the proteins separated in the gel were electrically transferred to a polyvinylidene difluoride membrane (Clear Blot P, ATTO, Japan) with a semi-dry method and the blot was blocked in TBS (pH 7.4) containing 4% skimmed milk (Wako) and 1% ovalbumin (Sigma) overnight at 4°C. The blots were then incubated with first antibodies [1:1000 diluted polyclonal anti-MAPK antibody (Upsate Biotechnology), phospho-specific MAPK antibody, or 1:2000 diluted anti-STAT1 antibody, anti-STAT3 antibody (Transduction Laboratories)] for 1 h at room temperature. After being washed in TBS containing 1% Triton X-100, the blots were then incubated with 1:2000 diluted second antibodies [goat anti-rabbit or anti-mouse IgG conjugated to horseradish peroxidase (Amersham)] for 30 min at room temperature. After the blots were washed three times, they were exposed to X-ray films by the chemiluminescence method (ECL, Amersham).

2.10 Immunoprecipitation
The cell-free lysates were prepared in the same way as Western blot analysis described above. Aliquots were mixed with 5 µg of mouse anti-phosphotyrosine monoclonal antibody (Upstate Biotechnology) in immunoprecipitation buffer (1% Triton X-100, 150 mM NaCl, 10 mM Tris–HCl, pH 7.4, 1 mM EGTA, 0.2 mM sodium vanadate, 0.2 mM PMSF, 0.5% NP-40). The mixtures were incubated at 4°C for 1 h. After additional incubation with 5 µg of the rabbit anti-mouse IgG antibody (Wako) for 30 min, 50% protein A agarose (Gibco) of 20 µl was added. The mixtures were incubated with agitation for 30 min at 4°C, and washed three time with the immunoprecipitation buffer described above. The pellets were resuspended in the loading buffer described above, boiled for 5 min, and centrifuged for 5 min. The supernatants were then electrophoresed in 10% SDS-PAGE.

2.11 Measurements of [Ca2+]i and ICa
[Ca2+]i was measured with Ca2+ fluorescence dye indo-1 as previously described [29]. Briefly, cells on the coverslip in 100-mm dishes were incubated in the serum-free media containing 5 µM indo-1 acetoxymethyl ester (indo-1/AM; Dojin Kagaku, Japan) at 37°C for 15 min, and then washed in the dye-free serum-free medium for 10 min. After the dye loading, the cells were placed in a flow-through chamber and continuously superfused at 37°C with normal Tyrode solution (137 mM NaCl, 3.7 mM KCl, 0.5 mM MgCl2, 1.8 mM CaCl2, 5.6 mM glucose, 4.0 mM Hepes, pH 7.35). The fluorescence from the cells was collected and divided with a dichroic mirror to permit a simultaneous measurement of 410-and 480-nm wavelengths. The ratio of 410-nm fluorescence intensity/480-nm fluorescence intensity was used as an indicator of [Ca2+]i.

L-type Ca2+ channel current (ICa) was recorded with the whole-cell variant of the patch clamp technique (Axopatch 200A, Axon Instruments) as previously described [29]. Cells were cultured sparsely so as not to contact each other on 25 mm coverslips. Suction micropipettes, with tip diameters of 1 to 2 µm (5–10 M{Omega} resistance when filled), were pulled (model P-87, Sutter Instrument) from borosilicate capillary tubings (Corning 7052, 1.65 mm O.D., 1.2 mm I.D., A-M Systems) and lightly fire-polished. For voltage clamp experiments, microelectrodes were filled with the dialyzing solution containing 130 mM CsCl, 10 mM NaCl, 0.5 mM MgCl2, 5 mM K2ATP, 5.5 mM dextrose and 10 mM Hepes. The pH was adjusted to 7.1 with KOH. We added 150 mM nystatin to the pipette solution just before use. We obtained giga seals by using the conventional method, and then waited for 10 min to obtain intracellular access with perforated patch. We recorded the membrane current during depolarizing steps from a holding potential of –40 mV to test potentials (–30, –20, –10, 0, +10, +20, +30, +40 mV), which indicated ICa.

