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(Circulation Research. 1999;85:985.)
© 1999 American Heart Association, Inc.

Molecular Medicine

Distinct Role of cAMP and cGMP in the Cell Cycle Control of Vascular Smooth Muscle Cells

cGMP Delays Cell Cycle Transition Through Suppression of Cyclin D1 and Cyclin-Dependent Kinase 4 Activation

Shinya Fukumoto, Hidenori Koyama, Masayuki Hosoi, Kenjiro Yamakawa, Shinji Tanaka, Hirotoshi Morii, Yoshiki Nishizawa

From the Second Department of Internal Medicine (S.F., H.K., M.H., K.Y., S.T., H.M., Y.N.), Osaka City University Medical School, Osaka, Japan. Dr Hosoi’s current address is Department of Internal Medicine, Osaka City General Hospital, Osaka, Japan.

Correspondence to Hidenori Koyama, MD, PhD, Second Department of Internal medicine, Osaka City University Medical School, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585, Japan. E-mail hidekoyama{at}med.osaka-cu.ac.jp

	Abstract

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Abstract—cAMP and cGMP are known to suppress vascular smooth muscle cell (SMC) proliferation. In this study, our aim was to delineate the molecular mechanism underlying cAMP and cGMP suppression of cell cycle transition in human SMCs. cAMP inhibits both platelet-derived growth factor–stimulated cyclin-dependent kinase (cdk) 2 and cdk4 activation through upregulation of the cdk2 inhibitor p27^Kip1 and downregulation of cyclin D1 expression, which leads to a complete arrest of the cells in phase G₁. In contrast, cGMP inhibits cyclin D1 expression, inhibits cdk4 activation, and delays platelet-derived growth factor–mediated cdk2 activation, resulting in a delay in G₁/S transition. A transient increase in p27^Kip1 in cdk2 immunoprecipitates, without changes in total cellular p27^Kip1 levels, correlates with the delay in cdk2 activation caused by cGMP. Thus, cAMP and cGMP differentially affect cell cycle through distinct regulation of cell cycle molecules in human SMCs.

Key Words: p27^Kip1 • platelet-derived growth factor • kinase

	Introduction

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Vascular smooth muscle cells (SMCs) in the normal arterial media are quiescent, contractile, and resistant to growth factor stimuli. However, injury to the arterial wall results in a phenotypic change in the SMCs that enables them to respond to growth factors and to proliferate.¹ The change in SMC phenotype that leads to proliferation is a key event in the progression of atherosclerotic lesions and in restenosis after angioplasty. The isolation and culture of SMCs are associated with a similar phenotypic change from a contractile to a synthetic, proliferative phenotype.² The molecular mechanisms of modulation from the quiescent to the proliferative phenotype are not fully understood.

Cyclic nucleotides (cAMP and cGMP) are known to inhibit SMC proliferation. The proliferation of SMCs is regulated by several intracellular signaling pathways, and cAMP is known to inhibit many of these pathways, including mitogen-activated protein kinase cascade,^{3 4 5} p70 S6 kinase,^{6 7} and cyclin-dependent kinase (cdk) 4,⁸ which can ultimately inhibit cell cycle progression. Little is known about the cGMP-mediated signaling system in the regulation of SMC proliferation. However, agents that increase intracellular cGMP can attenuate mitogenesis induced by growth factors.^{9 10 11}

Cell proliferation is regulated at the level of the cell cycle by cell cycle–regulatory proteins.¹² Under the appropriate stimulation, quiescent cells travel through the cell cycle as a consequence of the activation of specific cdks.¹³ Two families of cdk inhibitors regulate cyclin/CDK complexes.¹² Cyclin D1/cdk4 and cyclin E/cdk2 are known to be required for G₁/S transition and DNA synthesis.^{14 15 16 17}

In this study, we compared the suppressive effect of cAMP and cGMP on SMC proliferation stimulated by platelet-derived growth factor (PDGF)-BB. Our goal was to delineate the molecular mechanisms underlying the cAMP- and cGMP-mediated suppression of SMC proliferation.

	Materials and Methods

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Materials
All reagents, unless otherwise stated, were purchased from Sigma Chemical Co. Recombinant PDGF-BB was purchased from Genzyme. Antibodies against cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG) were obtained from Calbiochem Corp. cdk2, cdk4, and cyclin E were purchased from Santa Cruz Biochemicals. Antibodies to cyclin D1 were from Upstate Biotechnology Inc. Antibodies to p27^Kip1 and p21^Cip1/Waf1 were obtained from Pierce Chemical and Santa Cruz Biochemicals.

Cells and Cell Culture
Human SMCs (neonatal umbilical artery origin), obtained from Cell System Co, were cultured in Dulbecco’s modified Eagle’s medium containing 10% FCS (Life Technologies, Inc) and used for experiments within 3 to 10 passages. Before mitogenic stimulation, subconfluent cells were arrested in the quiescent state with a culture in Dulbecco’s modified Eagle’s medium containing 0.2% FCS for 72 hours. 8-Bromo-cAMP (8-Br-cAMP) and 8-bromo-cGMP (8-Br-cGMP) were simultaneously added with mitogenic stimulation.

