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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:27.)
© 2000 American Heart Association, Inc.

Vascular Biology

Nitric Oxide–Induced Increase in p21^{Sdi1/Cip1/Waf1} Expression During the Cell Cycle in Aortic Adventitial Fibroblasts

Miaofen Gu; Peter Brecher

From the Department of Biochemistry, Boston University School of Medicine, Boston, Mass.

	Abstract

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Abstract—This study was performed to investigate whether the expression of p21^{Sdi1/Cip1/Waf1}, one of the cyclin-dependent kinase inhibitor proteins, could be regulated by nitric oxide (NO) and might account for the antiproliferative effect of NO. Quiescent adventitial fibroblasts were stimulated to proliferate by serum addition and by NO donors added during different phases of the cell cycle. [³H]Thymidine incorporation was markedly reduced by S-nitroso-N-acetyl-penicillamine (SNAP) added either with serum at quiescence or at later time point in the cell cycle. Northern and Western blot analyses showed that addition of SNAP either at quiescence or 15 hours after serum addition induced a rapid induction of p21 mRNA and protein. Immunoprecipitation studies and electrophoretic mobility shift analysis indicate that the treatment of cells with SNAP induced the phosphorylation of p53 (a tumor suppressor protein) and enhanced the ability of p53 to bind DNA when SNAP was added during the cell cycle. The increased expression of p21 mRNA or p53 activation during late G₁ or S phase was also caused by addition of 8-bromo-cGMP and effectively blocked by a specific inhibitor of the soluble guanylate cyclase. Furthermore, this response to SNAP was blocked by an inhibitor of protein kinase G. These studies implicate NO as a potential regulator of the cell cycle in aortic adventitial fibroblasts through a cGMP-mediated transcriptional mechanism involving the induction of p21.

Key Words: nitric oxide • p21^{Sdi1/Cip1/Waf1} • cell cycle • adventitial fibroblasts

	Introduction

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In vascular biology, the control of cell proliferation is an important aspect of the changes occurring in atherosclerosis, restenosis, and hypertension. Recent work has implicated nitric oxide (NO) as a potentially important regulatory substance with regard to vascular growth and proliferation, because increased NO production has been associated with antiproliferative effects, countering the responses to a variety of growth-promoting agonists, including angiotensin II, endothelin, and platelet-derived growth factor. Many studies have documented the antiproliferative effects of NO on vascular smooth muscle cells, and this phenomenon has been ascribed to mechanisms that are both cGMP dependent¹ and cGMP independent² and include inhibition of thymidine kinase³ and ribonucleotide reductase.^{4 5} Additionally, activation of a cAMP-dependent protein kinase by cGMP has been implicated.⁶ A recent study⁷ has used cultured vascular smooth muscle cells to show that NO addition results in p21 mRNA and protein induction, which might account for the antiproliferative effect of NO. The p21/Waf1/Cip1 gene encodes a protein classified as a cyclin-dependent kinase inhibitor that acts by either directly suppressing the activity of cyclin-dependent kinase complexes or by forming a complex with proliferating cell nuclear antigen. The overall effect is to arrest cell proliferation during the cell cycle by preventing DNA replication in S phase.^{8 9} The results described in vascular smooth muscle cells⁷ appeared to preclude p53 activation, and the inhibitor was relatively selective for p21 expression when compared with many other cell cycle regulatory proteins.

We have been interested in the role of the adventitia in vascular remodeling processes, in view of the fact that several studies have shown that during experimental hypertension, proliferation of adventitial fibroblasts is a relatively rapid response.^{10 11} Recent in vivo and in vitro studies have implicated the adventitial fibroblast as a major site for superoxide production in response to angiotensin II administration,^{12 13} and we have recently shown that adventitial fibroblasts are a major source of the inducible form of NO synthase in aortic tissue after the in vivo administration of endotoxin to produce an inflammatory response.¹⁴

To begin to evaluate the possible role of NO in modulating vascular remodeling during hypertension, we have used procedures for obtaining adult rat adventitial fibroblasts in culture and have studied the effects of NO on cell proliferation under standardized conditions in which quiescent cells were stimulated with FCS. In the present study, we have determined whether the expression of p21 could be regulated by NO and might account for the antiproliferative effect of NO. We have shown that NO inhibits cell proliferation in aortic adventitial fibroblasts by a mechanism involving the p53-dependent induction of p21. This novel effect of NO (which occurs during the cell cycle) involves the activation of p53 and the subsequent transcriptional effect on p21 produced by NO during the cell cycle, requires the presence of serum, and occurs rapidly through a cGMP-dependent mechanism.

