Induction of the Cyclin-dependent Kinase Inhibitor p21Sdi1/Cip1/Waf1 by Nitric Oxide-generating Vasodilator in Vascular Smooth Muscle Cells*

(Received for publication, July 9, 1996, and in revised form, January 22, 1997)

Akio Ishida Dagger §, Toshiyuki Sasaguri Dagger , Chiya Kosaka Dagger , Hiroshi Nojima par and Jun Ogata Dagger

From the Dagger  Department of Bioscience, National Cardiovascular Center Research Institute, 5-7-1 Fujishiro-dai, Suita, Osaka 565, the § Third Department of Internal Medicine, University of the Ryukyus School of Medicine, Okinawa 903, and the par  Research Institute for Microbial Diseases, Osaka University, Osaka 565, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Nitric oxide-generating vasodilators inhibit vascular smooth muscle cell proliferation. To elucidate the mechanism underlying this process, we investigated the effect of S-nitroso-N-acetylpenicillamine (SNAP), a nitric oxide-releasing agent, on the smooth muscle cell cycle. When G0 cells were stimulated with fetal bovine serum and basic fibroblast growth factor, DNA synthesis assessed by [3H]thymidine incorporation started about 15 h later. SNAP dose-dependently inhibited this incorporation, and this effect was maximal at 100 µM. This inhibition was attenuated when SNAP was added after 9-12 h. SNAP inhibited the activity of cyclin-dependent kinase 2 (Cdk2) and phosphorylation of the retinoblastoma protein, both of which usually increased from about 9 h, whereas it did not inhibit the activities of cyclin D-associated kinase(s), Cdk4, and Cdk6, which normally increased from 0-3 h. Although SNAP reduced the mRNA levels of cyclins E and A, it neither reduced their protein levels nor impaired their association with Cdk2. SNAP did not reduce the mRNA levels of cyclins G, C, and D1, Cdk2, Cdk4, and Cdk5, which were normally elevated from 0-3 h. The mRNA and protein levels of the Cdk inhibitor p21 were high in the early G1 phase, peaking at 3 h and then rapidly decreasing after 6 h. In the presence of SNAP, however, p21 expression was enhanced, and moreover, the later decrease disappeared. SNAP also increased the amount of Cdk2-associated p21. These results suggested that nitric oxide inhibits the G1/S transition by inhibiting Cdk2-mediated phosphorylation of the retinoblastoma protein and that p21 induction is involved in the Cdk2 inhibition.


INTRODUCTION

The proliferation of vascular smooth muscle cells (VSMCs)1 plays a crucial role in the formation of vascular lesions, such as fibrous plaques in atherosclerosis and intimal thickening after balloon angioplasty (1, 2). The increase in smooth muscle mass in hypertensive vascular walls may be related to an increase in cell number and DNA content (3). Therefore, to understand the etiology of these disorders and to develop new therapeutic strategies, it is essential to clarify the molecular mechanism controlling VSMC proliferation.

The endothelium is the source of a variety of substances that control vascular functions, such as nitric oxide (NO). NO inhibits platelet adhesion and aggregation, leukocyte adhesion, and smooth muscle contraction (4) and may also regulate VSMC proliferation. Vasodilators that release NO, such as sodium nitroprusside, isosorbide dinitrate, and S-nitroso-N-acetylpenicillamine (SNAP), inhibit the proliferation of cultured VSMCs, probably by releasing NO (5). NO may also inhibit VSMC proliferation in vivo because intimal thickening after balloon angioplasty is prevented by L-arginine, the metabolic precursor of NO (6), and by transferring plasmids that express endothelial constitutive NO synthase into the vascular wall (7). However, little is known about the signal transduction involved in the antiproliferative effect of NO, although the involvement of cGMP has been investigated (5, 8-11).

The eukaryotic cell cycle is regulated by cyclins and cyclin-dependent kinases (Cdks). In mammalian cells, cyclin D, cyclin E, and possibly cyclin A play important roles in controlling the transition from G1 to S phase (12, 13). These cyclins activate their catalytic partners Cdk4, Cdk6, and Cdk2 to hyperphosphorylate the retinoblastoma protein (pRb) in the late G1 phase (14), which then triggers entry into S phase. Cdk activities are also controlled by the Cdk inhibitor proteins p21, p27, p16, and p15 (13, 15).

Several extra- and intracellular signals inhibit cell proliferation. Among them, transforming growth factor-beta (16-22), cAMP (23, 24), protein kinase C (25, 26), and prostaglandin A2 (27, 28) have been studied relatively in depth. Most of them inhibit G1/S transition by preventing pRb phosphorylation, which may result from reduced Cdk activities. To elucidate the mechanism underlying the NO-induced inhibition of VSMC proliferation, we examined the effects of SNAP on cellular events during the G1 and S phases, including pRb phosphorylation, Cdk activation, and the expression of cyclins, Cdks, and Cdk inhibitors, using VSMCs from human umbilical arteries.


MATERIALS AND METHODS

Chemicals

SNAP was purchased from Research Biochemicals International (Natick, MA) dissolved in Me2SO4, and stored at -20 °C until use. 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (cPTIO) was obtained from Dojindo Laboratories (Kumamoto, Japan). 8-Bromoguanosine 3',5'-cyclic monophosphate (8-bromo-cGMP) was from Sigma. Rp-8-bromoguanosine-3',5'-monophosphorothioate (Rp-GMPS) and Rp-8-bromoadenosine-3',5'-monophosphorothioate (Rp-AMPS) were from BioLog Life Sciences Institute (Bremen, Federal Republic of Germany). Other common chemicals were of reagent grade.