Cell Cycle Transition
Cell cycle transition was determined through bromodeoxyuridine (BrdU) or ³H-thymidine incorporation. BrdU (10 µmol/L) was added to SMCs just before PDGF stimulation, and the cells were cultured for an indicated number of hours. The cells were fixed in 70% ethanol, and incorporated BrdU was detected on the basis of fluorescein isothiocyanate–labeled anti-BrdU antibody. The total number of nuclei was determined on the basis of staining with propidium iodide, and the cells that had entered S phase were calculated as the percentage of BrdU-incorporated cells. ³H-Thymidine incorporation was determined as described previously.¹⁸

Northern Blot Analysis
Northern blot analysis for cell cycle molecules was performed as described previously.¹⁹ cDNAs for mouse cyclin D1, cdk2, and cdk4 were kindly provided by Dr H. Matsushime (Tokyo University, Tokyo, Japan). Rat GAPDH cDNA was a generous gift of Dr P. Fort (Université des Sciences et Techniques du Languedoc, Montpellier Cedex, France).²⁰

Immunoprecipitation, Western Blot Analysis, and Measurements of cdk2 or cdk4 Activity
Cell lysate preparation and Western blot analyses were performed as previously described.¹⁸ Cyclin/cdk complexes were determined through immunoprecipitation with antibodies against cdk4 and cdk2, followed by immunoblotting with specific antibodies as described previously.²¹ Both cdk2 and cdk4 activities were determined with immunoprecipitation and in vitro kinase assay as previously described.^{21 22} Cell lysates were precleared with immobilized protein G–Sepharose, and cdk2 or cdk4 was immunoprecipitated with anti-cdk2 or anti-cdk4 monoclonal antibody (100 µL/sample), respectively. Histone H1 (Boehringer Mannheim) was used for a substrate for cdk2, and retinoblastoma protein (Santa Cruz Biochemicals) was used for a substrate for cdk4.

Statistical Analysis
Statistical analysis was performed with ANOVA with multiple comparisons (Scheffé test) with the use of StatView IV software.

	Results

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Inhibitory Effect of cAMP and cGMP on Cell Cycle Transition in SMCs
We first compared the effect of two cyclic nucleotides, 8-Br-cAMP and 8-Br-cGMP, on PDGF-stimulated cell cycle transition in human SMCs. G₁/S transition in the cell cycle was determined with BrdU incorporation, in which BrdU was added with 10 ng/mL PDGF stimulation. When SMCs were treated with the indicated concentrations of 8-Br-cAMP or 8-Br-cGMP, PDGF-stimulated S-phase entry at 24 hours was dose-dependently and equally inhibited (Figure 1). However, the kinetics of S phase accumulation reveal that the suppressive effect of 8-Br-cGMP was transient (Figure 2). At 30 and 36 hours after PDGF stimulation, no significant inhibitory effects of cGMP were observed, whereas cAMP inhibited S-phase transition by >50% during a period of 24 to 36 hours.

View larger version (26K): [in this window] [in a new window]   Figure 1. Suppression of S-phase entry by both cAMP and cGMP. Human SMCs were serum starved for 72 hours, and cells were treated with 10 ng/mL PDGF. Indicated concentrations of 8-Br-cAMP or 8-Br-cGMP were added together with PDGF stimulation. G1/S transition in cell cycle was determined by BrdU incorporation, in which BrdU was added together with PDGF stimulation. Each column represents mean±SD values. *P<0.05 vs 0 mmol/L (multiple comparison, Scheffé test). This experiment was performed twice, and representative data are shown.

View larger version (17K): [in this window] [in a new window]   Figure 2. cGMP delays G1/S transition. Quiescent SMCs were treated with PDGF (10 ng/mL) alone, PDGF plus 8-Br-cAMP (1 mmol/L), or PDGF plus 8-Br-cGMP (1 mmol/L) for indicated hours. S-phase transition was determined by BrdU incorporation as in legend to Figure 1. Each plot represents mean±SD values. *P<0.05 vs PDGF alone (multiple comparison, Scheffé test). This experiment was performed twice and was reproducible.

To further analyze the possible distinct roles of cAMP and cGMP on SMC proliferation, we examined the effect of combinatory treatment with cAMP and cGMP on SMC DNA synthesis. The suppressive effect of 1 mmol/L cGMP on SMC DNA synthesis was additive to 0.1 to 1.0 mmol/L cAMP (Figure 3A). Moreover, the suppressive effect of 0.3 mmol/L cAMP on SMC proliferation was further augmented by cotreatment with as little as 0.1 mmol/L cGMP (Figure 3B).

View larger version (23K): [in this window] [in a new window]   Figure 3. Additive suppressive effect of cGMP and cAMP on SMC DNA synthesis. Quiescent SMCs were treated with PDGF (10 ng/mL) alone, PDGF plus 8-Br-cAMP, PDGF plus 8-Br-cGMP, or PDGF plus cAMP plus cGMP for 24 hours. SMC DNA synthesis was determined with 3H-thymidine incorporation assay. Each plot represents mean±SD values. This experiment was performed twice and was reproducible.

It has been reported that PKG is downregulated in SMCs in culture.²³ To examine the possibility that the difference between the effects of cAMP and cGMP is due to downregulation of PKG in our SMC system, the expression of PKG was determined through Western blot analysis. As shown in Figure 4, both PKA and PKG were abundantly expressed in this SMC system. These results led us to further analyze the distinct molecular mechanism underlying cAMP and cGMP suppression of SMC proliferation.

