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Published ahead of print on March 11, 2004, doi:10.1165/rcmb.2003-0397OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 31, pp. 184-192, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0397OC

p42/44 Mitogen-Activated Protein Kinase Regulated by p53 and Nitric Oxide in Human Pulmonary Arterial Smooth Muscle Cells

Shiro Mizuno, Maiko Kadowaki, Yoshiki Demura, Shingo Ameshima, Isamu Miyamori and Takeshi Ishizaki

Third Department of Internal Medicine, and Department of Fundamental Nursing, University of Fukui, Fukui, Japan

Address correspondence to: Shiro Mizuno M.D., Third Department of Internal Medicine, University of Fukui, 23-3 Matsuoka-cho Yoshida-gun, Fukui, Japan. E-mail: shirotan{at}qf6.so-net.ne.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although nitric oxide (NO) is known to inhibit vascular smooth muscle cell proliferation, the subcellular molecular mechanisms involved with the inhibitory signal transduction pathways are uncertain. We investigated the effect of exogenous NO on cell proliferation and the expression of p53, p21, and phosphorylated p42/44 mitogen-activated protein kinase (MAPK) in human pulmonary arterial smooth muscle cells (HPASMC). Both S-nitroso-N-acetyl penicillamine and diethylenetriaminelNONOate dose-dependently suppressed [3H]-thymidine incorporation in cultured HPASMC, and induced the expression of p53 and p21 protein. Further, the NO donors transiently increased the phosphorylation of p42/44 MAPK and then suppressed it. Although MAPK kinase inhibitors suppressed [3H]-thymidine incorporation by the cells, no significant change was observed in the expression of p53 and p21. The NO donors also suppressed the activation of p42/44 MAPK evoked by transient transfection of the wild-type p53 gene; however, they failed to suppress the activation of p42/44 MAPK in constitutive-active mutations of the Ras or Raf genes trasnsfected from HPASMC. These results indicate that exogenous NO is able to transiently activate p42/44 MAPK via the induction of p53, and then suppress it via inactivation of the Ras and Raf cascades.

Abbreviations: cyclin-dependent protein kinase, CDK • 2-(4-carboxy-phenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide, cPTIO • diethylenetriaminelNONOate, DETANO • Dulbecco's modified Eagle's medium, DMEM • extracellular-regulated kinase1/2, ERK1/2 • fetal bovine serum, FBS • human pulmonary arterial smooth muscle cells, HPASMC • mitogen-activated protein kinase, MAPK • nitric oxide, NO • NO synthase, NOS • phosphate-buffered saline, PBS • polymerase chain reaction, PCR • smooth muscle cells, SMC • S-nitroso-N-acetyl penicillamine, SNAP • vascular smooth muscle cells, VSMC


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Nitric oxide (NO) is synthesized from L-arginine via nitric oxide synthase (NOS), and endothelial NOS plays an important regulatory role in hypertrophic and hyperplastic growth of vascular smooth muscle cells (VSMC) in vivo and in vitro. Clinically, pulmonary muscular arteries and arterioles in patients with primary or secondary pulmonary hypertension show several degrees of vascular remodeling, including smooth muscle hypertrophy and proliferation (1, 2). Basically, several studies have suggested that NO derived from endothelial NOS has a protective effect toward arterial smooth muscle cell proliferation (3–5).

In accordance with those studies, procedures using chemical NO donors and NOS gene transfer have been shown to inhibit cultured smooth muscle cell (SMC) proliferation, and the mechanisms of NO-induced inhibition of SMC proliferation are suggested to involve an increase of the cyclin-dependent protein kinase (CDK) inhibitor p21 (6–10). Further, p21 was discovered in genes induced by tumor suppressor p53, transcriptionally upregulated by p53, and is thought to be involved in p53-mediated growth arrest (11, 12).

Recent studies have established that some mitogens stimulate the synthesis of DNA and cell proliferation by activating the phosphorylation cascade of mitogen-activated protein kinase (MAPK). Phosphorylation of p42/44 MAPK, known as an extracellular-regulated kinase1/2 (ERK1/2), is increased in response to epidermal growth factor and platelet-derived growth factor (13, 14), and activation of p42/44 MAPK seems to be associated with numerous nuclear transcription factors and cell proliferation. Although NO has antiproliferative effects on VSMC, several studies have noted that p42/44 MAPK is activated by NO exposure (15–18). In addition, recent investigations reported that a sustained activation of MAPK cascade could be induced by p53, and this activation of MAPK is mediated at a level of upstream of Ras (19, 20). Furthermore, p53 overexpressed VSMC expressed higher levels of phosphorylated p42/44 MAPK at baseline compared with p53 knockout VSMC, and exposure to NO reduced this expression (21). These results indicate that p53, induced by NO, may affect the activation of MAPK to some degree.