	Methods

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Materials
S-Nitroso-N-acetyl-penicillamine (SNAP), (R)-p-bromoadenosine 3^',5^'-cyclic monophosphorothioate (Rp-8Br-cAMPS), and (R)-p-bromoguanosine 3^',5^'-cyclic monophosphorothioate (Rp-8Br-cGMPS) were obtained from Alexis Corp. Sodium nitroprusside, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), dibutyryl cAMPs, type IV collagenase and elastase, and actinomycin D were from Sigma Chemical Co. 8-Bromo-cGMP was from Calbiochem. [³H]Methylthymidine, [-³²P]dCTP, and [-³²P]ATP were from Dupont/NEN. Antibodies for p21 were purchased from Pharmingen.

Isolation and Culturing of Aortic Adventitial Fibroblasts
The procedure adopted was based on methods described previously.^{15 16} Aortic tissue was removed aseptically from male Wistar rats weighing between 250 to 300 g and rapidly placed in ice-cold DMEM/F-12 supplemented with penicillin (100 U/mL), streptomycin (100 U/mL), and amphotericin B (25 µg/mL). Loosely adhering connective tissue was rapidly removed from the aorta, and the luminal surface was exposed by a longitudinal cut. Endothelial cells were removed by gently rubbing the lumen with the blunt side of dissecting scissors, and the medial layer was removed by successively peeling off sections with the use of 2 forceps. The remaining tissue, which was predominantly adventitia, was cut into segments 2 mm² and placed into the DMEM/F-12 solution supplemented with antibiotics for subsequent enzymatic digestion and dispersion of cells. That solution also contained type IV collagenase (205 U/mL) and elastase (10 U/mL). Adventitial segments from 4 to 6 rats were incubated for up to 5 hours in 10 mL of the enzymatic digestion medium at 37°C. At hourly intervals, the tissue was subjected to repetitive pipetting for 1 minute. The resulting suspension was centrifuged at 200g for 10 minutes, and the cell pellet was resuspended in 10 mL of DMEM/F-12 supplemented with antibiotics and containing 10% FCS. The cells were transferred to a T75 tissue culture flask and allowed to adhere and divide for 48 hours. Medium was replaced every 48 hours. Cells reached confluence within 7 days and were subsequently passed by harvesting with a trypsin (0.05%) and EDTA (0.02%) solution. Cells were diluted and serially passed at a 1:5 ratio. Cells in the third to fifth passages were routinely used for subsequent studies. The morphology and growth characteristics of the cells were typical of fibroblasts and were distinguished from smooth muscle cells on the basis of the absence of "hill and valley" growth pattern and the lack of smooth muscle -actin staining.

Measurement of Labeled Thymidine Incorporation
For analysis of [³H]thymidine incorporation, cells were plated in 24-well plates at 5000 cells per well. After 1 day, the subconfluent cells were made quiescent in a 0.2% FCS–containing medium for 48 hours. The cells were incubated for the designated time with tritiated thymidine (0.5 to 1 µCi/mL) and the other designated additions. At the end of the incubation period, incorporation of labeled thymidine was determined essentially as described previously by us.¹⁷ All experiments were performed in quadruplicate and are representative of 2 or 3 separate experiments with different preparations of cells.

RNA Isolation, Gel Electrophoresis, and Analysis
Total cellular RNA was isolated by the acid guanidinium thiocyanate–phenol–chloroform method.¹⁸ Northern blot analysis was carried out as previously described.^{17 19} The cDNA probe for rat p21 containing a 0.7-kb insert from the open reading frame was kindly provided by Dr B. Schreiber (Boston University School of Medicine). The p53 cDNA probe was a 1-kb insert from mouse p53 cDNA obtained by treatment with SacII and XhoI of a plasmid obtained from Dr K. Ravid (Boston University School of Medicine). The rat ß-actin probe was purchased from Ambion. The cDNAs were labeled with [-³²P]dCTP by a random prime labeling method (Amersham). Densitometric analysis of the blots was performed with a PDI scanner (model 420 oe), and the data are reported as fold increase over control cells.