View larger version (54K): [in this window] [in a new window]   Figure 4. Expression of PKA and PKG in human SMCs, which were cultured until confluency and serum deprived for 2 days. Expression of PKA and PKG was determined by Western blot analysis. Lane 1 represents PKA; lane 2, PKG. This experiment was repeated with SMCs with different passages (6 and 9), and the two kinases were similarly detected.

cGMP Preferably Inhibits cdk4 Activities, and cAMP Inhibits Both cdk4 and cdk2 Activities
To understand the molecular basis underlying cyclic nucleotide suppression of cell cycle transition, we examined the effect of the cyclic nucleotides on cdk4 and cdk2 activities, the activation of which is shown to be essential for the G₁/S transition.¹³ The kinetics of cdk2 and cdk4 activation after PDGF stimulation revealed that cdk2 was activated later than 18 to 24 hours, whereas cdk4 activation reached a maximal level at 18 hours (Figure 5A). The activation of cdk4 was preceded by an increase in cyclin D1 mRNA and protein levels. After PDGF stimulation, cyclin D1 mRNA was induced as early as 3 hours and reached a maximal level at 6 hours, whereas cyclin D1 protein levels were gradually increased up until 18 hours (Figure 5B).

View larger version (28K): [in this window] [in a new window]   Figure 5. Kinetics of cdk2 and cdk4 activation and cyclin D1 expression after PDGF stimulation. Quiescent SMCs were treated with PDGF (10 ng/mL), and cdk2 activity (A), cdk4 activity (A), and cyclin D1 mRNA or protein levels (B) were determined as described in Materials and Methods. This experiment was repeated, and identical results were obtained.

Both 8-Br-cAMP and 8-Br-cGMP (Figure 6) equally and entirely suppressed PDGF-stimulated cdk4 activation at 18 hours. cdk4 protein levels were also increased by PDGF, and both cyclic nucleotides significantly suppressed its induction. Protein levels of cyclin D1 were induced by PDGF, and these were suppressed to basal levels by both 8-Br-cAMP and 8-Br-cGMP (Figure 6). The suppression of cyclin D1 protein expression by cyclic nucleotides was associated with significantly decreased levels of cyclin D1 mRNA, suggesting that both cyclic nucleotides inhibit cyclin D1 expression at an mRNA level.

View larger version (35K): [in this window] [in a new window]   Figure 6. Suppression of cdk4 activities by both cAMP and cGMP. Quiescent SMCs were treated with PDGF (10 ng/mL), PDGF plus 8-Br-cAMP (1 mmol/L), or PDGF plus 8-Br-cGMP (1 mmol/L) for 18 hours. cdk4 activity, cdk4 protein, cyclin D1 protein, and cyclin D1 mRNA levels were determined as described in Materials and Methods. B, Summary of results from 3 independent experiments. Each column represents mean±SD values. *P<0.05 vs control; **P<0.05 vs PDGF (multiple comparison, Scheffé test).

We next examined the effect of cyclic nucleotides on PDGF-stimulated cdk2 activation. cAMP completely suppressed cdk2 activation at 18 and 24 hours (Figure 7). cGMP suppressed PDGF-induced cdk2 activation at 18 hours, but its inhibitory effect was not continued until 24 hours (Figure 7). To explore the possibility that cGMP delays cdk2 activation, the kinetics of PDGF-stimulated cdk2 activation were examined in the presence of vehicle, cAMP, or cGMP. As shown in Figure 8, cGMP delayed, but did not block, the activation of cdk2 induced by PDGF. In contrast, cAMP significantly inhibited the PDGF-stimulated cdk2 activation at all time points. At 18 hours, the protein levels of cdk2 were increased 50% by PDGF, but its level was not suppressed by either cAMP or cGMP (Figure 7). The faster migrating form of cdk2 on Western blotting was phosphorylated on Thr¹⁶⁰, which has been shown to be essential for cdk2 activation.²⁴ The density of phosphorylated cdk2 was induced by PDGF at 18 hours, and this induction was significantly but partially suppressed by either cAMP or cGMP (Figure 7). Neither cAMP nor cGMP affected the protein levels of cyclin E, which forms a complex with cdk2 and contributes to cdk2 activation in the late G₁ phase.¹³

View larger version (42K): [in this window] [in a new window]   Figure 7. Effects of cGMP and cAMP on PDGF-stimulated cdk2 activity. Quiescent SMCs were treated with PDGF (10 ng/mL), PDGF plus 8-Br-cAMP (1 mmol/L), or PDGF plus 8-Br-cGMP (1 mmol/L) for 18 or 24 hours. cdk2 protein, cdk2 activity, and cyclin E protein levels were determined as described in Materials and Methods. B, Summary of results from 3 independent experiments. Each column represents mean±SD values. *P<0.05 vs control; **P<0.05 vs PDGF (multiple comparison, Scheffé test).