Cell Culture

VSMCs obtained from the media of human umbilical arteries by explant cultures were used at the third passage. Their origin was confirmed by immunostaining with anti-smooth muscle alpha -actin (Dako Corp.). The cells were maintained in Dulbecco's modified Eagle's medium containing 20% (v/v) fetal bovine serum (HyClone), 10 ng/ml human recombinant basic fibroblast growth factor (Amersham Corp.), 100 units/ml penicillin, 100 µg/ml streptomycin, and 1 µg/ml amphotericin B (growth medium). The cells were cultured in Dulbecco's modified Eagle's medium with 0.1% bovine serum albumin in the absence of fetal bovine serum and basic fibroblast growth factor for 48 h to synchronize them in a quiescent state (G0). Thereafter, they were stimulated with growth medium to reenter the cell cycle. Cell numbers were determined using a Coulter counter (Z1).

Flow Cytometry

To measure the DNA content, the cells were dispersed with trypsin, suspended in phosphate-buffered saline, and fixed with 85% (v/v) ethanol (-20 °C) for 30 min. The ethanol was removed and the cells were incubated in phosphate-buffered saline containing RNase (172 Kunitz units/ml, Sigma) for 30 min at 37 °C, and then stained for 30 min with 0.005% propidium iodide dissolved in phosphate-buffered saline. The fluorescence of the DNA was measured using a flow cytometer (CytoACE150, Japan Spectroscopic).

DNA Synthesis

DNA synthesis was assessed by the level of thymidine (TdR) incorporation. Cells seeded in 24-well plates (1 × 104 cells/well) were cultured in growth medium for 48 h. They were washed three times with serum-free Dulbecco's modified Eagle's medium containing 0.1% bovine serum albumin and then incubated in the same medium for 48 h for synchronization in G0. The cells were labeled with 37 kBq/ml [6-3H]TdR (0.74-1.1 TBq/mmol, Amersham Corp.) in growth medium for the indicated periods. The medium was discarded and the cells were washed three times with 0.5 ml of ice-cold phosphate-buffered saline containing 1 mM MgCl2 and 1 mM CaCl2. The cells were precipitated with 0.5 ml of 5% trichloroacetic acid for 10 min, and then the acid was removed with 0.5 ml of ethanol:diethyl ether (3:1, v/v). The precipitates were lysed with 0.5 ml of 0.3 M NaOH, neutralized with 2 M HCl, and mixed with 5 ml of scintillation fluid (Aquasol-2, DuPont NEN Research Products). Radioactivity was determined using a liquid scintillation counter (LS5801, Beckman Instruments).

Immunoprecipitation and Western Blotting

To prepare pRb immunoprecipitates, about 3 × 106 cells were lysed in 1 ml of buffer A (10 mM Tris/HCl, pH 7.4, 100 mM NaCl, 1 mM EDTA, 50 mM NaF, 0.2 mM Na3VO4, 20 µg/ml leupeptin, 20 µg/ml phenylmethylsulfonyl fluoride, and 0.5% (v/v) Nonidet P-40), followed by centrifugation at 16,000 × g to remove the insoluble pellet. The supernatant was incubated with 50 µl of 50% (v/v) suspension of protein G-conjugated Sepharose (Pharmacia Biotech Inc.) for 30 min on ice, followed by centrifugation to remove the beads. The supernatant was incubated with an anti-human pRb monoclonal antibody (2 µg/ml, XZ104, PharMingen) for 1 h on ice. Thereafter, 50 µl of a 20% (v/v) suspension of protein G-Sepharose was added, and the incubation was continued for 1 h. Immunoprecipitates of cyclin A, cyclin E, Cdk2, p21, p27, and p53 were obtained in the same manner except for the use of polyclonal antibodies to human cyclin A (Santa Cruz Biotechnology), cyclin E (Upstate Biotechnology), Cdk2 (Upstate Biotechnology), p21 (Santa Cruz Biotechnology), and p27 (Santa Cruz Biotechnology), and a monoclonal antibody to human p53 (G59-12, PharMingen). Cyclin D and Cdk4 immunoprecipitates were prepared using polyclonal antibodies to human cyclin D (Upstate Biotechnology) and Cdk4 (PharMingen), respectively, after cells were lysed by sonication for 10 s in buffer B (50 mM Hepes/NaOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10 mM NaF, 0.1 mM Na3VO4, 20 µg/ml leupeptin, 20 µg/ml phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 0.1% Tween 20, and 10% glycerol).

After five washes with buffer A or B, the precipitates were fractionated by SDS-polyacrylamide gel electrophoresis (PAGE), electroblotted onto a polyvinylidene difluoride membrane, and immunoblotted with the indicated antibodies. Second antibodies (horseradish peroxidase-linked anti-mouse or anti-rabbit immunoglobulins) were visualized using the ECL Western blotting detection system (Amersham Corp.).

Protein concentrations were measured by the modified Lowry's method using a protein assay kit (Bio-Rad).