Electrophoretic Mobility Shift Assay
The procedures followed were similar to those described previously by us.¹⁹ Nuclear extracts were prepared by the method of Schreiber et al²⁰ from monolayer cultures in 100-mm Petri dishes, and 5 to 20 µg protein was used for the electrophoretic mobility shift assay (EMSA). The p53 double-stranded oligonucleotide with the consensus sequence of 5'-TAC AGA ACA TGT CTA AGC ATG CTG GGG ACT-3' was end-labeled with [-³²P]ATP and T4 polynucleotide kinase and purified by P-25 Biogel columns. Nuclear extracts were added to ³²P-labeled p53 oligonucleotide (3 to 4x10⁴ cpm per reaction) in a binding buffer containing 3 µg poly dI · dC (Pharmacia), 20 mmol/L HEPES (pH 7.8), 10% glycerol, 1 mmol/L EDTA, 5 mmol/L MgCl₂, 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L dithiothreitol, and 100 mmol/L NaCl. Reaction mixtures were incubated for 45 minutes on ice, and the DNA-protein complexes were resolved on a 4.0% nondenaturing polyacrylamide gel. In competition experiments, unlabeled oligonucleotide was added to the nuclear extracts for 15 minutes before addition of the radiolabeled probe. Antibodies (2 µg per reaction) were added after the oligonucleotide had reacted for 45 minutes with the nuclear extracts on ice and then incubated for an additional 60 minutes at ambient temperature.

Immunoprecipitation and Western Blot Analysis
For analysis of p53, the cells were maintained and treated in 10-cm Petri dishes. After treatment, the cells were scraped and then suspended in 0.6 mL of a lysis buffer containing 10 mmol/L Tris (pH 7.4), 150 mmol/L NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, 50 mmol/L NaF, 0.2 mmol/L sodium vanadate, and 20 µg/mL leupeptin. The total cell lysate was obtained after sonication for 10 seconds and centrifugation at 14 000g for 30 minutes at 4°C. Protein concentration was determined by using the Bio-Rad DC protein assay. Aliquots of each fraction (0.8 mg protein for total cell lysate) were incubated with 0.5 µg of anti-p53 mouse monoclonal antibody (Ab-1, Oncogene) for 1 hour at 4°C. Addition of protein A–Sepharose and incubation for 16 hours at 4°C was followed by centrifugation and repetitive washing of the Sepharose beads. After the final wash, the beads were resuspended in 35 µL of 2x sample buffer, and the samples were boiled and then separated by 10% SDS-polyacrylamide gel electrophoresis. Protein was transferred to nitrocellulose membranes, and Western blot analysis was performed with phospho-p53 (Ser 15) antibody (BioLabs). The membrane was then treated with an appropriate second antibody conjugated with horseradish peroxidase and visualized with enhanced chemiluminescence (BioLabs).

For analysis of p21, cells were lysed in 1 mL of a buffer containing 10 mmol/L Tris (pH 7.4), 100 mmol/L NaCl, 2% NP-40, 0.2% SDS, 0.5% sodium deoxycholate, 50 mmol/L NaF, 0.2 mmol/L sodium vanadate, 10 µg/mL leupeptin, 1 mmol/L phenylmethylsulfonyl fluoride, and 10 µg/mL aprotinin. The suspension was sonicated for 10 seconds and centrifuged at 14 000g for 30 minutes at 4°C. The resulting supernatant was analyzed directly for p21 protein by Western blot analysis using aliquots containing 150 µg protein. After separation by 12% SDS-polyacrylamide gel electrophoresis and transfer onto nitrocellulose membranes, the blot was treated with anti-human monoclonal p21 antibody (Pharmingen) and then treated with a second antibody and visualized with enhanced chemiluminescence.

Statistical Analysis
The thymidine incorporation data and the data for the time course of p21 mRNA in the presence and absence of SNAP are expressed as mean±SE. The data in Figure 4B representing the phosphorylation of p53 is expressed as mean±SD. Statistical analysis was performed by 1-way ANOVA and the Student test for unpaired data with the use of StatView version 4.01 (Abacus Concepts Inc). A value of P<0.05 was considered to be significant.