View larger version (28K): [in this window] [in a new window]   Figure 8. Effects of cGMP and cAMP on kinetics of cdk2 activity after PDGF stimulation. Quiescent SMCs were treated with PDGF (10 ng/mL), PDGF plus 8-Br-cAMP (1 mmol/L), or PDGF plus 8-Br-cGMP (1 mmol/L) for indicated hours. cdk2 activity was determined as described in Materials and Methods. B, Summary of results from 3 independent experiments. Each plot represents mean±SD values. *P<0.05 vs control; **P<0.05 vs PDGF alone (multiple comparison, Scheffé test).

cAMP, but Not cGMP, Inhibits PDGF-Mediated Downregulation of p27^Kip1
To investigate how cAMP and cGMP differentially regulate cdk activation and cell cycle transition, we examined regulation of the candidates for cdk inhibition: p21^Cip1/Waf1 and p27^Kip1.¹² We examined the levels of cdk inhibitors complexed with cdk4 or cdk2 at 18 hours, when cdk2 and cdk4 activities were significantly suppressed by both cAMP and cGMP. Western blot analysis showed that PDGF treatment for 18 hours downregulated p27^Kip1 protein levels and that cAMP prevented this p27^Kip1 suppression (Figure 9). Consistent with the high level of total cellular p27^Kip1, p27^Kip1 levels associated with cdk4 and cdk2 were significantly higher in the presence of cAMP (Figure 9). The effect of cAMP on total cellular and cdk2-bound p27^Kip1 levels was consistent up to 30 hours (Figure 9).

View larger version (33K): [in this window] [in a new window]   Figure 9. cAMP, but not cGMP, prevents PDGF-stimulated p27 downregulation. Quiescent SMCs were treated with PDGF (10 ng/mL), PDGF plus 8-Br-cAMP (1 mmol/L), or PDGF plus 8-Br-cGMP (1 mmol/L) for 18 or 30 hours. Total cellular and cdk4- or cdk2-bound p27Kip1 levels were determined as described in Materials and Methods. . B, Summary of results from 3 independent experiments. Each plot represents mean±SD. *P<0.05 vs control; **P<0.05 vs PDGF (multiple comparison, Scheffé test).

In contrast to cAMP, cGMP did not have a significant effect on total cellular p27^Kip1. Instead of increasing, it decreased cdk4-associated p27^Kip1 levels at 18 hours. However, p27^Kip1 levels associated with cdk2 in cGMP-treated cells were higher than those in vehicle-treated cells, suggesting a dynamic shift in p27^Kip1 from cdk4 to cdk2 (Figure 9). The cGMP-induced increase in cdk2-bound p27^Kip1 levels was transient and was not observed at 30 hours (Figure 9). p21^Cip1/Waf1 expression in this cell was hardly detected in this SMC, and neither cyclic nucleotide had a significant effect on its level (Figure 9).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
We examined the molecular basis underlying cAMP and cGMP regulation of SMC proliferation and clearly showed that the main effect of cGMP is suppression of cyclin D1 and cdk4 activity, which leads to a delay in cdk2 activation and G₁/S transition in the cell cycle. In contrast, cAMP inhibits both cdk4 and cdk2 activities, leading to complete cell cycle arrest in G₁.

It is well documented that cGMP-generating reagents have an antimitogenic effect on SMC proliferation in vitro and in vivo.^{9 10} Because cAMP is also known to strongly suppress cell cycle transition in vascular SMCs,^{25 26 27} we sought to examine and delineate the molecular basis for cAMP and cGMP suppression of SMC proliferation. Interestingly, SMCs treated with cGMP do not enter the S phase 24 hours after PDGF stimulation, whereas at 30 hours, a significant portion of the cells travel to the S phase. This result suggests that cGMP delays, but does not block, the G₁/S transition, probably through the prolongation of G₁. In contrast, cAMP appears to arrest the cells in G₁ as previously described,⁸ because the percentage of cells that have entered the S phase after PDGF stimulation is proportionally suppressed at each time examined. It has been reported that PKG is downregulated in SMCs in culture.²³ It is unlikely that the distinct effect of cAMP and cGMP is due to the downregulation of PKG, because both PKG and PKA are abundantly expressed in our human SMC system. Because even the lower concentrations of cAMP and cGMP have additive suppressive effects on SMC proliferation (Figure 3), several antimitogenic agents, including vasodilators, may have a cooperative role in the regulation of SMC proliferation with the use of distinct PKA and PKG systems. These findings led us to examine possible distinct regulation of cell cycle molecules by cAMP and cGMP.

It has been proposed that cdk activities control the G₁/S transition in mammalian cells.¹³ Both D- and E-type cyclins are known to be important regulators in G₁/S control, even though some reports raise the possibility that they have distinct roles.^{17 28} In many cell systems, the induction of cyclin D after growth factor stimulation precedes that of cyclin E. Thus, cyclin D–associated kinase is maximally activated earlier than cyclin E–associated kinase.¹³ cdk4 is a main catalytic partner of cyclin D, and cdk2 is known to form complexes with cyclin E and cyclin A. In the present study in human SMCs, cdk4 activation after PDGF stimulation occurs as early as 12 hours and reaches a maximal level at 18 hours, and cdk2 activities follow those of cdk4. Thus, the distinct kinetics of the activation of these cdks support the idea that they have different roles in cell cycle regulation. Cyclin D1 expression precedes the activation of cdk4, and cdk4 protein levels are also significantly increased after PDGF stimulation. Although cdk2 activities are dramatically induced after treatment with PDGF in human SMCs, cyclin E protein levels do not oscillate. These results in human SMCs confirm previous findings in various SMC systems concerning the growth factor regulation of cell cycle molecules.^{19 21 29 30}