2.12 Statistical analysis
Values are expressed as mean±S.E. Statistical analysis was performed by use of Student’s t test. A value of P<0.05 was considered statistically significant.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Characterization of cell types in cultured cells
Before investigating the effect of PDGF, we characterized cell types in our cultured cells by immunocytochemistry. The cells were simultaneously stained with ethidium bromide (Fig. 1A) and anti-sarcomeric {alpha}-actin antibody (Fig. 1B). We considered that the nuclei of all the cells should be stained with ethidium bromide, and that all cardiac myocytes should be stained with anti-sarcomeric {alpha}-actin antibody. Most (>95%) of the cells, the nuclei of which were stained with ethidium bromide, were positive for sarcomeric {alpha}-actin. On the other hand, we observed no significant fluorescence in the staining with anti-smooth muscle {alpha}-actin, which can recognize the smooth muscle isoform of {alpha}-actin (Fig. 1C). These results demonstrated that our culture exclusively consisted of cardiac myocytes.


Figure 1
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  		Fig. 1 Characterization of cell types in cultured cells. To characterize cell types in our culture, we performed immunocytochemistry (n=4). (A) The cells were stained with ethidium bromide. Ethidium bromide can stain the nuclei of all types of cells; therefore, these stainings were considered to show all the cells in the field. (B) The cells in the same field as (A) were stained simultaneously with anti-sarcomeric {alpha}-actin antibody. Most (>95%) of the cells, the nuclei of which were stained with ethidium bromide, were positive for sarcomeric {alpha}-actin. (C) No significant fluorescence was observed in the staining with anti-smooth muscle {alpha}-actin.

 
3.2 Acceleration of cellular hyperplasia induced by PDGF
To examine the effect of PDGF on growth of cardiac myocytes, we assayed viable cell number by MTT assay. We assayed this parameter after 72 h exposure to PDGF-AA, -AB, or -BB in various concentrations (0.1~100 ng/ml). PDGF-AA did not significantly affect the viable cell number at any concentrations (Fig. 2A left). Both PDGF-AB and -BB significantly increased the viable cell number dose-dependently (Fig. 2A center, right). The concentration of PDGF-BB (~5 ng/ml), at which maximal mitogenic effect was achieved, appeared to be lower than that concentration of PDGF-AB (~50 ng/ml). However, we observed no significant difference between the maximal mitogenic responses to PDGF-AB and -BB. Moreover, we examined DNA synthesis by BrdU incorporation assay. Cells were incubated with BrdU for 3 h after 12 h exposure to PDGF. In accordance with the results of MTT assay, 5 ng/ml PDGF-AB and -BB significantly increased DNA synthesis (202*±18%, 295*±25%, respectively, n=4, *P<0.05 vs. control), but 5 ng/ml PDGF-AA did not affect DNA synthesis (104±11%) (Fig. 3). These data revealed that PDGF-AB and -BB, but not AA, induced cellular growth in cardiac myocytes.


Figure 2
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  		Fig. 2 Growth effects and binding sites of three PDGF isoforms. (A) After 72 h exposure to each PDGF isoform (0.1~100 ng/ml), viable cell number was estimated by MTT assay. PDGF-AA did not significantly affect viable cell number at any concentrations (left). PDGF-AB significantly increased viable cell number dose-dependently at concentrations of more than 5 ng/ml (center). PDGF-BB also significantly increased viable cell number dose-dependently at concentrations of more than 0.25 ng/ml (right). Ratio of absorbance at 570/630 nm was corrected by those of untreated myocytes, which were set as 100%. Data are means±S.E. of four separate experiments. *P<0.05 vs. control. (B) Representative Scatchard plots for each PDGF isoform are shown. By Scatchard analysis, Bmax of PDGF-AA, -AB, -BB were 5±1, 63±12, 126±24 fmol/106 cells, and Kd was 0.04±0.01, 0.63±0.10, 1.62±0.23 nM, respectively. These values are means±S.E. of three separate experiments.

 

Figure 3
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  		Fig. 3 Acceleration of DNA synthesis by PDGF. DNA synthesis was measured by BrdU incorporation assay. After 12 h exposure to each PDGF isoform (5 ng/ml), cells were incubated with BrdU for 3 h. PDGF-AA did not significantly affect BrdU incorporation. PDGF-AB and -BB significantly accelerated BrdU incorporation. Ratio of absorbance at 405/490 nm was corrected by those of untreated myocytes, which were set as 100%. Data are means±S.E. of four separate experiments. *P<0.05 vs. control; (C) indicates control.