View larger version (35K): [in this window] [in a new window]   Figure 4. Characterization of the activation of p53 by SNAP. A, SNAP (100 µmol/L) was added to cells pretreated for 15 hours with 10% FCS, and then cell extracts were obtained 2 hours after SNAP addition for EMSA of p53. The specificity of the p53 complex was shown by using a competition assay. The extracts were incubated with the labeled oligonucleotide in the presence of a 10- or 100-fold excess of unlabeled probe or with a 100-fold excess of an oligonucleotide containing the consensus sequence for the unrelated transcription factor SP1. The lanes on the far right indicate results when extracts were incubated with labeled p53 oligonucleotide in the presence of 2 µg antibody per reaction against either p53 or the unrelated protein C/EBP. B, Cells were pretreated for 15 hours with 10% FCS, and then SNAP (100 µmol/L) was added for an additional 15 minutes to 4 hours. Total cell lysates were obtained for immunoprecipitation and Western Blot analysis of p53 by using procedures described in Methods. The density of phosphorylated p53 bands were measured with a PDI scanner (model 402 oe), and the values are expressed as mean±SD (n=3). *P<0.01 vs control (10% FCS). C, Either 10 µmol/L ODQ (O), 50 µmol/L Rp-8Br-cGMP (G), or 50 µmol/L Rp-8Br-cAMP (A) was added 30 minutes before SNAP addition. After 30 minutes of SNAP (S) treatment, total cell lysates were extracted and analyzed for phosphorylated p53. The results shown are representative of 2 independent experiments. Carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (CBZ) is a proteasome inhibitor and was incubated with cells for 30 minutes at 5 µmol/L.

	Results

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SNAP Inhibits Thymidine Incorporation Into DNA in Growing Cells
Figure 1A shows the effect of SNAP addition on thymidine incorporation into DNA in subconfluent adventitial fibroblasts growing in the presence of 10% FCS. In the absence of SNAP, thymidine incorporation increased progressively with time after a lag phase, with the major changes occurring 12 hours after FCS was added to the quiescent cells. When SNAP was added, thymidine incorporation was markedly attenuated to the levels seen when quiescent cells were maintained in 0.2% FCS. This inhibitory effect of SNAP on DNA synthesis was dose dependent between 25 and 100 µmol/L under conditions in which SNAP was added with the 10% FCS and in which thymidine incorporation was measured in the 18- to 24-hour interval after the addition of serum (Figure 1B). When SNAP was added at different times after the addition of serum to the quiescent cells, thymidine incorporation was inhibited to a maximal extent even when added 12 hours after serum, indicating that the inhibitory effect was occurring in late G₁ or S phase. Even when SNAP was added 21 hours after serum, the DNA synthesis occurring between 24 and 36 hours was inhibited by almost 40% (Figure 1C). Because NO, at high concentration, has potent cell-killing activity, the effect of SNAP to inhibit adventitial fibroblast proliferation could be due to NO-dependent cell death. This did not appear to be the case, because in all experiments with NO donors or other treatments, cell viability was confirmed by trypan blue exclusion and, in selected experiments, by measuring the release of lactate dehydrogenase into the culture medium; no evidence of cell damage was found (data not shown).

View larger version (22K): [in this window] [in a new window]   Figure 1. SNAP inhibits thymidine incorporation into rat adventitial fibroblasts. Subconfluent cells were made quiescent by incubation for 48 hours in a medium containing 0.2% FCS. A, After addition of 0.5 µCi/mL [3H]thymidine, cells were maintained for the indicated time in either 0.2% FCS or 10% FCS plus or minus 100 µmol/L SNAP. [3H]Thymidine incorporation was measured as described in Methods. B, Ten percent FCS either without or with the designated concentration of SNAP was added to quiescent cells and incubated for 24 hours. Labeled thymidine incorporation was measured between 18 and 24 hours after addition of serum. C, Ten percent FCS was added to quiescent cells, and 100 µmol/L SNAP was added at the designated times after addition of serum. Labeled thymidine incorporation was measured between 24 and 36 hours after initial addition of 10% FCS. Values are mean±SE (n=4 for panels A and C and n=3 for panel B). *P<0.0001 vs control (10% FCS).

SNAP Increases p21 mRNA During Cell Cycle
To determine whether the induction of p21, an inhibitor of cyclin-dependent kinases, was involved in the antimitotic effects of SNAP, steady-state mRNA levels for this protein were measured. Figure 2 shows representative data for p21 mRNA in cells treated with 10% FCS alone or in the presence of 100 µmol/L SNAP (Figure 2A) over a 24-hour period. In the absence of SNAP, there was an acute but reproducible increase in p21 mRNA 2 hours after serum addition. Subsequently, this level decreased to amounts slightly lower than that found in quiescent cells. In contrast, when SNAP was present, steady-state mRNA levels increased between 4 and 8 hours after addition and remained high for up to 24 hours. The bar graph shown in Figure 2B summarizes densitometric data for the ratio between p21 and ß-actin mRNA in cells treated with 10% FCS either in the absence or presence of SNAP and clearly illustrates the SNAP-induced increase in p21 mRNA at the later time points.