Cyclin-dependent Kinase Assay

To measure the activity of Cdk2, histone H1 and a pRb fragment were used as the substrates. The Cdk2 immunoprecipitate was suspended in 40 µl of 20 mM Tris/HCl (pH 7.4) containing 10 mM MgCl2, 1 mM dithiothreitol, 50 µM [gamma -32P]ATP (3.7 MBq/ml, Amersham Corp.), and 100 µg/ml histone H1 (Boehringer Mannheim) or 20 µg/ml glutathione S-transferase-fused murine pRb carboxyl-terminal (GST-Rb) (amino acid residues 769-921, Santa Cruz Biotechnology), and then incubated for 30 min at 30 °C with occasional mixing. The reaction was terminated with an equal volume of 2 × loading buffer (2% SDS, 10% 2-mercaptoethanol, 125 mM Tris/HCl, pH 6.8, 20% glycerol, and 0.04% bromphenol blue). The sample was boiled for 3 min, and then the beads were precipitated by centrifugation. The supernatant was fractionated by SDS-PAGE and transferred to a membrane. Phosphorylated proteins were visualized using a bioimage analyzer (BAS-2000, Fuji Photo Film Co.).

To assay the activities of cyclin D-associated kinase(s), Cdk4, and Cdk6, GST-Rb (amino acid residues 754-921) prepared as described (29) was the substrate. The immunoprecipitates of cyclin D, Cdk4, and Cdk6 were suspended in 40 µl of 50 mM Hepes/NaOH (pH 7.5) containing 10 mM MgCl2, 1 mM dithiothreitol, 20 µg/ml GST-Rb, and 50 µM [gamma -32P]ATP (3.7 MBq/ml), and the mixtures were incubated for 40 min at 30 °C with occasional mixing. The following procedures were as described for the Cdk2 assay.

Northern Blotting

The cDNAs for human Cdc2, Cdk2, Cdk4, Cdk5, and cyclins C, D1, E, and G were produced as described (26). The cDNA for p21 was produced by means of the reverse transcription-polymerase chain reaction, using RNA extracted from human VSMCs as described (27) and a pair of oligonucleotides synthesized according to the EMBL data base, and then it was subcloned into pBluescript II SK(+) (Stratagene). The clone was sequenced to confirm the identity of the product. The cDNAs for human cyclins A and B were gifts from Jonathon Pines.

Equal amounts of total cellular RNA (10 µg/lane) were resolved by electrophoresis on a 1% agarose gel containing 20 mM 3-(N-morpholino)propanesulfonic acid (pH 7.0), 5 mM sodium acetate, 0.5 mM EDTA, and 6% formaldehyde. After staining with ethidium bromide, RNA was transferred to a nylon membrane and fixed by means of ultraviolet irradiation. The membranes were incubated with cDNA probes labeled with 32P by random-prime DNA labeling in 50% formamide, 10% polyethylene glycol, 7% SDS, 1 mM EDTA, 0.25 M NaCl, 0.25 M NaHPO4 (pH 7.2), and 100 µg/ml denatured salmon sperm DNA for 16 h at 50 °C. They were washed with 15 mM NaCl containing 1.5 mM sodium citrate and 0.1% SDS at 65 °C and analyzed with a BAS-2000 bioimage analyzer.

Statistics

The results are expressed as the means ± standard deviation (S.D.) of the number of observations. The statistical significance was assessed by Student's t test for paired or unpaired values.


RESULTS

SNAP Reversibly Inhibited VSMC Proliferation

First we determined whether or not SNAP inhibits cell proliferation. In growth medium, the cell number increased exponentially until they reached confluence around day 7 (Fig. 1). SNAP (100 µM) markedly inhibited the increase, and removing SNAP from the medium recovered the proliferation. We confirmed by trypan blue exclusion that the antiproliferative effect of SNAP did not result from cytotoxicity (data not shown). Flow cytometry showed that hypodiploid cells did not accumulate in the presence of 100 µM SNAP, indicating that this concentration did not induce apoptosis (data not shown).


Fig. 1. The effect of SNAP on VSMC proliferation. VSMCs seeded in 6-well plates (3 × 104/well) were cultured in growth medium (open circle ). The medium was changed every other day and the cells were counted every day. Two-thirds of the cultures (bullet ) were exposed to SNAP (100 µM) added every other day simultaneously with the changing medium (filled arrows). On day 5 (open arrow), one-half of the cultures treated with SNAP were placed in SNAP-free growth medium after several washes (triangle ), and the other half were continuously exposed to SNAP. Data represent the means ± S.D. (n = 3). *, p < 0.01 versus control.
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To examine whether the G1 and S phases are influenced by SNAP, we synchronized cells in G0 phase by serum starvation and stimulated them with growth medium. DNA synthesis, assessed by measuring [3H]TdR incorporation, started about 15 h after stimulation and reached a plateau at about 33 h (Fig. 2a). When SNAP (100 µM) was added at 0-6 h, over 90% of the DNA synthesis was inhibited, but this was attenuated when added later. These findings suggested that SNAP inhibited the cell cycle in the late G1 phase. However, SNAP inhibited 15-20% of the incorporation when added after 18 h, indicating that SNAP also inhibited S phase to some extent. SNAP dose-dependently inhibited the incorporation to levels of over 90% at concentrations above 100 µM (Fig. 2b). We then examined the effect of cPTIO, an NO scavenger (30), on [3H]TdR incorporation, to test whether the antiproliferative effect of SNAP is mediated by NO released from the compound (Fig. 2c). Because a high concentration of cPTIO and long exposure to cPTIO were cytotoxic, we pulse-labeled unsynchronized cells with [3H]TdR in the presence of a relatively low concentration (1-10 µM) of this compound. Although cPTIO added alone reduced [3H]TdR incorporation, it prevented the SNAP-induced inhibition of the incorporation.