 
3.3 Binding sites of PDGF isoforms in cardiac myocytes
Next, we performed binding assay of three PDGF isoforms to clarify which subtype of the PDGF receptors was involved in the PDGF-induced myocardial growth. By Scatchard analysis, Bmax of PDGF-AA, -AB, -BB was 5±1, 63±12, 126±24 fmol/106 cells, and that Kd was 0.04±0.01, 0.63±0.10, 1.62±0.23 nM, respectively (Fig. 2B). These data suggest that PDGF-β receptor is expressed dominantly in the surface of cardiac myocytes compared with PDGF-{alpha} receptor.

Thus, from the results of the binding assay, the number of the binding sites for PDGF-BB was 20-fold greater than that for PDGF-AA, or was 2-fold greater than that for PDGF-AB. Moreover, the receptor subtypes activated by PDGF-BB wholly include those activated by PDGF-AA and -AB [6]. Therefore, we used PDGF-BB in order to further examine which intracellular signals contribute to chick embryonic myocardial growth.

3.4 Induction of c-fos mRNA by PDGF-BB
We examined the induction of an immediate-early gene, c-fos, mRNA by Northern blot analysis. The c-fos mRNA was induced transiently after a 30 min exposure to 5 ng/ml PDGF-BB (Fig. 4A). A PKC inhibitor, calphostin C (Sigma, 250 nM, pretreatment for 30 min), did not affect the c-fos induction by PDGF-BB (Fig. 4B). On the other hand, the induction of c-fos mRNA was markedly attenuated by either a MEK1 inhibitor, PD98059 (New England Biolabs, 10 µM, pretreatment for 1 h)(Fig. 4C), or a tyrosine kinase inhibitor, genistein (Sigma, 100 µM, pretreatment for 1 h)(Fig. 4D). These results demonstrated that PDGF-BB induced c-fos mRNA through the activation of tyrosine kinase and MAPK. In contrast, the c-fos induction by PDGF-BB seems to be independent of the activation of PKC.


Figure 4
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  		Fig. 4 Induction of c-fos mRNA by PDGF. Upper panels show representative Northern blots for c-fos mRNA and lower panels show densitometric scores obtained from several independent experiments. (A) 5 ng/ml of PDGF-BB induced c-fos mRNA transiently after 30 min exposure (n=4). (B) PKC inhibitor, 250 nM calphostin C (Cal), did not affect the c-fos induction by PDGF (n=2). (C) On the other hand, a MEK1 inhibitor, 10 µM PD98059 (PD), markedly attenuated the c-fos induction by 52*±2% at 30 min after exposure to 5 ng/ml PDGF (n=2). (D) A tyrosine kinase inhibitor, 100 µM genistein (Gen), also markedly attenuated the c-fos induction by 63*±2% at 30 min after exposure to 5 ng/ml PDGF-BB (n=2). The ethidium bromide stainings of 28S and 18S ribosomal RNAs are also shown. Bar graphs represent mean values, and error bars represent S.E. C indicates control, P indicates PDGF-BB (5 ng/ml). * P<0.05 vs. control.

 
3.5 Increases in binding activity to the AP-1 site by PDGF-BB
c-Fos protein is a major component of AP-1, which acts as a transcriptional factor by being bound to its consensus sites. We examined the AP-1 binding activity by electrophoretic mobility shift assay. The band designated with a half bracket was efficiently competed for by increasing concentrations of a cold AP-1 oligonucleotide but not by a mutant AP-1 (Fig. 5A). Therefore, this band was considered to represent specific AP-1 binding activity. Although considerable AP-1 activity was observed in the control samples, 5 ng/ml of PDGF-BB significantly increased the AP-1 binding activity time-dependently (Fig. 5A, E). Calphostin C (250 nM, pretreatment for 30 min) significantly attenuated the AP-1 binding activity at baseline. The PDGF-BB-induced increases in the AP-1 binding activity were attenuated by calphostin C to some extent (Fig. 5B, F). When cells were pretreated with PD98059 (10 or 50 µM) for 1 h, these increases were significantly attenuated in a dose-dependent manner (Fig. 5C, F). The PDGF-induced increases were also completely inhibited by genistein (100 µM, pretreatment for 1 h) (Fig. 5D, F). Thus, we consider that PDGF-BB increases the AP-1 binding activity through the activation of MAPK and tyrosine kinase as observed in the c-fos induction.