View larger version (38K): [in this window] [in a new window]   Figure 2. SNAP increases steady-state mRNA levels of p21. A, Ten percent FCS was added to quiescent cells either alone or with 100 µmol/L SNAP. At indicated times, total RNA was obtained, and Northern blot analysis was performed with use of cDNA probes for either p21 or ß-actin. B, Densitometric analysis of the p21 and ß-actin mRNA ratio for cells treated either without or with SNAP is shown. Values are mean±SE (n=2 for p21 mRNA in 10% FCS and n=3 for p21 mRNA in 10% FCS+100 µmol/L SNAP). *P<0.0001 vs corresponding control (10% FCS) at the same time point. To characterize p21 mRNA increases by NO when added to cells during the cell cycle, SNAP (100 µmol/L) was added to cells pretreated with 10% FCS for 15 hours. C, After incubation with SNAP for the designated time, cells were analyzed for p21 and ß-actin mRNA by Northern blot analysis. D, Western blot analysis shows an increase in p21 protein in cells treated with SNAP (100 µmol/L) for either 15 or 19 hours or for the 4-hour interval after 15 hours of pretreatment with 10% FCS.

To determine whether SNAP could influence p21 mRNA when added during the cell cycle rather than during quiescence, we added the NO donor 15 hours after the addition of 10% FCS and analyzed RNA from cells during a 4-hour treatment, corresponding to 19 hours after serum was added to the quiescent cells (Figure 2C). The changes in p21 mRNA levels were relatively slight during the first hour but were consistently increased 3- to 4-fold after 3 to 4 hours of treatment with 100 µmol/L SNAP. The changes in steady-state mRNA levels corresponded to concomitant changes in protein, as indicated by Western blot analysis of cells treated with SNAP for either 15 hours or 19 hours or for the time interval between 15 and 19 hours. In all cases, there was a 3- to 5-fold increase in protein over that found when the cells were treated solely with 10% FCS (Figure 2D). Thus, NO appears to increase the expression of p21 in adventitial fibroblasts when added during the cell cycle. Additional experiments using this protocol for SNAP-induced increases in p21 mRNA during the cell cycle (15 to 19 hours after addition of 10% serum) showed that the increase was dose dependent with respect to SNAP addition, that it also occurred when an alternate NO donor, sodium nitroprusside, was added, and that the effect was abolished when the NO scavenger hydroxocobalamin was added (data not shown).

SNAP Activates p53 in a Serum-Dependent Manner
The increase in p21 mRNA induced by SNAP between 15 and 19 hours was observed when cells were either quiescent or actively growing. The Northern blot shown in Figure 3A compares the effect of SNAP on cells that were initially quiescent and then maintained for 15 hours in the presence of high serum (10%) or low serum (0.2%). SNAP was then added to the cells for the next 4 hours with either high or low serum in the medium. In all combinations, SNAP increased p21 mRNA levels during the 4-hour treatment period, indicating that the effect was independent of serum concentration. Because one mechanism established for the induction of p21 mRNA is via the transcription factor p53, studies were performed to determine whether SNAP addition activated p53. Figure 3B shows the effect of serum concentration on the activation of p53 by a 1-hour treatment with SNAP using an EMSA. SNAP (100 mmol/L) was added to cells treated for 15 hours with either 0.2% serum (lanes 2 and 3) or 10% serum (lanes 4 and 5), and nuclear extracts were obtained 1 hour later. Only when 10% serum was present (lane 5) did SNAP addition result in a protein-DNA complex consistent with p53 activation.

View larger version (35K): [in this window] [in a new window]   Figure 3. Effect of serum concentration on the activation of p21 mRNA and p53 by SNAP when added during the cell cycle. A, Effect of serum on p21 mRNA. Cells were made quiescent by incubation in 0.2% serum for 24 hours. Cells were then incubated for a further 15 hours either with 0.2% or 10% serum in the absence of SNAP (100 µmol/L). After 15 hours of pretreatment with either 0.2% or 10% serum, the medium was changed as indicated for an additional 4 hours either with or without SNAP addition. The cells were then analyzed by Northern blot analysis for p21 mRNA or ß-actin mRNA. B, Effect of SNAP on p53 activation. EMSA was performed for extracts from cells treated for 15 hours with either 0.2% FCS (lanes 2 and 3) or 10% FCS (lanes 4 and 5) to which 100 µmol/L SNAP was added (lanes 3 and 5) for an additional 1 hour. Extracts were incubated with a labeled oligonucleotide containing the consensus sequence for p53 and EMSA performed as described in Methods. NS refers to a nonspecific band that was consistently found in all experiments in which cell extracts were incubated with the labeled oligonucleotide. The graphs represent typical results from 3 or 4 individual experiments.