Fig. 2. The effect of SNAP on DNA synthesis. a, cells seeded in 24-well plates (1 × 104 cells/well) were cultured in growth medium for 48 h and then in serum-free Dulbecco's modified Eagle's medium containing 0.1% bovine serum albumin for 48 h to synchronize the cells in G0 phase. The cells were stimulated with growth medium to reenter the cell cycle in the presence of [3H]TdR (37 kBq/well), and the amount of accumulated radioactivity was measured at the indicated times (open circle ). Closed circles (bullet ) represent the SNAP-induced inhibition (%) of [3H]TdR incorporation measured at 33 h against the values obtained with solvent alone (0.1% Me2SO4). SNAP (100 µM) or vehicle was added at the indicated times. Data represent the means ± S.D. (n = 3). b, G0 cells were labeled with [3H]TdR in growth medium in the presence of various concentrations of SNAP added at time 0, and then [3H]TdR incorporation was measured at 33 h. Data represent the means ± S.D. (n = 4). *, p < 0.01 versus the value obtained in the absence of SNAP. c, Cells seeded in 24-well plates (1 × 104/well) were cultured in growth medium for 48 h and then labeled with [3H]TdR (37 kBq/well) for 4 h in the presence or absence of various concentrations of cPTIO and 100 µM SNAP. Data represent the means ± S.D. (n = 3). *, p < 0.05 versus the value obtained in the absence of cPTIO.
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SNAP Inhibited pRb Phosphorylation

To determine how it inhibits DNA synthesis, we examined the effect of SNAP on pRb phosphorylation because this is essential for cells to enter S phase (14). We precipitated pRb with an antibody that recognizes both the underphosphorylated and hyperphosphorylated forms and then fractionated the precipitates by SDS-PAGE (Fig. 3a). The faster- and slower-migrating bands (110 and 114 kDa) are hypophosphorylated and hyperphosphorylated pRb, respectively. In G0 cells, the faster-migrating band was dominant. The mobility shift of pRb from 110 to 114 kDa began at about 9 h after stimulation and appeared to have been completed by about 18 h. SNAP (100 µM) inhibited this shift, suggesting that it blocked a G1 event that preceded pRb phosphorylation. Like DNA synthesis, pRb phosphorylation was inhibited dose-dependently on SNAP (Fig. 3b). To exclude the possibility that pRb immunoprecipitates were unevenly loaded into the lanes and the SNAP-induced reduction of the hyperphosphorylated pRb resulted from a diminished loading, alpha -tubulin was immunoblotted as a protein loading control. As shown in Fig. 3c, there was no difference in the amount of alpha -tubulin between control and SNAP-treated cells, whereas in the same samples, SNAP down-regulated the hyperphosphorylated and up-regulated the hypophosphorylated pRb. In addition, SNAP had no significant effect on the total protein level (5.78 ± 0.34 µg/ml/104 cells in control and 6.40 ± 0.34 µg/ml/104 cells in SNAP-treated cells).


Fig. 3. The effect of SNAP on pRb phosphorylation. a, subconfluent G0 cells were stimulated with growth medium in the presence or absence of SNAP (100 µM) and lysed at the indicated times. Immunoprecipitates prepared with the antibody to pRb were resolved by SDS-7.5% PAGE and immunoblotted with another monoclonal antibody to pRb (1.5 µg/ml, G3-245, PharMingen). The arrows indicate hypophosphorylated (110 kDa) and hyperphosphorylated (114 kDa) forms of pRb (pRb and pRbphos., respectively). b, G0 cells were stimulated with growth medium in the presence or absence of various concentrations of SNAP. Cell lysates prepared at 24 h were analyzed as described in a. NS and S, not stimulated and stimulated with growth medium, respectively. c, pRb was blotted as described in b in the absence or presence of SNAP (100 µM). Equal proportions of the supernatant that remained after pRb immunoprecipitation were fractionated by SDS-10% PAGE, transferred to a membrane, and immunoblotted with a monoclonal antibody to alpha -tubulin (5 µg/ml, DM1A, Oncogene Research Products).
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The Effects of SNAP on Cdk Activities

To elucidate the mechanism by which SNAP inhibits pRb phosphorylation, we measured the activities of Cdks because pRb is the putative substrate for Cdk2, Cdk4, and Cdk6, which are activated during G1 phase (14). Fig. 4a shows the effect of SNAP on Cdk2 activity measured using histone H1 and GST-Rb as the substrates. The activity of histone H1 kinase was low in the G0 and early G1 phases. It began to increase about 9 h after stimulation and continued to increase until 24 h. SNAP (100 µM) added at time 0 markedly suppressed the activities measured at 12 and 24 h. SNAP also inhibited the GST-Rb kinase activity of Cdk2, the time course of which was parallel to that of histone H1 kinase activity.