The precise mechanisms and interactions between the pathways activated by MAPK, as well as the antiproliferative effects of tumor suppressor p53 and CDK inhibitor p21 during exposure to NO in pulmonary arterial SMC, remain uncertain. The purpose of this study was to clarify the inhibitory effect of NO in cultured human pulmonary arterial SMC (HPASMC), with special attention given to the interactions of p42/44 MAPK, CDK inhibitor p21, and tumor suppressor p53. For the present examination, we assessed the effect of S-nitroso-N-acetyl penicillamine (SNAP) and diethylenetriaminelNONOate (DETANO), known as short-acting and long-acting NO donors, on cell proliferation and the expression of p21, p53, and phosphorylated p42/44 MAPK in cultured HPASMC, which were transfected with plasmids encoding wild-type and mutant p53, Ras, or Raf genes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals
Chemicals and materials were obtained from the following sources. Humedia SG medium, recombinant human EGF and FGF, gentamycin, streptomycin, and amphotericin B came from Kurabou Ltd. (Osaka, Japan); DETANO from Dojindo Labratories (Kumamoto, Japan); [3H]-thymidine and an ECL system from Amersham (Buckinghamshire, UK); moloney murine leukemia virus reverse transcriptase from Toyobo Co. Ltd. (Osaka, Japan); a Quantitech SYBAR Green PCR kit from Qiagen (Santa Clarita, CA); lipofectamine, lipofectamine-plus reagent, 4–12% Bis-Tris Nupage gels, and MES-SDS running buffer from Invitrogen (Carlsbad, CA); a DC protein assay kit and polyvinylidene difluoride (PVDF) membranes from Bio-Rad Laboratories (Richmond, CA); rabbit anti-p21 polyclonal antibody, mouse anti-p53 monoclonal antibody, mouse anti-phospho-p42/p44 MAPK monoclonal antibody, rabbit anti-p42/p44 MAPK polyclonal antibody, mouse anti-ß-actin monoclonal antibody, and horseradish peroxidase–conjugated goat anti-mouse and rabbit IgG from Santa Cruz Biotechnology Inc. Nitrate/nitrite colorimetric assay kit was purchased from Cayman chemical company. All other chemicals were purchased from Sigma (St. Louis, MO).

Cell Culture
HPASMC were supplied by Kurabou Ltd., and grown in Humedia SG medium containing 5% fetal bovine serum with 50 µg/ml of gentamycin, 50 ng/ml of amphotericin B, 1 ng/ml of recombinant human EGF, and 1 ng/ml of recombinant human FGF. The cells were cultured in 75-cm2 tissue culture flasks (Corning, Corning, NY) in a cell-culture incubator (37°C, 5% CO2, and 95% air) and used at the seventh passage after trypsinization in all of the experiments.

Assay of [3H]-Thymidine Incorporation of HPASMC
HPASMC were seeded in a 24-well culture disk at a density of 3,000 cells/cm2 and incubated for 24 h, after which the medium was changed to Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) and antibiotics. Various concentrations of SNAP, DETANO, or the vehicle were added to the wells with or without 20 µM of 2-(4-carboxy-phenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (cPTIO), known as a scavenger of NO, and they were incubated for another 24 h. Cells were labeled with 0.5 µCi/ml of [3H]-thymidine during the last 20 h of incubation, after which some of the medium was aspirated and saved for analysis of the nitrite contents. After labeling, the cells were washed three times with cold phosphate-buffered saline (PBS) and treated with 98% ethanol, then air dried and treated with 5% trichloroacetic acid on ice. After aspiration with trichloroacetic acid, the cells were solubilized with 0.1 N of NaOH and 0.2% SDS. Radioactivity in the cell lysate aliquots was measured using a liquid scintillation counter (LSC-5300; Aloca, Tokyo, Japan).