p53 Is Phosphorylated by SNAP
In Figure 4A, cells were treated with SNAP for 2 hours during the time interval corresponding to 15 to 17 hours after addition of 10% FCS. SNAP addition caused a 3-fold increase in the band designated as the p53 complex. Addition of either a 10- or 100-fold excess of unlabeled oligonucleotide for the consensus sequence of p53 completely eliminated the slowly migrating complex, whereas an unrelated oligonucleotide sequence corresponding to that for SP1 binding sites had no effect, indicating that the complex observed contained p53. Furthermore, addition of an antibody specific for p53 to the incubation of nuclear extract and labeled p53 consensus oligonucleotide reduced the formation of the complex activated by SNAP addition, whereas addition of antibodies to another transcription factor (anti-C/EBP) had no effect. In separate experiments, antibodies to other transcription factors were also ineffective in altering the complex formation, although we were never able to effectively demonstrate a supershift with the use of several p53 antibodies available commercially (Pharmingen and Oncogene). Further evidence for p53 activation by SNAP is shown in Figure 4B. By use of the total lysate, cell extracts from cells treated for 15 minutes to 4 hours with 100 µmol/L SNAP were analyzed for p53 by immunoprecipitation and Western blot analysis. Phosphorylation of p53 was apparent 15 minutes after stimulation with SNAP, and maximal phosphorylation occurred 30 to 60 minutes after SNAP addition and was maintained for up to 4 hours. The p53 phosphorylation (measured 30 minutes after SNAP treatment) was clearly inhibited by pretreatment with either 10 µmol/L ODQ, 50 µmol/L Rp-8Br-cGMP, or 50 µmol/L Rp-8Br-cAMP, inhibitors of soluble guanylate cyclase, protein kinase G, and protein kinase A, respectively (Figure 4C). As an additional control, cells were exposed to carbobenzoxy-L-leucyl-L-leucyl-L-leucinal, a proteasome inhibitor, for 30 minutes. Dramatically increased levels of p53 were observed; this observation is consistent with previous studies implicating ubiquitin-dependent proteolysis in the normal turnover of p53.²¹

Response to NO Is Mediated in Part by cGMP
To determine the signaling pathway initiated by NO and leading to the activation of p53 and p21 expression, cells were treated with SNAP 15 hours after 10% FCS in the absence or presence of ODQ, a specific inhibitor of soluble guanylate cyclase. When cells were pretreated with ODQ, the induction of p21 mRNA during the 15- to 19-hour interval was essentially eliminated, whereas there was no change in either p53 mRNA or ß-actin mRNA (Figure 5A). Interestingly, p53 mRNA was not affected by SNAP addition; this finding is consistent with activation of the preexisting p53 protein. ODQ pretreatment also blocked the activation of p53, as determined by EMSA (Figure 5B), with partial inhibition seen at 1 µmol/L ODQ and complete inhibition seen at 10 µmol/L ODQ.

View larger version (38K): [in this window] [in a new window]   Figure 5. ODQ inhibits SNAP-induced changes in p21 mRNA and p53 activation. A, Cells were pretreated with 10% FCS for 15 hours. The designated concentration of ODQ (µmol/L), a specific inhibitor of soluble guanylate cyclase, was added 30 minutes before SNAP (100 µmol/L). Cells also were treated with ODQ alone (lanes 6 to 8). After 4 hours, RNA was extracted and analyzed for p21 and ß-actin mRNA. The bar graphs on the right represent the ratios of p21/ß-actin and p53/ß-actin. B, Cells were pretreated with 10% FCS for 15 hours. The designated concentration of ODQ (µmol/L) was added 30 minutes before SNAP (100 µmol/L), and the cells were then treated for an additional 2 hours. EMSA was performed on total cell extracts. The bar graph on the right shows the density of each band on the right. The graphs represent typical results from 3 or 4 individual experiments

Evidence implicating cyclic nucleotides in the signaling pathway activated by SNAP is shown in Figure 6. Rp-8Br-cGMPS and Rp-8Br-cAMPS, specific inhibitors of cGMP- and cAMP-dependent protein kinases, respectively, effectively inhibited the increase in p21 mRNA induced by SNAP, whereas no change in p53 mRNA was noted. Furthermore, when gel-shift analysis was used to measure p53 activation also, both drugs were effective inhibitors, thereby implicating both cAMP and cGMP in the process.