Figure 5
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  		Fig. 5 Increases in the AP-1 binding activity by PDGF. We performed electrophoretic mobility shift assay with an oligonucleotide probe containing the AP-1 site at least three times for each treatment and representative data are shown. (A) PDGF-BB (5 ng/ml) increased the AP-1 binding activity in a time-dependent manner. The AP-1 binding activity of PDGF-BB-treated cells (5 ng/ml, 30 min) in the presence of 10- and 100-fold molar excess of unlabeled AP-1 probe (x10, x100 cold) and 100-fold molar excess of unlabeled mutant AP-1 probe (x100 mut) were also shown. F indicates free probes. (B) Calphostin C (Cal, 250 nM) significantly attenuated the AP-1 binding activity at basal line. The PDGF-BB-induced increases in the AP-1 binding activity were slightly attenuated (at 30 min) by calphostin C. (C) When cells were pretreated with 10 or 50 µM PD98059 (PD), the increases in the AP-1 binding activity was attenuated dose-dependently. (D) Genistein (Gen, 100 µM) also inhibited the PDGF-BB-induced increases in the AP-1 binding activity. (E) Densitometric scores of the AP-1 binding activity in the time course of PDGF treatment were shown. The AP-1 activity was increased by 2.2-fold at 30 min after PDGF treatment. *P<0.05 vs. control. (F) Effects of various kinase inhibitors were shown in terms of PDGF-induced increases in AP-1 binding activity. *P<0.05 vs. PDGF-treated (without inhibitor) cells. {dagger}P<0.05 vs. control. The densitometric value of AP-1 binding activity in control cells is expressed as 100%. Bar graphs represent mean values, and error bars represent S.E. (–) indicates the cells without PDGF treatment. (+) indicates PDGF-treated cells.

 
3.6 Tyrosine phosphorylation and translocation into nuclei of MAPK by PDGF
MEK1 inhibitor attenuated both the induction of c-fos mRNA and the increased AP-1 binding activity by PDGF-BB. Therefore, we tested whether PDGF-BB activated MAPK or not. Only one isoform (~42000) of MAPK was observed in chick embryonic ventricular myocytes as described previously [21]. Western blot analysis with anti-phospho MAPK antibody revealed that MAPK was tyrosine-phosphorylated after 5 min exposure to 5 ng/ml PDGF (Fig. 6A). Moreover, immunocytochemical analysis with anti-phospho MAPK antibody demonstrated that phosphorylated MAPK was translocated into nuclei after 15 min exposure to 5 ng/ml PDGF-BB (Fig. 6B). Thus, PDGF induced tyrosine phosphorylation and translocation into nuclei of MAPK in embryonic cardiac myocytes.


Figure 6
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  		Fig. 6 MAPK activation by PDGF. (A) Representative Western blots are shown for three separate experiments. Western blot analysis with anti-phospho MAPK antibody, which could only recognize MAPK phosphorylated at Tyr 204, showed that MAPK was tyrosine-phosphorylated after 5 min exposure to 5 ng/ml PDGF-BB. The same samples were immunoblotted with polyclonal anti-MAPK antibody. (B) Representative immunostainings with anti-phospho MAPK antibody are shown for three separate experiments. After 15 min exposure to 5 ng/ml PDGF-BB (P), we observed significant increases in fluorescence especially in nuclei, indicating that phosphorylated MAPK was mostly translocated into nuclei by PDGF-BB. (C) indicates control.

 
3.7 Effects of PDGF-BB on STAT proteins
Recently, PDGF has been reported to activate STAT proteins [7]. Thereby, we analyzed the activation status of STAT proteins by Western blot analysis. We prepared Western blots for whole cell extracts (Fig. 7 top panels), nuclear extracts (Fig. 7 center panels), and the extracts that were immunoprecipitated with anti-phosphotyrosine antibodies (Fig. 7 bottom panels). Each blot was probed with anti-STAT1 antibody (Fig. 7 left row) or anti-STAT3 antibody (Fig. 7 right row). We observed that STAT3 was constitutively tyrosine-phosphorylated (Fig. 7 right, bottom) and translocated into nuclei (Fig. 7 right, center). This activated STAT3 was not significantly affected by 5 ng/ml PDGF-BB (Fig. 7 right, center and bottom). On the other hand, we could not detect the activation of STAT1 regardless of PDGF treatment (Fig. 7 left, center and bottom). Therefore, PDGF-BB does not seem to change the phosphorylation status of STAT1 or STAT3 in our cultured cells.