Measurement of Nitrate and Nitrite Contents
Nitrate and nitrite contents in the culture media were determined by Greiss assay with enzymatic modification using nitrate/nitrite colorimetric assay kit according to the manufacturer's protocol. Briefly, mixing 80 µl of medium with 10 µl of nitrate reductase and 10 µl of enzyme co-factors were incubated at room temperature for 2 h. Then 100 µl of Greiss reagent (0.5% sulfanilamide, 2.5% orthophosphoric acid, and 0.25% napthylenediamide) were added to the wells. Absorbance at 540 nm was measured and the nitrate and nitrite concentration was determined using sodium nitrite as the standard. Nitrite contents in the culture media were determined by Greiss assay without enzymatic modification.

Propidium Iodide Staining
To examine whether the G1 and S phases were influenced by SNAP or DETANO, flow cytometric analysis with propidium iodide staining was performed. HPASMC were seeded in a 6-well culture disk at a density of 3,000 cells/cm2 and incubated for 24 h, after which the medium was changed to DMEM with 10% FBS and antibiotics. Next, either 100 µM of SNAP, DETANO, or the vehicle was added to the wells and incubated for 24 h. To measure the DNA content, the cells were harvested by trypsin and EDTA, and fixed with 70% ethanol. The ethanol was removed and the cells were incubated in PBS containing RNase (172 kunits/ml) at 37°C for 30 min, and then stained with propidium iodide (50 µg/ml) and dissolved in PBS for 30 min on ice. DNA fluorescence was measured and flow cytometric analysis was performed using an EPICS XL (Beckman Coulter, Fullerton, CA).

Real-Time Reverse Transcriptase–Polymerase Chain Reaction Analysis of p21 and p53 mRNA Using LightCycler
HPASMC were cultured in a 6-well flat-bottom culture plate at a density of 3,000 cells/cm2 and cultured for 24 h. The cells were washed twice with PBS, then placed in DMEM supplemented with 10% FBS and antibiotics in the presence or absence of various concentrations of SNAP or DETANO for 24 h. The cells were then harvested by trypsinization, washed three times, and pelleted by centrifugation. Total cellular RNA was obtained from the cells by a single extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture (22). Reverse transcription (RT) was performed using 0.5 µg of total RNA. cDNA synthesis was done with 200 U of Moloney murine leukemia virus reverse transcriptase, 5 µM of oligoDT, 1 mM of dNTPs, and 3 mM of Mg2+ in a volume of 20 µl. The temperature profile consisted of annealing at room temperature for 5 min, extension at 44°C for 40 min, and termination at 99°C for 5 min.

Polymerase chain reaction (PCR) was performed with the resulting RT products using specific oligonucleotide primers for p21, p53, and GAPDH, which were designed using the computer software Primer 3 (Whitehead Institute for Biomedical Research, Cambridge, MA). The sequence of the forward primer for p21 was 5'-GGAAGACCATGTGGACCTGT-3' and that of the reverse primer was 5'-GGCGTTTGGAGTGGTAGAAA-3'. The sequence of the forward primer for p53 was 5'-GTTCCGAGAGCTGAATGAGG-3' and that of the reverse primer was 5'-TTATGGCGGGAGGTAGACTG-3'. The sequence of the forward primer for GAPDH was 5'-CAGCCTCAAGATCATCAGCA-3' and that of the reverse primer was 5'-GTCTTCTGGGTGGCAGTGAT-3'. All PCR reactions were performed with a LightCycler PCR system (Roche Diagnostics, Meylan, France) using DNA binding SYBR Green dye for the detection of PCR products. The cycling conditions were as follows: initial denaturation at 95°C for 15 min, followed by 50 cycles of denaturation at 94°C for 15 s, annealing at 55°C for 15 s, and extension at 72°C for 15 s. The GAPDH gene was used as the reference. The PCR products were isolated from the LightCycler glass capillaries and visualized by electrophoresis on 1.5% agarose gels with ethidium bromide staining to confirm the products. Each assay was performed in four independent experiments.