Fig. 4. The effects of SNAP on Cdk activities. a, G0 cells were stimulated with growth medium in the absence or presence of SNAP (100 µM). Cell lysates prepared at the indicated times were immunoprecipitated with the anti-Cdk2 antibody. After the kinase reaction using histone H1 or GST-Rb as the substrate, the assay mixture was fractionated by SDS-PAGE (acrylamide concentration was 12.5% for histone H1 and 10% for GST-Rb), electroblotted onto a membrane, and visualized by autoradiography. b, cell lysates prepared at the indicated times were immunoprecipitated with the anti-cyclin D antibody. After the kinase reaction using GST-Rb as the substrate, the assay mixture was resolved by SDS-10% PAGE and analyzed as described in a. c, the procedure was the same as described in b except that anti-Cdk4 and anti-Cdk6 antibodies were used for immunoprecipitation.
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Cyclin D and its major catalytic partners Cdk4 and Cdk6 may play important roles in pRb phosphorylation (12-14). To examine the effects of SNAP on the activities of cyclin D-associated kinase(s), Cdk4, and Cdk6, we measured the kinase activity of their immunoprecipitates using GST-Rb as the substrate. The activity of cyclin D-associated kinase(s) was detectable in G0 cells, and it increased during early G1 (3-7.5 h after stimulation). After it dropped during late G1 (at 9 h), the activity again increased from 12 until 24 h. SNAP did not affect the activation during early G1, although the increase after the entry into S phase (24 h) was inhibited (Fig. 4b). Correspondingly, SNAP did not inhibit the activities of Cdk4 and Cdk6 in the G1 phase (Fig. 4c).

The Effects of SNAP on the mRNA Expression of Cyclins and Cdks

We examined the effects of SNAP on the mRNA expression of cyclins by Northern blotting because association with a cyclin is required to activate Cdks (Fig. 5a). Cyclin G was maximally expressed in the G0 and early G1 phases. Cyclins C and D1 were expressed at low levels in quiescent cells, but they were induced during early G1. SNAP (100 µM) did not reduce the expression of any of these three cyclins at least for the first 12 h but rather tended to enhance their expression during G1.


Fig. 5. The effects of SNAP on the mRNA expression of cyclins and Cdks. a, G0 cells were stimulated with growth medium in the presence or absence of SNAP (100 µM), and then total RNA was extracted at the indicated times. Equal amounts of RNA (10 µg/lane) were resolved by electrophoresis and hybridized with 32P-labeled cyclin cDNA fragments. The sizes of the transcripts (in kilobases) were: cyclin G, 2.7; cyclin C, 2.4; cyclin D1, 4.5; cyclin E, 1.9; cyclin A, 2.9; cyclin B, 1.9. b, Northern blotting proceeded as described in a, using Cdk cDNA fragments. The sizes of the transcripts (in kilobases) were: Cdk2, 2.5; Cdk4, 1.7; Cdk5, 1.4; Cdc2, 1.6; beta -actin, 2.1.
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It was difficult to define when the expression of cyclin E began (Fig. 5a). Small amounts of the mRNA were detected in early G1, and the expression was enhanced from 12-15 h. The level of cyclin A mRNA was low for 12 h, and then it increased from about 15 h, which was around the G1/S border. Cyclin B mRNA levels appeared to begin increasing a few hours later than cyclin A, probably after entry into S phase. In the presence of SNAP, however, the levels of these three mRNAs were suppressed, particularly after advance into S phase.

Fig. 5b shows Cdk gene expression. The level of Cdk2 mRNA was elevated during early G1 and further increased after advancing into S phase. Cdk4 was markedly expressed even in quiescent cells, and the level was not dramatically altered throughout the G1 and S phases. Cdk5 expression was enhanced during the G1 phase, but rather attenuated after 12 h. Cdc2 mRNA was not clearly detected until 24 h, suggesting that it was expressed after entry into S phase. SNAP (100 µM) suppressed the expression of Cdc2, which seemed to result from inhibition of the G1/S transition. On the other hand, the expression of Cdk2, Cdk4, and Cdk5 was not inhibited at least for the first 12 h, indicating that SNAP did not inhibit their expression during the G1 phase.

The Effects of SNAP on the Protein Levels of Cyclins and Cdks

Because the mRNA levels of cyclins E and A were reduced in the presence of SNAP, we tested by means of Western blotting whether SNAP also reduces their protein levels. The Western blots showed that cyclins E and A were already present in quiescent cells (Fig. 6a). Mitogenic stimulation elevated the level of cyclin A, but did not alter that of cyclin E. SNAP did not reduce these levels, even at 24 h, at which time it obviously reduced those of the corresponding mRNAs. Because SNAP did not alter the protein level of Cdk2, we examined by immunoblotting the Cdk2 immunoprecipitates whether it inhibits the association of Cdk2 with these cyclins (Fig. 6b). However, there was no significant change in the levels of cyclins coprecipitated with Cdk2 in cells exposed or not to SNAP.