Figure 7
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  		Fig. 7 Effects of PDGF on STAT proteins. We performed Western blot analysis at least three times for each experiment and representative blots are shown. The top panels show Western blots for whole cell extracts. The center panels show Western blots for nuclear extract. The bottom panels show Western blots for the extracts that were immunoprecipitated with anti-phosphotyrosine antibody. The left three blots were probed with anti-STAT1 antibodies. The right three blots were probed with anti-STAT3 antibody. Each extract was prepared from the untreated cells (C) or the cells treated with 5 ng/ml PDGF-BB for 15 min (P). STAT 3 was constitutively tyrosine-phosphorylated (right bottom, C) and translocated into nuclei (right center, C). This activated STAT3 was not significantly affected by PDGF-BB (right center and right bottom). On the other hand, we could not detect the tyrosine-phosphorylation or the translocation into nuclei of STAT1 regardless of PDGF-BB treatment (left center and left bottom). Ig indicates immunoglobulin.

 
3.8 Effect of PDGF-BB on [Ca2+]i and ICa
To examine whether PDGF induced Ca2+ mobilization in cardiac myocytes or not, we measured intracellular Ca2+ concentration with Ca2+ fluorescent dye indo-1 and L-type Ca2+ channel current (ICa) with voltage clamp method. We observed no significant changes in intracellular Ca2+ concentration after 5 min exposure to 5 ng/ml of PDGF-BB (Fig. 8A). Moreover, ICa was neither altered by 5 ng/ml PDGF-BB (Fig. 8B, C). Thus, these data suggest that PDGF may not provoke Ca2+ mobilization in chick embryonic cardiac myocytes.


Figure 8
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  		Fig. 8 Effects of PDGF on [Ca2+]i and ICa. (A) Representative tracings of [Ca2+]i transient before (C) and 5 min after exposure to 5 ng/ml PDGF-BB (P) are shown for five separate experiments. PDGF did not affect [Ca2+]i transient. (B) Representative tracings of ICa before (C) and 5 min after exposure to 5 ng/ml PDGF-BB (P) are shown for three separate experiments. These tracings indicates ICa when cells were depolarized from –40 mV to +10 mV for 0.4 s. ICa was neither altered by PDGF-BB. (C) The voltage–current relationship during depolarizing steps from a holding potential of –40 mV to test potentials (–30, –20, –10, 0, +10, +20, +30, +40 mV) is also shown. There was no significant change in this relationship after PDGF exposure. Each point is the mean±S.E. of three separate experiments.

 
3.9 Inhibition of PDGF-induced myocardial growth by MEK1 inhibitor
The current analysis demonstrated that MAPK cascade might be one of the main signaling pathways induced by PDGF in cardiac myocytes. Finally, we examined whether MAPK cascade could lead to myocardial growth or not. We performed BrdU incorporation assay in the presence or absence of calphostin C, PD98059 as well as genistein (Fig. 9). In non-pretreated cells, 5 ng/ml of PDGF-BB significantly increased DNA synthesis (292*±19%, n=6, *P<0.05 vs. control). Calphostin C (250 nM, pretreatment for 30 min) did not significantly attenuate the acceleration of DNA synthesis by PDGF-BB (264±33%). PD98059 (10 or 50 µM, pretreatment for 1 h) significantly reduced the acceleration in a dose-depend manner (198*±14%, 168*±14%, respectively, n=6, *P<0.05 vs. PDGF-BB-treated cells). The acceleration was completely inhibited by pretreatment (1 h) with genistein (100 µM) (85*±5%, n=6, *P<0.05 vs. PDGF-BB-treated cells). These data suggest that MAPK cascade may contribute to PDGF-induced mitogenic responses.