To explore the possibility that cdk4 or cdk2 activities contribute to the distinct effects of cAMP and cGMP on cell cycle transition, we examined the effects of the cyclic nucleotides on cdk4 and cdk2 activities. cAMP is known to inhibit cdk4 activity in macrophages,⁸ and in SMCs, cyclin D1 expression is known to be partially inhibited by cAMP.²⁷ There was only one report of cGMP regulation of the cell cycle in rat SMCs, in which cyclin A and cyclin D expressions are suppressed by cGMP.³⁰ No reports, however, have compared the detail regulation of cell cycle molecules underlying cAMP and cGMP suppression of SMC proliferation. In the human SMC system, both cAMP and cGMP entirely suppress PDGF-stimulated cdk4 activity at 18 hours, which is associated with the suppression of cyclin D1 mRNA, cyclin D1 protein, and cdk4 protein levels. PDGF-stimulated cdk2 activation is completely inhibited by cAMP, whereas the kinetics of cdk2 activation are delayed by cGMP. Thus, altered kinetics in cdk2 activation may account for delayed G₁/S transition in cells treated with cGMP. Even though PDGF-stimulated cdk2 activities at 18 hours are almost entirely suppressed by cGMP or cAMP, the levels of cyclin E are not dramatically regulated by cyclic nucleotides. However, cAMP and cGMP significantly suppress cdk2 phosphorylation induced by PDGF. These data imply the involvement of cdk2 inhibitors in the inactivation of the cyclin E/cdk2 complex.³¹

A family of cdk inhibitors plays a major role in the cell cycle machinery.^{12 32 33} Two molecules, p21^Cip1/Waf1 and p27^Kip1, directly inhibit cdk activity and prevent its phosphorylation. p27^Kip1 has been shown to mediate cell cycle arrest in response to various factors, including transforming growth factor-ß,³⁴ rapamycin,³⁵ cAMP,⁸ and extracellular matrices.²¹ On the other hand, p21^Cip1/Waf1 appears to be involved in cell cycle arrest induced by irradiation or UV irradiation.^{36 37} p21^Cip1/Waf1 is also known to regulate senescence, apoptosis, or differentiation of various cells.^{38 39 40 41} The expression of p27^Kip1 and p21^Cip1/Waf1 in vascular injury was extensively studied and showed the possible roles of the inhibitors in SMC behavior in SMCs in vivo.^{42 43 44} Moreover, exogenous overexpression of the inhibitors in vessel wall successfully inhibits injury-induced SMC proliferation.^{45 46}

In this study, p21^Cip1/Waf1 is barely detected and regulated in SMCs. p27^Kip1 is abundantly expressed in serum-starved cells, and its level is downregulated by PDGF as previously described.^{29 47} cAMP successfully interferes with the PDGF-directed decrease in p27^Kip1 in total cellular lysates or cdk4 immunoprecipitates, which is in good agreement with previous observations.⁸ Because p27^Kip1 complexed with cdk2 or cdk4 is higher in cAMP-treated cells than in vehicle-treated cells, p27^Kip1 levels appear to account for the entire suppression of both cdk activities, which leads to G₁ arrest in the cell cycle. In contrast, cGMP fails to prevent PDGF-mediated decrease in total cellular p27^Kip1 levels; in addition, instead of increasing, it decreases the level of p27^Kip1 in cdk4 immunoprecipitates. However, p27^Kip1 associated with cdk2 in cGMP-treated cells is significantly higher than that in vehicle-treated cells at 18 hours, but this effect of cGMP is not maintained until 30 hours. Thus, this transient shift in p27^Kip1 to cdk2 may explain how cGMP delays the activation of cdk2. It is well documented that p27^Kip1 is mainly pooled in cyclin D1 complex in growing cells. Withn proliferation-inhibitory stimuli such as lovastatin treatment, cyclin D1 is degraded and p27^Kip1 is rapidly redistributed to cdk2, leading to the suppression of cdk2 activation and the inhibition of the cell cycle transition.⁴⁸ cGMP suppression of cyclin D1 levels could transiently redistribute p27^Kip1 to cdk2, resulting in a delay in cdk2 activation and G₁/S transition. In the present study, it was not clear why the shift of p27^Kip1 to cdk2 did not completely arrest the cell cycle. However, it may be possible that upregulation of total cellular p27^Kip1 is necessary for the permanent suppression of cdk2 activity and G₁/S transition. Taken together, p27^Kip1 upregulation appears to be a main target for cAMP-mediated signaling, whereas downregulation of cyclin D1 and cdk4 activities could account for a delay in cell cycle transition in cGMP-treated cells.