Western Blot Analysis
HPASMC were cultured in a 10-cm dish at a density of 3,000 cells/cm2 and cultured for 24 h. The cells were washed twice with PBS, and then placed in DMEM supplemented with 10% FBS and antibiotics. After that, various concentrations of SNAP or DETANO were added to the dishes, and they were cultured for 24 h in the presence or absence of PD098059 (30 µM) or U0126 (10 µM). After incubation, the cells were harvested and resuspended in protein lysis buffer (150 mM of NaCl, 20 mM of Tris-HCl, 1% NP40, 10 mM of EDTA, 10% glycerol, 1 mM of PMSF, 10 µg/ml of aprotinin, 1 µg/ml of leupeptin, 1 µg/ml of pepstatin), and then incubated for 30 min on ice. After incubation, the cell lysis buffers were centrifuged at 10,000 x g for 15 min at 4°C to remove the cell fragments, and the supernatants were analyzed for protein content using a DC protein assay kit. Each sample was quantified, and then 25 µg of protein was loaded onto each lane of a 4–12% Bis-Tris Nupage gel with MES SDS running buffer, according to the manufacturer's protocol. The gel was transferred to a PVDF membrane by electrophoresis at 100 V for 1 h. The membrane was blocked in PBS, 0.2% Tween 20 (PBS-T), and 5% nonfat milk at room temperature for 1 h. All antibodies were diluted in the same blocking buffer. The membrane was then probed with rabbit anti-p21 polyclonal antibody (1:1,000 dilution), mouse anti-p53 monoclonal antibody (1:2,000 dilution), mouse anti–phospho-p42/p44 MAPK monoclonal antibody (1:1,000 dilution), rabbit anti-p42/p44 MAPK polyclonal antibody (1:5,000 dilution), and mouse anti–ß-actin monoclonal antibody (1:5,000 dilution), and then incubated for 1 h at room temperature. After incubation, the membrane was washed with PBS-T and incubated with horseradish peroxidase–conjugated goat anti-rabbit or mouse IgG (1:2,000 dilution) for 2 h at room temperature. After washing with PBS-T, an ECL system was used for detection of the proteins. Each assay was performed in four independent experiments.

Transient Transfection of HPASMC
Plasmids encoding p53, Ras, and Raf-1 (wild-type p53, Ras, and Raf genes), p53 mt135, RasV12, and RafCAAX (dominant-negative mutants of p53, Ras, and Raf), and RasN17, RafS621A (constitutive-active mutants of Ras and Raf), and pCMV ß-galactosidase were purchased from Clontech (San Jose, CA). All plasmids were purified using Qiagen plasmid midi and maxi kits.

HPASMC were seeded into 6-cm dishes and incubated in DMEM with 10% FBS for 24 h, and reached ~ 70% confluence. After rinsing, the cells were incubated with 2.5 ml of liposome solution consisting of serum free Opti-MEM medium, Lipofectamine (10 µl/plate), Lipofectamine-Plus reagent (40 µl/plate), and plasmid DNA (3 µg/plate). After 5 h of incubation, the same amount of Opti-MEM medium containing 20% FBS was added to the dishes and the incubation was continued for 19 h. After 24 h of transfection, the cells were washed twice with PBS, and the liposome solution was replaced with DMEM with 10% FBS. Next, 100 µM of SNAP or DETANO was added to the dishes and cultured for 24 h. After incubation, the cells were harvested and analyzed using Western blot analysis.

We assessed the transfection efficacy by transfecting a pCMV ß-galactosidase and determining the number of cells stained blue with X-gal. After transfection with the pCMV ß-galactosidase, the cells were stained with X-gal solution. Briefly, cells on collagen-coated membranes were fixed with 0.25% glutaraldehyde for 10 min, washed twice with PBS containing 1 mM MgCl2, and stained with 1 mg/ml X-gal in a solution of 1 mM MgCl2, 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6 for 4 h. The transfection efficacy was routinely found to be more than 60%.

Statistical Analysis
Results are expressed as the means ± SE. Statistical analysis was performed by ANOVA with Bonferroni for multiple comparisons. Comparisons were considered statistically significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of SNAP and DETANO on Proliferation of HPASMC
SNAP and DETANO dose-dependently increased nitrate and nitrite concentrations in the culture media, though DETANO produced ~ 2-fold greater amounts of NO than SNAP (Figure 1A). Furthermore, SNAP-induced increase of nitrite concentrations reached a plateau after 6 h incubation, and DETANO-induced increase of nitrite concentrations lasted for 24 h (Figure 1B). SNAP dose-dependently suppressed DNA synthesis of HPASMC from a concentration of 30 µM, whereas DETANO significantly suppressed it at a concentration of 100 µM (Figure 1C). On the contrary, cPTIO significantly prevented both SNAP- and DETANO-induced inhibition of the incorporations (Figure 1D). SNAP and DETANO significantly decreased the percentage of S and M phase, and increased the percentage of G0/G1 phase. Apoptosis was not observed in the DNA fragmentation (Figure 2).