Fig. 6. The effects of SNAP on the protein expression of cyclins and Cdks. a, G0 cells were stimulated with growth medium in the presence or absence of SNAP (100 µM). Cells were harvested at the indicated times, and cell lysates were immunoprecipitated with the polyclonal antibodies to cyclin E or cyclin A. Proteins were fractionated by SDS-10% PAGE and immunoblotted with monoclonal antibodies to cyclin E (1.5 µg/ml, HE12, PharMingen) or cyclin A (1.5 µg/ml, C160, PharMingen). b, cell lysates extracted 24 h after growth stimulation were immunoprecipitated with the anti-Cdk2 antibody, fractionated as described in a, and immunoblotted with the same antibody to Cdk2 or the monoclonal antibodies to cyclin E or cyclin A, as indicated. c, cells stimulated as described in a were harvested at 7.5 h, and then cell lysates were immunoprecipitated with the antibodies to cyclin D or Cdk4. Proteins were fractionated as described in a and immunoblotted with the same antibodies to cyclin D or Cdk4 (1.5 µg/ml), as indicated.
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SNAP did not reduce the protein levels of cyclin D and Cdk4, in parallel with the results of the Northern blotting (Fig. 6c). SNAP did not reduce the amount of Cdk4 coprecipitated with cyclin D, either, indicating that it did not inhibit the association of Cdk4 with cyclin D.

The Effect of SNAP on the Expression of p21 and p27

Because p21 inhibits the activity of cyclin E-Cdk2, cyclin A-Cdk2, cyclin D1-Cdk4, and cyclin D2-Cdk4 complexes (31, 32), we investigated the effect of SNAP on its expression. We detected p21 mRNA in quiescent cells (Fig. 7a). The expression did not change, or rather increased during the first 3 h after stimulation but decreased after 6 h to levels that were much lower than those of quiescent cells. SNAP enhanced the expression at 3 h and, moreover, prevented the decrease in the later phase. As a result, high levels of expression continued up to 30 h in cells exposed to SNAP. Western blotting showed a considerable amount of p21 protein in G0 cells (Fig. 7b). The expression increased 3 h after stimulation but decreased thereafter. In the presence of SNAP, however, there was little decrease in the amount of the protein, resulting in continuous and elevated expression throughout the observation period. To determine whether p21 induced by SNAP is responsible for the inhibition of Cdk2 activity, we examined the effect of SNAP on the level of p21 associating with Cdk2 (Fig. 7c). The level of p21 coprecipitated with Cdk2 gradually increased in response to growth stimulation. Although SNAP did not significantly alter this level during the early G1 phase (3-6 h), it was elevated in the presence of SNAP from the late G1 phase (12-24 h).


Fig. 7. The effect of SNAP on the expression of p21 and p53. a, G0 cells were stimulated with growth medium in the presence or absence of SNAP (100 µM), and total RNA was extracted at the indicated times. Equal amounts of RNA (10 µg/lane) were resolved by electrophoresis and hybridized with 32P-labeled p21 cDNA fragment. b, G0 cells were stimulated as described in a. Cells were harvested at the indicated times, and then lysates were immunoprecipitated with the antibody to p21. Proteins were resolved by SDS-12.5% PAGE and immunoblotted with a monoclonal antibody to p21 (1.5 µg/ml, 6B6, PharMingen). c, procedures were the same as described in b except that lysates were immunoprecipitated with the antibody to Cdk2. d, cell lysates prepared as described in b were immunoprecipitated with a monoclonal antibody to p53 (1.5 µg/ml, G59-12, PharMingen). Proteins were fractionated by SDS-10% PAGE and then immunoblotted with the same antibody.
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On the other hand, the protein expression of p27, a Cdk inhibitor related to p21 (33), was not altered during the G1 and S phases, and there was no significant difference in its levels between cells exposed to SNAP and those not exposed (data not shown).

p21 is transcriptionally up-regulated by the tumor suppressor gene product p53 and is suggested to be involved in the p53-mediated growth arrest in response to stimuli that damage DNA, such as ultraviolet irradiation. To test whether SNAP induces p21 through the induction of p53, we examined the effect of SNAP on the level of p53. The expression of p53 increased after growth stimulation, but was not enhanced by SNAP (Fig. 7d).


DISCUSSION

The effects of SNAP in this study were likely to be mediated by NO released from the agent. First, SNAP spontaneously releases NO with a half-life of about 5 h when added to neutral media exposed to room air at 37 °C (34). Second, sodium nitroprusside, another NO-generating agent, also inhibited VSMC proliferation (data not shown). Third, the NO scavenger, cPTIO, attenuated the inhibitory effect of SNAP on DNA synthesis. High concentrations of NO may be cytotoxic or could induce apoptosis. Several reports have suggested that NO induces apoptosis in macrophages (35, 36). Recent evidence shows that NO released from VSMCs stimulated with interleukin-1 causes them to induce their own apoptosis by up-regulating Fas and that apoptosis is also induced in VSMCs by incubating them with 500 µM sodium nitroprusside for 48 h (37). However, SNAP up to 100 µM did not induce cell death as assessed by trypan blue exclusion, and cells resumed proliferating after removing SNAP. Flow cytometry showed that SNAP did not accumulate hypodiploid cells, indicating that the cells were not apoptotic. Therefore, we assumed that the effects of SNAP were mediated by NO and that concentrations up to 100 µM did not induce cytotoxicity or apoptosis.