Recently, NO donors sodium nitroprusside and S-nitroso-N-acetylpenicillamine have been shown to block cdk2 activation without affecting protein levels of cdk2 or cyclin E.^{11 49} Guo et al¹¹ showed that the suppressive effects of NO donors on cdk2 activities are sustained until 48 hours, and Ishida et al⁴⁹ suggested that p21^Cip1/Waf1 can be a target of NO. It is known that the effect of NO is transduced through cGMP.⁵⁰ Yu et al⁹ and Kronemann et al³⁰ showed in rat SMCs that cGMP completely suppressed epidermal growth factor– or FCS-induced DNA synthesis, respectively. In our human SMC system, 1 mmol/L cGMP delayed, but did not block, PDGF-stimulated cdk2 activation and DNA synthesis. Moreover, p21^Cip1/Waf is barely detected in our SMC system. The lack of data from the present study showing complete inhibition of SMC proliferation may be due to differences in species or SMC phenotype. It may also be possible that expression levels of PKG differ in rat SMCs.²³ Potential unidentified NO-mediated signaling other than cGMP could also contribute to differences in effect between NO donors and cGMP.

In summary, cGMP and cAMP differentially affect cell cycle through distinct regulation of cell cycle molecules in human SMCs. A main target of cGMP appears to be the suppression of cyclin D1 expression, which leads to cdk4 inactivation and a delay in cdk2 activation and G₁/S transition in the cell cycle.

	Acknowledgments

 
This work was supported in part by Japan Heart Foundation/Pfizer Pharmaceuticals Grant for Research on Coronary Artery Disease (Dr Koyama). The authors thank Masayo Monden for her excellent technical assistance.

Received June 15, 1999; accepted September 8, 1999.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993;362:801–809.[Medline] [Order article via Infotrieve]

2. Thyberg J, Hedin U, Sjolund M, Palmberg L, Bottger BA. Regulation of differentiated properties and proliferation of arterial smooth muscle cells. Arteriosclerosis. 1990;10:966–990.[Free Full Text]

3. Cook SJ, McCormick F. Inhibition by cAMP of Ras-dependent activation of Raf. Science. 1993;262:1069–1072.[Abstract/Free Full Text]

4. Graves LM, Bornfeldt KE, Raines EW, Potts BC, Macdonald SG, Ross R, Krebs EG. Protein kinase A antagonizes platelet-derived growth factor-induced signaling by mitogen-activated protein kinase in human arterial smooth muscle cells. Proc Natl Acad Sci U S A. 1993;90:10300–10304.[Abstract/Free Full Text]

5. Wu J, Dent P, Jelinek T, Wolfman A, Weber MJ, Sturgill TW. Inhibition of the EGF-activated MAP kinase signaling pathway by adenosine 3',5'-monophosphate. Science. 1993;262:1065–1069.[Abstract/Free Full Text]

6. Graves LM, Bornfeldt KE, Argast GM, Krebs EG, Kong X, Lin TA, Lawrence JC, Jr. cAMP- and rapamycin-sensitive regulation of the association of eukaryotic initiation factor 4E and the translational regulator PHAS-I in aortic smooth muscle cells. Proc Natl Acad Sci U S A. 1995;92:7222–7226.[Abstract/Free Full Text]

7. Monfar M, Lemon KP, Grammer TC, Cheatham L, Chung J, Vlahos CJ, Blenis J. Activation of pp70/85 S6 kinases in interleukin-2-responsive lymphoid cells is mediated by phosphatidylinositol 3-kinase and inhibited by cyclic AMP. Mol Cell Biol. 1995;15:326–337.[Abstract/Free Full Text]

8. Kato JY, Matsuoka M, Polyak K, Massague J, Sherr CJ. Cyclic AMP-induced G₁ phase arrest mediated by an inhibitor (p27Kip1) of cyclin-dependent kinase 4 activation. Cell. 1994;79:487–496.[Medline] [Order article via Infotrieve]

9. Yu SM, Hung LM, Lin CC. cGMP-elevating agents suppress proliferation of vascular smooth muscle cells by inhibiting the activation of epidermal growth factor signaling pathway. Circulation. 1997;95:1269–1277.[Abstract/Free Full Text]

10. Chen L, Daum G, Forough R, Clowes M, Walter U, Clowes AW. Overexpression of human endothelial nitric oxide synthase in rat vascular smooth muscle cells and in balloon-injured carotid artery. Circ Res. 1998;82:862–870.[Abstract/Free Full Text]

11. Guo K, Andres V, Walsh K. Nitric oxide-induced downregulation of Cdk2 activity and cyclin A gene transcription in vascular smooth muscle cells. Circulation. 1998;97:2066–2072.[Abstract/Free Full Text]

12. Sherr CJ, Roberts JM. Inhibitors of mammalian G1 cyclin-dependent kinases. Genes Dev. 1995;9:1149–1163.[Free Full Text]

13. Sherr CJ. Mammalian G₁ cyclins. Cell. 1993;73:1059–1065.[Medline] [Order article via Infotrieve]

14. Baldin V, Lukas J, Marcote MJ, Pagano M, Draetta G. Cyclin D1 is a nuclear protein required for cell cycle progression in G₁. Genes Dev. 1993;7:812–821.[Abstract/Free Full Text]

15. Quelle DE, Ashmun RA, Shurtleff SA, Kato JY, Bar-Sagi D, Roussel MF, Sherr CJ. Overexpression of mouse D-type cyclins accelerates G₁ phase in rodent fibroblasts. Genes Dev. 1993;7:1559–1571.[Abstract/Free Full Text]