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  		Figure 1. Effect of SNAP and DETANO on DNA synthesis of cultured HPASMC. Cultured HPASMC were exposed to various concentrations of SNAP or DETANO in the presence of [3H]-thymidine for 24 h with or without 20 µM of cPTIO. (A) Nitrate and nitrite concentrations in the cultured media were increased dose-dependently by SNAP (solid bars) and DETANO (shaded bars). Data are expressed as mean ± SE (n = 6). (B) Time course of nitrite concentrations in the culture mediums with 100 µM of SNAP (solid bars) or DETANO (shaded bars) were measured by Greiss assay. Although SNAP- induced increase of nitrite concentrations became plateau after 6 h incubation, DETANO-induced increase of nitrite concentrations continuously increased during 24 h. Data are expressed as mean ± SE (n = 6). Open bars, control. (C) [3H]-thymidine incorporation was suppressed by both SNAP (solid bars) and DETANO (shaded bars) dose-dependently. Data are expressed as mean ± SE (n = 6). *P < 0.05 versus control. (D) cPTIO (solid bars) prevented both 100 µM of SNAP- and DETANO-induced inhibition of the [3H]-incorporations. Data are expressed as mean ± SE (n = 6). *P < 0.05 versus the value obtained in the absence of cPTIO (vehicle; open bars).

 


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  		Figure 2. Cell cycle analysis of HPASMC treated with SNAP or DETANO. Cultured HPASMC were exposed to 100 µM of SNAP or DETANO. After 24 h, the cells were harvested and DNA fragmentations were analyzed using a flow cytometer with propidium iodide staining. Area definition of the DNA histogram: C: G0/G1 phase, D: S phase, E:M phase. The cell cycle was significantly suppressed at the S and M phases and increased at the G0/G1 phase by both SNAP and DETANO. Figures shown are representative histograms and bar graph shows data expressed as mean ± SE (n = 6). *P < 0.05 versus control. Solid bars, control; open bars, SNAP; shaded bars, DETANO.

 
Effects of SNAP and DETANO on Activation of p42/44 MAPK, and Expression of p21 and p53
Both SNAP and DETANO increased the expression of p21 mRNA. p21 expression was significantly increased at concentrations of 30, 100, and 300 µM of SNAP, and 100 and 300 µM of DETANO; however, no significant changes were detected in the expression of p53 mRNA (Figure 3A). The PCR products were confirmed by agarose gel electrophoresis and melt curve analysis (Figure 3B). Interestingly, both SNAP and DETANO increased the expression of p53 and p21 protein dose-dependently (Figure 4), whereas the expression of ß-actin proteins did not change after treatment with either NO donor. Conversely, phosphorylated p42/p44 MAPK protein, but not nonphosphorylated p42/44 MAPK, was dose-dependently decreased in the presence of both NO donors after 24 h (Figure 4). When the time-course of the increases of p21 and p53 proteins was studied, the presence of SNAP (100 µM) or DETANO (100 µM) caused them to rise after 6 h of exposure. In the case of SNAP, p53 protein expression peaked at ~ 12 h and then showed a declining trend at 18 h (Figure 5), which was also seen with DETANO. Further, the phosphorylation of p42/44 MAPK was transiently increased at ~ 6 h, and tended to decrease 18 h later after treatment with SNAP and DETANO (Figure 5).




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  		Figure 3. mRNA expressions of p21 and p53 in HPASMC treated with SNAP and DETANO. Cultured HPASMC were exposed to various concentrations of SNAP or DETANO for 24 h, and real-time RT-PCR analyses were performed using a LightCycler. (A) Bar graph shows the ratio of p21 (solid bars) and p53 (open bars) versus GAPDH. Both SNAP and DETANO increased the expression of p21 mRNA, whereas no significant changes were observed in the expression of p53 mRNA. Data are expressed as mean ± SE (n = 4). *P < 0.05 versus control. (B) Melt curve analysis of p21, p53, and GAPDH amplification reaction showed the distinct melting curves of the products. The same reactions were analyzed by agarose gel electrophoresis with EB staining, which revealed single amplification products of the predicted sizes (lane 1, DNA molecular weight marker; lane 2, p21, 146 bp; lane 3, p53, 123 bp; lane 4, GAPDH, 135 bp).