View larger version (34K): [in this window] [in a new window]   Figure 6. Effect of protein kinase A and protein kinase G inhibitors on SNAP-induced increase of p21 mRNA and p53 activation. Cells were made quiescent and then incubated with 10% FCS for 15 hours. SNAP (100 µmol/L) was added either with or without 25 µmol/L Rp-8Br-cGMPS or 50 µmol/L Rp-8Br-cAMPS and incubated for either 4 hours (A) for Northern blot analysis of p21 mRNA or 2 hours (B) for EMSA of p53. The graphs represent typical results from 2 or 3 individual experiments.

Further evidence for the involvement of the cyclic nucleotides is provided in Figure 7. Direct addition of either 8-bromo-cGMP or dibutyryl cAMP produced a strong increase in p21 mRNA (Figure 7A) when added during the cell cycle for 4 hours at concentrations between 0.1 and 1.0 mmol/L. In contrast to the effect of either nucleotide on p21 mRNA, only the addition of 8-bromo-cGMP activated p53 (Figure 7B). The results in Figure 7B are with the addition of 0.5 mmol/L of either nucleotide. In separate experiments, 0.1 mmol/L and 1.0 mmol/L dibutyryl cAMP was also ineffective in activating p53.

View larger version (50K): [in this window] [in a new window]   Figure 7. Direct effect of cAMP or cGMP on p21 mRNA or p53 activation when added during the cell cycle. Cells were made quiescent and then incubated with 10% FCS for 15 hours. Cyclic nucleotides were then added at concentrations of either 0.1 or 1.0 mmol/L, and cells were incubated for an additional 4 hours (A) for Northern blot analysis of p21 mRNA or 2 hours (B) for EMSA of p53. Ctrl indicates control; Bt2-cAMP, dibutyryl cAMP; and 8br-cAMP, 8-bromo-cAMP. In panel A, SNAP (100 µmol/L) was added for comparison, and RNA from separate samples was analyzed in the second and the last lane. The data shown are representative of at least 3 individual experiments.

	Discussion

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We have used adventitial fibroblasts from the rat aorta to examine the role of NO in modulating the serum-induced proliferation of these cells. The data indicated that the antiproliferative effects of NO were related to the increased expression of p21. We found that NO donors induce p21 expression when added to quiescent cells and also when added to cells that are actively dividing; this finding is consistent with the ability of NO to reduce thymidine incorporation into DNA when added either to quiescent cells or during the cell cycle. We have focused on the effects of NO that occur during the cell cycle because these mechanisms associated with the response to NO may differ between cycling cells and cells at quiescence and because NO exposure during the cell cycle is an event that could occur during pathophysiological conditions.

The antiproliferative effects of NO have been described for a variety of cell types, initially for vascular smooth muscle cells¹ and subsequently for other cell types, including fibroblasts.² However, the mechanisms involved are not clear; in fact, there are findings that are inconsistent among cell types and also in a given cell type. For example, the investigators who first described the inhibitory effects of NO on DNA synthesis in cultured vascular smooth muscle cells later showed that in primary cultures of those cells, NO enhanced fibroblast growth factor–induced mitogenesis, whereas in subsequent passages in vitro, NO inhibited mitogenesis.²² In smooth muscle cells, the antiproliferative effects were mimicked by cGMP, whereas in BALB/c3T3 fibroblasts, which lack soluble guanylate cyclase, addition of NO donors inhibited mitogenesis and proliferation, suggesting a mechanism that did not require cGMP in those cells.