16. Tsai LH, Lees E, Faha B, Harlow E, Riabowol K. The cdk2 kinase is required for the G₁-to-S transition in mammalian cells. Oncogene. 1993;8:1593–1602.[Medline] [Order article via Infotrieve]

17. Ohtsubo M, Theodoras AM, Schumacher J, Roberts JM, Pagano M. Human cyclin E, a nuclear protein essential for the G₁-to-S phase transition. Mol Cell Biol. 1995;15:2612–2624.[Abstract/Free Full Text]

18. Fukumoto S, Nishizawa Y, Hosoi M, Koyama H, Yamakawa K, Ohno S, Morii H. Protein kinase C delta inhibits the proliferation of vascular smooth muscle cells by suppressing G₁ cyclin expression. J Biol Chem. 1997;272:13816–13822.[Abstract/Free Full Text]

19. Koyama H, Nishizawa Y, Hosoi M, Fukumoto S, Kogawa K, Shioi A, Morii H. The fumagillin analogue TNP-470 inhibits DNA synthesis of vascular smooth muscle cells stimulated by platelet-derived growth factor and insulin-like growth factor-I: possible involvement of cyclin-dependent kinase 2. Circ Res. 1996;79:757–764.[Abstract/Free Full Text]

20. Fort P, Marty L, Piechaczyk M, el Sabrouty S, Dani C, Jeanteur P, Blanchard JM. Various rat adult tissues express only one major mRNA species from the glyceraldehyde-3-phosphate-dehydrogenase multigenic family. Nucleic Acids Res. 1985;13:1431–1442.[Abstract/Free Full Text]

21. Koyama H, Raines EW, Bornfeldt KE, Roberts JM, Ross R. Fibrillar collagen inhibits arterial smooth muscle proliferation through regulation of Cdk2 inhibitors. Cell. 1996;87:1069–1078.[Medline] [Order article via Infotrieve]

22. Matsushime H, Quelle DE, Shurtleff SA, Shibuya M, Sherr CJ, Kato JY. D-type cyclin-dependent kinase activity in mammalian cells. Mol Cell Biol. 1994;14:2066–2076.[Abstract/Free Full Text]

23. Cornwell TL, Lincoln TM. Regulation of intracellular Ca²⁺ levels in cultured vascular smooth muscle cells: reduction of Ca²⁺ by atriopeptin and 8-bromo-cyclic GMP is mediated by cyclic GMP-dependent protein kinase. J Biol Chem. 1989;264:1146–1155.[Abstract/Free Full Text]

24. Gu Y, Rosenblatt J, Morgan DO. Cell cycle regulation of CDK2 activity by phosphorylation of Thr160 and Tyr15. EMBO J. 1992;11:3995–4005.[Medline] [Order article via Infotrieve]

25. Bornfeldt KE, Campbell JS, Koyama H, Argast GM, Leslie CC, Raines EW, Krebs EG, Ross R. The mitogen-activated protein kinase pathway can mediate growth inhibition and proliferation in smooth muscle cells: dependence on the availability of downstream targets. J Clin Invest. 1997;100:875–885.[Medline] [Order article via Infotrieve]

26. Indolfi C, Avvedimento EV, Di Lorenzo E, Esposito G, Rapacciuolo A, Giuliano P, Grieco D, Cavuto L, Stingone AM, Ciullo I, Condorelli G, Chiariello M. Activation of cAMP-PKA signaling in vivo inhibits smooth muscle cell proliferation induced by vascular injury. Nat Med. 1997;3:775–759.[Medline] [Order article via Infotrieve]

27. Vadiveloo PK, Filonzi EL, Stanton HR, Hamilton JA. G₁ phase arrest of human smooth muscle cells by heparin, IL-4, and cAMP is linked to repression of cyclin D1 and cdk2. Atherosclerosis. 1997;133:61–69.[Medline] [Order article via Infotrieve]

28. Resnitzky D, Reed SI. Different roles for cyclins D1 and E in regulation of the G₁-to-S transition. Mol Cell Biol. 1995;15:3463–3469.[Abstract/Free Full Text]

29. Rao GN. Differential regulation of p27 kip1 levels and CDK activities by hypertrophic and hyperplastic agents in vascular smooth muscle cells. Biochim Biophys Acta. 1999;1448:525–532.[Medline] [Order article via Infotrieve]

30. Kronemann N, Nockher WA, Busse R, Schini-Kerth VB. Growth-inhibitory effect of cyclic GMP- and cyclic AMP-dependent vasodilators on rat vascular smooth muscle cells: effect on cell cycle and cyclin expression. Br J Pharmacol. 1999;126:349–357.[Medline] [Order article via Infotrieve]

31. Aprelikova O, Xiong Y, Liu ET. Both p16 and p21 families of cyclin-dependent kinase (CDK) inhibitors block the phosphorylation of cyclin-dependent kinases by the CDK-activating kinase. J Biol Chem. 1995;270:18195–18197.[Abstract/Free Full Text]

32. Hunter T, Pines J. Cyclins and cancer, II: cyclin D and CDK inhibitors come of age. Cell. 1994;79:573–582.[Medline] [Order article via Infotrieve]