 


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  		Figure 4. Protein expressions of p21 (open bars), p53 (solid bars), and phosphorylated ERK1/2 (shaded bars) in HPASMC treated with SNAP and DETANO. Cultured HPASMC were exposed to various concentrations of SNAP or DETANO for 24 h. Western blot analyses showed that the expressions of p21 and p53 were increased by both SNAP and DETANO, whereas the expression of phosphorylated p42/44 MAPK (p-ERK1/2) was suppressed by the NO donors. Photomicrographs shown are representative of four similar experiments, and the bar graph shows the density ratios of p21, p53, and p-ERK1/2 protein bands versus those of ß-actin protein bands. Data are expressed as mean ± SE (n = 4). *P < 0.05 versus control.

 


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  		Figure 5. Time-course of expression of p21 (open bars), p53 (solid bars), and phosphorylated ERK1/2 (shaded bars) proteins. Cultured HPASMC were exposed to 100 µM of SNAP or DETANO and harvested at the indicated times. Western blot analyses showed that increases of p21 and p53 expression were caused by both SNAP and DETANO after 6 h. Both NO donors increased p-ERK1/2 transiently and then suppressed it. Photomicrographs shown are representative of four similar experiments, and the bar graph shows the density ratios of p21, p53, and p-ERK1/2 protein bands versus those of ß-actin protein bands. Data are expressed as mean ± SE (n = 4). *P < 0.05 versus control.

 
Effects of MEK Inhibitor on Proliferation of HPASMC
PD98059 (30 µM), a specific inhibitor of MAPK kinase, tended to suppress the DNA synthesis of HPASMC, whereas U0126 (10 µM), another specific inhibitor of MAPK kinase, significantly inhibited it. The addition of PD98059 and U0126 accelerated the inhibitory effect of the NO donors (Figure 6a). Although the MAPK kinase inhibitors PD98059 and U0126 inhibited the phosphorylation of p42/44 MAPK protein, no significant increase was noted in the expression of p21 or p53 protein (Figure 6B).




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  		Figure 6. Effect of MEK inhibitors on DNA synthesis and expression of p21, p53, and phosphorylated ERK1/2 proteins in cultured HPASMC. (A) Cultured HPASMC were exposed to 100 µM of SNAP or DETANO in the presence or absence of the MEK inhibitors PD098059 (solid bars) and U0126 (shaded bars) for 24 h, and then [3H]-thymidine incorporation was measured using a liquid scintillation counter. Both PD098059 and U0126 significantly suppressed [3H]-thymidine incorporation during exposure to SNAP and DETANO. Data are expressed as mean ± SE (n = 6). *P < 0.05 versus vehicle (open bars). (B) Cultured HPASMC were exposed to 100 µM of SNAP or DETANO in the presence or absence of the MEK inhibitors PD098059 (PD) or U0126 (U) for 24 h. The cells were then harvested and Western blot analyses were performed. Although the expression of p-ERK1/2 was significantly suppressed by the addition of PD098059 or U0126, no significant change was observed in the expression of p21 and p53 with the addition of either MEK inhibitor. Photomicrographs are representative of four similar experiments, and the bar graph shows the density ratios of p21 (open bars), p53 (solid bars), and p-ERK1/2 (shaded bars) protein bands versus those of ß-actin protein bands. Data are expressed as mean ± SE (n = 4). *P < 0.05 versus control. {dagger}P < 0.05 versus SNAP or DETANO.

 
Effect of Transient Transfection of p53, Ras, and Raf Genes on p42/44 MAPK Phosphorylation and p21 Expression
Transient transfection of wild-type p53 significantly increased p42/44 MAPK phosphorylation and p21 expression, whereas both SNAP and DETANO inhibited those increases in HPASMC. On the other hand, the dominant-negative mutant transfected p53 did not affect the basal state of p42/44 MAPK phosphorylation, whereas the addition of SNAP or DETANO to those transfected cells significantly inhibited phosphorylation. Further, p21 expression did not change with the addition of the NO donors to cells with transfected dominant-negative mutant p53 genes (Figure 7A). In contrast, SNAP and DETANO significantly inhibited p42/44 MAPK phosphorylation cells transfected with wild-type Ras or Raf genes. However, neither SNAP nor DETANO suppressed the phosphorylation of p42/44 MAPK following trasnsfection with constitutive-active mutant Ras or Raf genes (Figure 7B). In addition, induction of p21 and p53 expression by SNAP and DETANO was not affected by transfection with the wild-type and constitutive-active mutant Ras or Raf genes.