The antiproliferative effect of NO-generating agents is not limited to VSMCs. They also inhibit the proliferation of some other mammalian cells, such as fibroblasts, renal mesangial cells, and venous endothelial cells (9, 38, 39). This suggests that NO interferes with cellular events that are common among some cell types. SNAP inhibited the cell cycle of VSMCs in the G1 phase and, probably, also those in the S phase. This is because its inhibitory effect on DNA synthesis was attenuated when added in the late G1 phase, but some effect remained when added after the advance into S phase. Some mechanisms have been proposed for the S phase inhibition by NO. NO gas and NO generated by activated macrophages inhibit ribonucleotide reductase, a rate-limiting enzyme in DNA synthesis (40, 41). SNAP (over 100 µM) inhibits thymidine kinase, another enzyme important for DNA synthesis, in VSMCs (42). However, the mechanism underlying NO-induced G1 inhibition is unknown.

Several afferent signals that control cell cycle converge onto pRb (14). Hypophosphorylated pRb binds to and inhibits several proteins involved in initiating DNA replication, such as E2F transcription factors. The pRb hyperphosphorylated by Cdks during late G1 loses this ability and allows the cell to advance into S phase. Most of the antiproliferative signals so far studied inhibit pRb phosphorylation by reducing the expression levels and the activities of cyclin-Cdk complexes responsible for pRb phosphorylation. Therefore, we examined the effects of SNAP on pRb, cyclins, and Cdks.

SNAP inhibited late G1 events, namely Cdk2 activation and pRb phosphorylation. Because Cdk2 phosphorylates pRb in vitro (43) and may be a mediator of G1/S transition (44), the inhibition of Cdk2 activity may explain the SNAP-induced inhibition of pRb phosphorylation and the G1/S transition. On the other hand, SNAP did not inhibit the cellular events that started in early G1, such as the expression of cyclins G, C, and D1, Cdk2, Cdk4, and Cdk5, or the activation of cyclin D-associated kinase(s), Cdk4, and Cdk6. Cdk4 and Cdk6 may also be involved in pRb phosphorylation (14). According to a recent report (45), pRb has multiple Cdk consensus phosphorylation sites and each may have a specific function for regulating pRb binding to various proteins, such as SV40 large T-antigen, cAbl, and E2F. This implies that Cdk4 and Cdk6 phosphorylate specific sites distinct from those phosphorylated by Cdk2. If phosphorylation by Cdk2 is essential for pRb inactivation, the SNAP-induced inhibition of Cdk2 would cause G1 arrest despite the presence of active Cdk4 and Cdk6.

Associating with cyclin E or cyclin A is prerequisite for Cdk2 activation (12, 13). Because SNAP reduced the mRNA levels of cyclins E and A, we initially considered that this suppressed the Cdk2 activity. However, SNAP did not reduce the protein levels of these cyclins. Because the cyclin proteins were detected in quiescent cells, they may undergo little degradation and their levels may be maintained even if low levels of mRNA are expressed during the early G1 phase. Therefore, we concluded that other mechanisms are involved in the SNAP-induced inhibition of Cdk2 activity.

Cdk activities are negatively regulated by Cdk inhibitor proteins. p21, also known as senescent cell-derived inhibitor 1 (Sdi1) (46), Cdk-interacting protein 1 (Cip1) (31), and wild-type p53-activated fragment 1 (Waf1) (47), binds to cyclin-Cdk2 complexes to inhibit their activities (32). The overexpression of p21 inhibits pRb phosphorylation and the proliferation of mammalian cells, including VSMCs (32, 48). In vivo transfection with an adenovirus encoding p21 inhibits neointimal hyperplasia after balloon angioplasty of the rat carotid artery (48). Apart from injurious stimuli, such as ultraviolet irradiation, however, only a few physiological substances are known to induce p21. These include transforming growth factor-beta (21, 22) and prostaglandin A2 (28).

We found that SNAP induces p21. p21 protein was abundant during early G1, but its level decreased from about 9 h. A similar time course of p21 expression during G1 and S phases has been identified in IMR90 fibroblasts (49). In the presence of SNAP, however, p21 expression was enhanced and did not decline. On the other hand, Cdk2 activity was low up to 6 h after mitogenic stimulation, after which the level increased from about 9 h. SNAP suppressed this activation. This mirror image between p21 expression and Cdk2 activity suggested that p21 controls Cdk2 activity during the G1/S progression. However, it is unclear whether p21 contributes to the suppression of Cdk2 activity during the early G1 phase because the level of p21 associated with Cdk2 was relatively low during the early G1 phase. SNAP elevated the level of Cdk2-associated p21 from the late G1 phase, suggesting that p21 is involved in the SNAP-induced Cdk2 inhibition.

SNAP did not influence the activities of cyclin D-associated kinase(s), Cdk4, and Cdk6, although p21 reportedly inhibits the activity of cyclin D-Cdk4 complex (31, 32). However, a recent report showed that recombinant p21 nearly completely inhibits Cdk2 activity but only partially affects Cdk4 and has no effect on Cdc2, suggesting that sensitivity to p21 is different among different Cdks (50). Therefore, p21 induced by SNAP may inhibit Cdk2 much more effectively than Cdk4 and possibly Cdk6. Although the precise mechanism for p21-induced Cdk2 inhibition is unknown, p21 may prevent phosphorylation of Cdk2 on threonine-160 by Cdk-activating kinase (51). Additional study is needed to determine whether SNAP inhibits the phosphorylation of Cdk2.