33. Peter M, Herskowitz I. Joining the complex: cyclin-dependent kinase inhibitory proteins and the cell cycle. Cell. 1994;79:181–184.[Medline] [Order article via Infotrieve]

34. Polyak K, Kato JY, Solomon MJ, Sherr CJ, Massague J, Roberts JM, Koff A. p27Kip1, a cyclin-Cdk inhibitor, links transforming growth factor-beta and contact inhibition to cell cycle arrest. Genes Dev. 1994;8:9–22.[Abstract/Free Full Text]

35. Nourse J, Firpo E, Flanagan WM, Coats S, Polyak K, Lee MH, Massague J, Crabtree GR, Roberts JM. Interleukin-2-mediated elimination of the p27Kip1 cyclin-dependent kinase inhibitor prevented by rapamycin. Nature. 1994;372:570–573.[Medline] [Order article via Infotrieve]

36. Dulic V, Kaufmann WK, Wilson SJ, Tlsty TD, Lees E, Harper JW, Elledge SJ, Reed SI. p53-dependent inhibition of cyclin-dependent kinase activities in human fibroblasts during radiation-induced G₁ arrest. Cell. 1994;76:1013–1023.[Medline] [Order article via Infotrieve]

37. Brugarolas J, Chandrasekaran C, Gordon JI, Beach D, Jacks T, Hannon GJ. Radiation-induced cell cycle arrest compromised by p21 deficiency. Nature. 1995;377:552–557.[Medline] [Order article via Infotrieve]

38. Deng C, Zhang P, Harper JW, Elledge SJ, Leder P. Mice lacking p21CIP1/WAF1 undergo normal development, but are defective in G₁ checkpoint control. Cell. 1995;82:675–684.[Medline] [Order article via Infotrieve]

39. Wang J, Walsh K. Resistance to apoptosis conferred by Cdk inhibitors during myocyte differentiation. Science. 1996;273:359–361.[Abstract]

40. Brown JP, Wei W, Sedivy JM. Bypass of senescence after disruption of p21CIP1/WAF1 gene in normal diploid human fibroblasts. Science. 1997;277:831–834.[Abstract/Free Full Text]

41. Levkau B, Koyama H, Raines EW, Clurman BE, Herren B, Orth K, Roberts JM, Ross R. Cleavage of p21Cip1/Waf1 and p27Kip1 mediates apoptosis in endothelial cells through activation of Cdk2: role of a caspase cascade. Mol Cell. 1998;1:553–563.[Medline] [Order article via Infotrieve]

42. Yang ZY, Simari RD, Perkins ND, San H, Gordon D, Nabel GJ, Nabel EG. Role of the p21 cyclin-dependent kinase inhibitor in limiting intimal cell proliferation in response to arterial injury. Proc Natl Acad Sci U S A. 1996;93:7905–7910.[Abstract/Free Full Text]

43. Ihling C, Menzel G, Wellens E, Monting JS, Schaefer HE, Zeiher AM. Topographical association between the cyclin-dependent kinases inhibitor P21, p53 accumulation, and cellular proliferation in human atherosclerotic tissue. Arterioscler Thromb Vasc Biol. 1997;17:2218–2224.[Abstract/Free Full Text]

44. Tanner FC, Yang ZY, Duckers E, Gordon D, Nabel GJ, Nabel EG. Expression of cyclin-dependent kinase inhibitors in vascular disease. Circ Res. 1998;82:396–403.[Abstract/Free Full Text]

45. Chen D, Krasinski K, Sylvester A, Chen J, Nisen PD, Andres V. Downregulation of cyclin-dependent kinase 2 activity and cyclin A promoter activity in vascular smooth muscle cells by p27(KIP1), an inhibitor of neointima formation in the rat carotid artery. J Clin Invest. 1997;99:2334–2341.[Medline] [Order article via Infotrieve]

46. Chang MW, Barr E, Lu MM, Barton K, Leiden JM. Adenovirus-mediated over-expression of the cyclin/cyclin-dependent kinase inhibitor, p21 inhibits vascular smooth muscle cell proliferation and neointima formation in the rat carotid artery model of balloon angioplasty. J Clin Invest. 1995;96:2260–2268.

47. Agrawal D, Hauser P, McPherson F, Dong F, Garcia A, Pledger WJ. Repression of p27 kip1 synthesis by platelet-derived growth factor in BALB/c 3T3 cells. Mol Cell Biol. 1996;16:4327–4336.[Abstract/Free Full Text]

48. Poon RY, Toyoshima H, Hunter T. Redistribution of the CDK inhibitor p27 between different cyclin: CDK complexes in the mouse fibroblast cell cycle and in cells arrested with lovastatin or ultraviolet irradiation. Mol Biol Cell. 1995;6:1197–1213.[Abstract]

49. Ishida A, Sasaguri T, Kosaka C, Nojima H, Ogata J. Induction of the cyclin-dependent kinase inhibitor p21(Sdi1/Cip1/Waf1) by nitric oxide-generating vasodilator in vascular smooth muscle cells. J Biol Chem. 1997;272:10050–10057.[Abstract/Free Full Text]

50. McDonald LJ, Murad F. Nitric oxide and cGMP signaling. Adv Pharmacol. 1995;34:263–275.

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