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  		Figure 7. Effect of SNAP and DETANO on the expression of p21 (open bars), p53 (solid bars), and phosphorylated ERK1/2 (shaded bars) proteins in cells transfected with p53, Ras, or Raf genes. (A) HPASMC were transiently transfected with plasmids encoding the wild-type (Wt) or dominant-negative (DN) mutation of the p53 gene in the presence or absence of SNAP or DETANO for 24 h, and Western blot analyses were performed. DN-p53 gene transfer suppressed the induction of p21 by the NO donors. The NO donors also suppressed p-ERK1/2 in both Wt and DN mutation transfected cells. Photomicrographs shown are representative of four similar experiments, and the bar graph showes the density ratios of p21, p53, and p-ERK1/2 protein bands versus those of ß-actin protein bands. Data are expressed as mean ± SE (n = 4). *P < 0.05 versus mock. {dagger}P < 0.05 versus Wt or DN. (B) HPASMC were transiently transfected with plasmids encoding the Wt, DN, or constitutive-active (CA) mutation of Ras or Raf genes in the presence or absence of SNAP or DETANO for 24 h, after which the cells were harvested and Western blot analyses were performed. The NO donors failed to suppress p-ERK1/2 in the cells transfected with the CA mutation of the Ras and Raf genes. Photomicrographs shown are representative of four similar experiments, and the bar graph shows the density ratios of p21, p53, and p-ERK1/2 protein bands versus those of ß-actin protein bands. Data are expressed as mean ± SE (n = 4). *P < 0.05 versus mock. {dagger}P < 0.05 versus Wt.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we confirmed that exogenous NO suppresses the proliferation of HPASMC through inhibition of the G1/S transition phases, and also induces the expression of the tumor suppressor p53 and the CDK inhibitor p21. Our results also demonstrated that exogenous NO is able to activate p42/44 MAPK transiently via induction of p53, and then inhibit it via inactivation of the Ras and Raf cascades.

The inhibitory effect of NO on cell proliferation is considered to be related to G1 arrest, which is mediated by a high level of the CDK inhibitor p21. Because cell cycle progression is mediated by CDK, and because activity of CDK2 in complex with cyclin E and cyclin A regulates the progression in G1 phase and entry S phase, we would insist that p21, a CDK inhibitor, which suppress CDK activity by binding cyclin-CDK2 complex (23, 24), and lowering proteins of CDK2 and cyclin A in aortic VSMC (9), may be a potential cellular mechanisms of NO-induced cell suppression. The present results regarding p21 induction and antiproliferative effects were compatible with our and other previous studies using endothelial NOS gene transfer to porcine or pig coronary arterial SMC (7, 25).

In the present study, NO induced p53 protein expression was increased without affecting mRNA levels. Several studies indicate that p53 protein expression relies on post-transcriptional events (26). The p53 protein is a very short-lived protein because of its fast proteasomal degradation, and stabilization of the protein in response to variety stress, such as radiation or cytotoxic agents, results in a rapid rise in p53 levels. Haupt and coworkers reported that p53 protein is upregulated by the decreased degradation and that this degradation is regulated by Mdm2 protein, which bind to p53 protein and promote the degradation (27). Therefore, the increased p53 protein expression by NO donors might be regulated by the post-transcriptional events without affecting mRNA levels.

For the extrinsic NO-induced induction of p53 before p21 expression (and suppressing of p21 expression by the transfer of p53 dominant-negative mutant in our study), Ishida and colleagues demonstrated that p53 was involved in the expression of p21 induced by exogenous NO using p53 knockout mice and A321 cells that had a mutation in the p53 gene (28). Similarly, transfer of NOS genes caused the induction of p53 in rat aortic VSMC (10, 29). These might be the fair reason for the efficacy of endogenous NO on p53 induction.