Figure 9
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  		Fig. 9 Effects on PDGF-induced myocardial growth by protein kinase inhibitors. DNA synthesis was measured by BrdU incorporation assay in the presence or absence of calphostin C, PD98059 as well as genistein. Calphostin C (Cal, 250 nM) did not significantly attenuate the acceleration of DNA synthesis by PDGF-BB (5 ng/ml). PD98059 (PD, 10 or 50 µM) significantly reduced the acceleration in a dose-dependent manner. Genistein (Gen, 100 µM) completely inhibited the acceleration by PDGF-BB. Ratio of absorbance at 405/490 nm was corrected by those of control cells, which were set as 100%. Data are means±S.E. of six separate experiments. *P<0.05 vs. PDGF-treated (without inhibitor) cells.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
In this study, we demonstrated that PDGF-AB and -BB, but not PDGF-AA, could directly affect chick embryonic ventricular myocytes and that they stimulated their growth in a dose-dependent manner. PDGF-BB induced tyrosine phosphorylation and translocation into nuclei of MAPK. PDGF-BB also induced c-fos mRNA and increased the AP-1 binding activity. These changes were shown to depend on the activation of tyrosine kinase and MAPK. On the other hand, PDGF-BB did not significantly affect the phosphorylation states of STATs, [Ca2+]i, or ICa in chick embryonic ventricular myocytes.

PDGF has been shown to stimulate growth of various types of cells, including smooth muscle cells, fibroblasts, and endothelial cells [1]. Therefore, when we examine the effect of PDGF on ventricular myocytes, contamination of these other types of cells is often problematic. However, as for the cultured chick embryonic ventricular myocytes, it was reported that this cell culture contained negligible amounts of non-myocytes [30, 31]. Moreover, in the present study, we also demonstrated by immunocytochemistry that our culture exclusively consisted of cardiac myocytes. Accordingly, we may neglect the effects of those contaminating cells in this study.

Three different isoforms of PDGF, which are named as PDGF-AA, -AB, and -BB, are known to be present. On the other hand, three different isoforms of PDGF receptors, which are {alpha}{alpha}, {alpha}β and ββ, have been found. PDGF-BB can be bound to all types of receptors, PDGF-AB can be bound to {alpha}{alpha} and {alpha}β receptors, and PDGF-AA can be bound to only {alpha}{alpha} receptor [6]. In our study, PDGF-AB and -BB, but not -AA, induced myocardial growth. This result is in agreement with the observations of the PDGF-B gene-deficient mice. Moreover, the result seems to indicate that the PDGF-induced cellular growth is mediated by only the β-receptor. However, PDGF-AA has been reported to induce mitogenic effect in smooth muscle cells and other cell types [5, 32, 33], indicating that mitogenic effect can be also mediated by {alpha}-receptor. These earlier studies have demonstrated the predominant expression of PDGF-β receptor relatively to PDGF-{alpha} receptor as well as the predominant effect of PDGF-BB relatively to PDGF-AA. On the other hand, they have demonstrated no apparent difference between the intracellular signaling pathways of these PDGF isoforms. Therefore, the extent of mitogenic effect by each PDGF isoform may be dependent on the expression number of the individual receptor subtypes rather than the difference in their intrinsic signaling pathways. The number of PDGF receptors in cardiac myocytes has not been reported until now. In our study, the binding sites of PDGF-AA in chick embryonic cardiac myocyte are five-fold less than those in smooth muscle cells [5]. Lack of growth promoting effect by PDGF-AA may be a result of the scarce expression of {alpha}{alpha} receptors in cardiac myocytes.

According to the earlier studies [5, 6], the PDGF receptors have intrinsic tyrosine kinase domains and the binding of PDGF causes dimerization of the PDGF receptors and autophosphorylation on their tyrosine residues. Activated PDGF receptors physically associate with phosphorylated signaling molecules containing SH2 domains, including Grb2 and phospholipase C {gamma}. Grb2 activates Ras, and triggers a kinase cascade of Raf, MAPK kinase (MEK) and MAPK. On the other hand, activation of phospholipase C {gamma} leads to phosphatidylinositol turnover, which results in Ca2+ mobilization and PKC activation. It has been reported that the activation of MAPK cascade by PDGF induced DNA synthesis and proliferation in smooth muscle cells. In contrast, phosphatidylinositol turnover with Ca2+ mobilization by PDGF stimulated migration and chemotaxis of these cells [5]. In this study, we demonstrated that PDGF-BB induced c-fos mRNA and accelerated DNA synthesis through the activation of tyrosine kinase and MAPK, but that it did not induce Ca2+ mobilization. Thus, PDGF-BB seems to stimulate mainly a Ras–Raf–MEK–MAPK cascade, which can lead to proliferation of ventricular myocytes.