The increase in p21 mRNA levels that we noted when SNAP was added 15 hours after the addition of 10% FCS, a time when the cells were dividing, is a novel effect not described previously, and it was this response that we examined in greater detail. Under those conditions, we found that p53 activation also occurred in response to SNAP, a distinction between the effect of SNAP in adventitial fibroblasts and the studies reported in vascular smooth muscle cells in which the increase in p21 was not accompanied or preceded by p53 activation.⁷

We observed that p53 was rapidly phosphorylated as early as 15 minutes after SNAP treatment and that the binding activity was enhanced at 1 hour after SNAP treatment and was localized to the nuclear fraction immunochemically (data not shown). However, p21 mRNA accumulation was detected after only 3 to 4 hours of treatment with SNAP, suggesting that p53 activation could occur before increasing p21 expression. During this time frame, steady-state levels of p53 mRNA did not change. It was reported that NO induced nuclear accumulation of p53 protein in a dose- and time-dependent manner and that its DNA binding activity depended on the concentration of NO. At low concentrations (0.25 to 0.5 mmol/L), NO donors stimulated p53 accumulation as well as its DNA binding activity, whereas at higher concentrations (2 to 5 mmol/L), NO donors significantly decreased the DNA binding activity.²³ In the present study, when EMSA was used, p53 binding activity increased with concentrations of SNAP between 50 and 500 µmol/L, but at higher levels (1 mmol/L), less binding activity was found (data not shown). A high concentration of NO can cause DNA damage and mutation,^{24 25} resulting in subsequent accumulation of p53, as found in rodent macrophage, pancreatic cell lines, murine thymocytes, and human cancer cells.^{26 27} With our experimental conditions, we did not observe any morphological or biochemical changes in the adventitial fibroblasts treated with SNAP or sodium nitroprusside, as assayed by lactic dehydrogenase or trypan blue exclusion.

Transcription of the p21 gene can be activated both by p53-dependent and -independent mechanisms. Our findings in the adventitial fibroblast indicating that the presence of serum was not necessary for p21 mRNA increases but was required for p53 activation, suggest that p53 was not essential for p21 activation but was probably involved when serum was present. In the studies using vascular smooth muscle cells, p21 was reported to be regulated by NO by a p53-independent mechanism.⁷ In other studies, DNA damage produced by irradiation induced p21 gene activation in a p53-dependent manner in human diploid fibroblasts and thyroid epithelial cells,¹⁴ whereas growth factors, cytokines, oxidative stress, phorbol esters, retinoic acid, and vitamin D₃ all induced increased p21 gene expression by p53-independent mechanisms.^{28 29 30}

We found that the addition of cyclic nucleotide to adventitial fibroblasts could activate p53 and increase p21 mRNA. Our data using ODQ and inhibitors of protein kinase G suggest that NO activates p53 by a mechanism involving cGMP. It is generally felt that p53 is maintained within cells at low concentrations, has a short half life (6 to 30 minutes), and is activated by a variety of interactions, including phosphorylation by protein kinases.³¹ Previous studies have shown that colocalization of p53, protein kinase A, and protein kinase G in the nucleus provide access for these 2 kinases to phosphorylate p53.^{32 33} Because NO can induce p53 activation and nuclear localization and can activate protein kinase G through the ability to increase guanylate cyclase activity, it is reasonable to propose that the NO-induced activation of p53 is related to increased cGMP levels. At high levels of NO, it is presumed that protein kinase A can be activated by the inordinately high concentrations of cGMP produced,⁶ and because the catalytic subunit of protein kinase A can translocate to the nucleus, the possibility exists that either or both protein kinases might mediate an effect of cGMP by nuclear localization and subsequent p53 activation. Furthermore, because protein kinases A and G can phosphorylate transcriptional factors related to cAMP response element binding protein,³² other diverse effects could promote either p53-dependent or -independent increases in p21 transcription.

In an earlier study,¹⁷ we had shown that SNAP would inhibit angiotensin-induced thymidine uptake by cardiac fibroblasts when added at quiescence, yet the NO donor did not prevent cell changes characteristic of transition from G₀ to G₁, suggesting an effect within the cell cycle. SNAP addition was actually more effective in reducing thymidine incorporation into DNA when added after the G₀ to G₁ transition than when added at quiescence. Other studies using vascular smooth muscle cells have examined the effects of NO donors on cell cycle events subsequent to G₁.⁷ Those studies have indicated that NO donors effectively inhibited DNA synthesis when added to cells in S phase, but the mechanism for this S-phase arrest appeared to be independent of cGMP, in contrast to the effects we observed in adventitial fibroblasts, and relied, at least in part, by influencing ribonucleotide reductase activity, possibly by direct interactions between NO and ribonucleotide reductase.^{4 5}

	Acknowledgments

 
This study was supported by National Institutes of Health grants HL-55001 and HL-55620. We thank Cynthia Curry for technical assistance.

	Footnotes

 
Correspondence to Peter Brecher, PhD, Boston University School of Medicine, 715 Albany St, Boston, MA 02118.

Received June 11, 1999; accepted August 24, 1999.

	References

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