NO has also been reported to activate p42/44 MAPK in several types of cells, and it has been proposed the MAPK cascade interacts with cell proliferation via induction of cyclin D1, which is thought to be one of the key regulators of G1 progression (14). Further, several studies have also indicated that early (~ 15 min) activation of p42/44 MAPK is mediated by cGMP and/or cGMP-dependent protein kinase (PKG), which is a major receptor protein for cGMP (16, 30). This early transient activation of MAPK is in line with previous findings using endothelial NOS transfer to U937 cells (17) and rat mesangial cells exposed to NO derived from GSNO or inducible NOS (15). Although activation of p42/44 MAPK was transiently observed after 9 h of exposure to NO in the present study, delayed suppression of p42/44 MAPK was observed after 24 h, and such suppressive effect by NO was more enhanced in the presence of PD98059 or U0126.

This activation of the MAPK cascade at 9 h exposure of NO donors may be induced by p53, and p42/44 MAPK might have a compensatory effect on the response of cell cycle arrest induced by p53 and p21. A recent experiment using aortic VSMC obtained from p53 knockout and overexpression mice showed that MAPK activation occurs in a p53-dependent manner in response to NO exposure (21). Further, Lee and coworkers noted that p53-mediated MAPK activation requires functional Ras and Raf (19), and also showed that EGF-like growth factor induced by p53 activates MAPK and Akt signaling (20). In the present study, NO donors ultimately inhibit p42/44 MAPK activity, and this effect was not affected by transfection of dominant-negative p53 gene. It means that p53 plays a minor role in SNAP and DETANO-induced inhibition of p42/44 MAPK activity. In addition, our results showing that inhibition of the MAPK cascade using PD09859 and U0126 accelerated the antiproliferative effects of exogenous NO may be explained by inhibition of the compensatory effect of MAPK.

Preceding studies have suggested that NO can suppress cell proliferation via inhibition of the MAPK cascade including the Ras and Raf pathways, therefore, we evaluated the role of Ras and Raf in our NO study. Using wild-type and mutant p53, Ras, and Raf gene transfer to HPASMC, we demonstrated that NO-induced late suppression of p42/44 MAPK was prevented by Ras and Raf activation, which was independent of p53. In accordance with our results, Yu and colleagues reported that p42/44 MAPK activated by EGF was suppressed by exogenous NO and a cGMP elevating agent in rat aortic VSMC (31). Kibbe and coworkers also reported that p42/44 MAPK was activated in p53 overexpression mice and the activity was suppressed by SNAP (21). Furthermore, DETANO suppressed cyclin D1 production, which was a transcriptional product of the activation of MAPK cascades in a human breast cancer cell line (32). Taken together, these findings support the idea that NO or products derived from NO have another novel mechanism that can suppress activated p42/44 MAPK. Interestingly, we also found that this inhibitory effect of NO was related to inactivation of the Ras and Raf cascades, based on our results using transfer of constitutive-active mutant Ras and Raf genes to HPASMC.

Several studies have shown that induction of p21 by NO is increased through the activation of p42/44 MAPK (16, 18). Conversely, we demonstrated that inhibition of MEK did not affect the expression of p21 and p53. Further, transfer of constitutive active mutant Ras and Raf genes did not modify the expression of p21 and p53. In addition, we showed that p53 induction by NO is essential for p21 induction using transfer of a dominant-negative mutant of the p53 gene. It is well known that Ras is activated by various stimuli to grow and differentiate, and that activated Ras evokes the phosphorylation cascade of protein kinases, including Raf and MAPK. Surprisingly, using specific conditions (serum-starved or Rho inhibited), it has been shown that activated Ras inhibits this proliferation via the induction of p21 (33). Further, high levels of activated Ras and Raf cause cell cycle arrest and p21 induction in a p53-independent manner (34, 35). Therefore, we consider that this novel effect of MAPK toward cell proliferation may be partially explained by the interaction and differentiation of signals from Ras and Rho GTPases.

In summary, we showed that exogenous NO has an inhibitory effect on cell growth via induction of p21 and p53. Further, our results suggest that Ras and Raf signaling is a prerequisite for NO-mediated suppression of p42/44 MAPK in HPASMC. We believe that clarification of the cellular mechanisms of pulmonary smooth muscle proliferation can lead to improved modes of therapy for pulmonary hypertension and aid in remodeling of pulmonary circulation.

Received in original form November 6, 2003

Received in final form March 9, 2004


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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