In cardiac myocytes, mechanical stretch [10–12], norepinephrine [13, 14], angiotensin II [15–17], and endothelin-1 [18–20]have been shown to stimulate both phosphatidylinositol turnover and MAPK cascade leading to hypertrophic response. In those studies, the crosstalks of phosphatidylinositol turnover and MAPK cascade have also been suggested. In contrast, acidic fibroblast growth factor stimulates MAPK cascade through PKC-independent pathways in cardiac myocytes [18]. In the present study, PDGF-BB induced c-fos mRNA through MAPK activation, but independently of PKC. Growth factors, which are bound to tyrosine kinase receptors, including fibroblast growth factor and PDGF, seem to stimulate MAPK cascade in PKC-independent manners.

MAPK cascade is widely considered to mediate cell proliferation. Recently, it has been reported that transient transfection of a plasmid encoding a constitutively active form of MEK1 into epithelial cells increased MAPK activity and cellular growth, and that MEK1 inhibitor reduced both of them [34]. This report has ensured that the activation of MAPK cascade leads to cellular growth. We also demonstrated that MEK1 inhibitor could significantly inhibit PDGF-induced cellular growth. These data suggest that the activation of MAPK cascade by PDGF can lead to myocardial growth. However, the inhibition of PDGF-induced cellular growth by the MEK1 inhibitor was not complete. Therefore, MAPK-independent pathways may also contribute to PDGF-induced cellular growth, and further studies are needed to clarify this point.

On the other hand, calphostin C slightly attenuated the PDGF-BB-induced increases in the AP-1 binding activity in this study. These data suggest that PKC activation may contribute to the PDGF-BB-induced increases in the AP-1 binding activity without c-fos induction. However, it is also possible that the apparent inhibition of the PDGF-BB-induced increases in the AP-1 binding activity by calphostin C merely reflected the attenuation of the basal AP-1 activity by this drug. Therefore, further studies will also be needed to elucidate the precise role of PKC in the PDGF-induced myocardial growth.

PDGF has been known to activate both STAT1 and STAT3 [7]. In contrast, the present study demonstrated that STAT3 was constitutively activated at basal level and that PDGF-BB did not affect the phosphorylation status of STAT1 or STAT3 in chick embryonic cardiac myocytes. Although the reason for this discrepancy remains uncertain, differences in species, tissue, or developmental stage may have affected the results. On the other hand, a transient transfection experiment using c-fos promoters lacking sis-inducible element, which is bound to STATs, indicated that sis-inducible element might play only a minor role in the induction of c-fos mRNA by EGF in Hela cells [35]. In this regard, STATs may not be indispensable for the induction of c-fos mRNA, although we cannot exclude the possibility that constitutive activation of STAT3 is necessary for the c-fos mRNA induction in embryonic cardiac myocytes.

We consider that PDGF may have an important role in chick embryonic myocardial development. PDGF has also been reported to stimulate DNA synthesis in adult newt cardiac myocytes [3], although it is unknown whether PDGF affects adult mammalian cardiac myocytes. PDGF receptor mRNA species have been shown to be expressed in adult rat heart tissues [36]or in cardiac myocytes of human allograft [37]. These data suggest that PDGF can also exert some direct actions in adult cardiac myocytes, but further studies will be needed to elucidate this important point.

We conclude that PDGF induces cellular growth in chick embryonic ventricular myocytes. The signaling cascade through the activation of MAPK may play an important role in the PDGF-induced growth responses of these cardiac myocytes.

Time for primary review 22 days.


   Acknowledgements
 
We thank Dr. H. Fujie for his technical assistance in the BrdU incorporation assay. This study was supported in part by Grants-in-Aid (B)(2) 08457203 from the Ministry of Education, Culture and Science Sports of Japan.


   Notes
 
1 Presented in part at the 69th Scientific Sessions of the American Heart Association, New Orleans, LA, November 10–13, 1996, and published in an abstract form (Circulation, 94 [suppl. I]: I–469, 1996). Back


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

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