In addition to its ability to inhibit Cdks, p21 binds to the cofactor of DNA polymerase delta , proliferating cell nuclear antigen, and inhibits proliferating cell nuclear antigen-dependent DNA replication (52, 53). These two distinct inhibitory activities of p21 reside in separate domains of the protein (54, 55). The amino- and carboxyl-terminal domains of p21 interact with and inhibit Cdk2 and proliferating cell nuclear antigen, respectively. Therefore, not only the G1 inhibition but also the SNAP-induced inhibition of S phase may involve p21, although other mechanisms have been proposed for the NO-induced S phase inhibition, as described above.

p21 is induced by p53 and is thought to be important for cell cycle arrest following DNA damage. However, recent studies indicate that p21 is up-regulated through p53-independent mechanisms in several situations, including during normal tissue development, during cell differentiation, following serum stimulation, and following treatments with transforming growth factor-beta and prostaglandin A2 (56, 22, 28). SNAP did not elevate the level of p53 protein. This suggested that the growth arrest by SNAP is not secondary to DNA damage and that p21 induction by SNAP does not depend on p53. Further investigation is needed to determine whether SNAP stimulates the transcription of p21 independently of p53 or stabilizes the mRNA.

cGMP produced by soluble guanylate cyclase mediates NO-induced vasorelaxation (57). NO-releasing agents stimulate cGMP formation, and exogenous cGMP analogues mimic their effects on Ca2+ mobilization and smooth muscle tone (58). cGMP may also be involved in the NO-induced inhibition of VSMC proliferation (5, 8, 10). The cGMP-dependent activation of cAMP-dependent protein kinase is partly responsible for the NO-mediated inhibition of VSMC proliferation (11). Nevertheless, NO-generating vasodilators inhibit the proliferation of BALB/c 3T3 fibroblasts that lack soluble guanylate cyclase (9). In our VSMCs, high concentrations of 8-bromo-cGMP slightly inhibited [3H]TdR incorporation (data not shown). We examined whether cGMP mediates the effect of SNAP using inhibitors of cyclic nucleotide-dependent protein kinases. The effect of SNAP was not reversed by Rp-GMPS or Rp-AMPS, cGMP and cAMP analogues that inhibit cGMP- and cAMP-dependent kinases, respectively (data not shown). Therefore, whether or not cGMP is involved in the antiproliferative effect of NO remains inconclusive.

VSMC hyperplasia is involved in the development of atherosclerotic lesions, restenosis after balloon angioplasty, and vascular wall thickening in hypertension (1-3). VSMCs in normal arteries are abundant in contractile proteins and cannot proliferate (contractile type). However, the VSMCs in the neointima formed during atherosclerosis and restenosis after angioplasty have de-differentiated properties (59). Endothelial dysfunction may trigger a phenotypic change in the VSMCs and induce their abnormal proliferation. This implies that normal endothelium releases substances that differentiate VSMCs and inhibit their proliferation. The putative substances involved in this role include NO, as well as other factors produced by the endothelium, such as prostacyclin, adrenomedullin, C-type natriuretic peptide, transforming growth factor-beta , and heparan sulfate proteoglycans. The role of NO is evidenced by the observation that nitro-L-arginine reduces the antiproliferative effect of endothelial cells on cocultured VSMCs (60) and that intimal thickening after balloon angioplasty is prevented by L-arginine and NO synthase (6, 7).

Here, we proposed a biochemical background for the NO-induced inhibition of VSMC proliferation. However, there remain several unresolved issues, particularly the role of NO in VSMC differentiation. Because the cells we used here had the synthetic phenotype, we did not study the effect of NO on reciprocal changes between contractile and synthetic phenotypes. Recent reports suggest that p21 is involved in the differentiation of skeletal muscle cells and macrophages (61, 62). Therefore, it would be of interest to examine whether or not NO is involved in regulating VSMC differentiation through p21 induction.


FOOTNOTES

*   This study was supported in part by grants from the Ministry of Health and Welfare (Research Grants for Cardiovascular Diseases 6B-1 and 8A-1), the Science and Technology Agency (Special Coordination Funds for Promoting Science and Technology (Encouragement System of Center of Excellence)), and the Japan Cardiovascular Research Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
   To whom correspondence should be addressed. Tel.: 81-6-833-5012; Fax: 81-6-872-7485; E-mail: sasaguri{at}ri.ncvc.go.jp.
1   The abbreviations used are: VSMC, vascular smooth muscle cell; NO, nitric oxide; SNAP, S-nitroso-N-acetylpenicillamine; Cdk, cyclin-dependent kinase; pRb, retinoblastoma protein; cPTIO, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide; 8-bromo-cGMP, 8-bromoguanosine 3',5'-cyclic monophosphate; Rp-GMPS, Rp-8-bromoguanosine-3',5'-monophosphorothioate; Rp-AMPS, Rp-8-bromoadenosine-3',5'-monophosphorothioate; TdR, thymidine; PAGE, polyacrylamide gel electrophoresis; S.D., standard deviation; GST-Rb, glutathione S-transferase-fused murine pRb carboxyl-terminal.

ACKNOWLEDGEMENTS

We thank Koshiro Fukiyama for giving A. I. an opportunity to work with T. S., C. K., and J. O. in the National Cardiovascular Center Research Institute. We are also indebted to Jonathon Pines for providing the cDNAs of cyclins A and B and Hitoshi Matsushime for the plasmids encoding GST-Rb.


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