Effect of Nitric Oxide-cGMP-Dependent Protein Kinase Activation on Advanced Glycation End-Product–Induced Proliferation in Renal Fibroblasts

Reagents

FBS, DMEM, antibiotics, molecular weight standards, trypsin-EDTA, trypan blue stain, and all medium additives were obtained from Life Technologies, Inc. (Glasgow, UK). Anti-JAK2, -STAT5, -iNOS, -p42/p44 MAPK, -cyclin D1, -cdk4, and p21^Waf1/Cip1 antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antiphospho-p42/p44 MAPK antibody was purchased from New England Biolabs (Beverly, MA). Antiphospho-JAK2 and antiphospho-STAT5 antibodies were obtained from Upstate Biotechnology Inc. (Lake Placid, NY). Protein A/G-coupled agarose beads, SNAP, SNP, 8-para-chlorophenylthio-cGMP (8-pCPT-cGMP), KT5823, and anti-PKG-I antibody were purchased from Calbiochem (La Jolla, CA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit or anti-mouse secondary antibody, [γ-³²P]ATP, and the enhanced chemiluminescence (ECL) kit were obtained from Amersham Corp. (Piscataway, NJ). N,N′-methylenebisacrylamide, acrylamide, SDS, ammonium persulfate, Temed, and Tween 20 were purchased from Bio-Rad Laboratories (Richmond, CA). cGMP enzymatic immunoassay (EIA) set was purchased from Amersham Pharmacia Biotech (Buckinghamshire, UK). Bovine serum albumin, anti–β-actin antibody, DMSO, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay kit, and all other chemicals were obtained from Sigma Chemical Company (St. Louis, MO).

Culture Conditions

NRK-49F cells were obtained from the American Type Culture Collection (Manassas, VA). Cells were grown in culture flasks and maintained in DMEM (5.5 mM D-glucose) supplemented with 100 IU/ml penicillin, 100 μg/ml streptomycin, and 5% FBS in a humidified 5% CO₂ incubator at 37°C. In some experiments, cells were exposed to serum-free (0.1% FBS) DMEM supplemented with the specific PKG inhibitor, KT5823, for 16 h before timed exposure to FBS and AGE. KT5823 was dissolved in DMSO. Cell viability was assessed by the trypan blue exclusion test and was routinely >92%. For cell number analysis, cells (1.5 × 10⁵ cells/well) were cultured in 6-well culture plates (Nunclon) and grown in the added test agents. Cells were harvested and counted with a hemocytometer. Cells between passages 10 and 25 were used in all experiments. Each experimental data point represents the mean of duplicate wells from three independent experiments.

Preparation of AGE

AGE-BSA was prepared according to standard procedures: 20% (wt/vol) fatty acid-free BSA was incubated at 37°C under sterile phosphate-buffered saline (PBS), pH 7.2, supplemented with 250 mM glucose-6-phosphate, 100 μg/ml gentamycin, and 10 μg/ml ceftriaxone for 8 wk. Unincorporated glucose-6-phosphate and antibiotics were then removed by dialysis against PBS. Control nonglycated BSA was incubated in the same conditions with the exception of the absence of glucose-6-phosphate. They were then sterile-filtered through 0.2-μm nylon filters. Preparations were tested for endotoxin using the Endospecy ES-20S system; no endotoxin was detectable. The AGE content of the preparations was determined spectrofluorometrically with excitation set at 390 nm and emission set at 450 nm, and expressed as the percentage of relative fluorescence compared with nonincubated aliquots of the same batch of BSA in glucose-6-phosphate solution, stored frozen immediately after preparation.

NO Analysis

NO levels were measured with the Griess method. In brief, 1.2 × 10³ cells were plated in each well of a 96-well plate in DMEM medium with 5% FBS. After being passed through 50-kD ultrafilters, 20 μl of the medium was diluted with 120 μl assay buffer and mixed with 5 μl cofactor and 5 μl nitrate reductase (NO colorimetric assay kit, Calbiochem). After the medium had been kept at room temperature for 2 h to convert nitrate to nitrite, total nitrite was measured at 540 nm absorbance by reaction with Griess reagent (sulfanilamide and naphthalene–ethylene diamine dihydrochloride). Amounts of nitrite in the medium were estimated by a standard curve obtained from enzymatic conversion of NaNO₃ to nitrite.

cGMP and Protein Determination

Cells were grown in 24-well plates, 1.2 × 10⁴ cells/well. Sixteen hours before stimulation, culture media were replaced by serum-free DMEM supplemented with the specific inhibitors or other drugs. Basal cGMP level was checked in the wells to which a corresponding volume of incubation medium without activators was added. Reaction was terminated by adding 10% (vol/vol) ice-cold lysis buffer A from cGMP EIA kit. After 30 min of extraction on ice, the supernatant was collected to the Eppendorf tubes and stored at −20°C until cGMP assay. cGMP was determined using the acetylation method in a cGMP immunoassay system as described in the manual provided by the manufacturer. For protein measurement, the cells were rinsed twice with PBS and solubilized in 500 μl solution of SDS in 0.01 N NaOH. The 50-μl aliquots were used for Bio-Rad protein assay. Measurements were performed in triplicate.

Preparation of Nuclear Extracts

Nuclear extracts were prepared as in our previous study (13). Briefly, cells were maintained in 5% FBS medium for 1 d. After fasting (0.1% FBS) for 48 h, fresh DMEM (5% FBS) with different concentrations of AGE or agents were added. After stimulation, cells were harvested and vigorously vortexed. Then cell lysates were centrifuged and nuclear pellets were resuspended in nuclear extraction buffer. Nuclear proteins were measured for protein by using a Bio-Rad protein assay kit. The extracts were stored at −70°C for further use.

Immunoprecipitation and Western Blotting

For protein analysis, 1.5 × 10⁷ serum-deprived cells were treated with agents or AGE as described above. Total cell lysates were harvested, resolved by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to Protran membranes (0.45 μm; Schieicher & Schuell, Keene, NH). The membranes were blocked in blocking solution and subsequently probed with primary antibody (1 μg/ml). The membrane was incubated in 4000× diluted HRP-conjugated goat anti-rabbit or anti-mouse secondary antibody. The protein bands were detected using the ECL system, and the percentage of phosphorylated form of protein was determined using a scanning densitometer.

For p42/p44 MAPK and JAK/STAT activation assays, cell extracts were immunoprecipitated with p42/p44 MAPK and JAK/STAT monoclonal antibodies and with protein A/G-agarose beads. Then the samples were resolved by SDS-PAGE, and transferred to Protran membranes. The membranes were probed with anti-phospho-p42/p44 MAPK (0.75 μg/ml), antiphospho-JAK2 (0.75 μg/ml), anti-phospho-STAT5 (0.75 μg/ml), anti-p42/p44 MAPK (1:1000), anti-JAK2 (1:1000), and anti-STAT5 (1:1000) antibodies. The anti-phospho-STAT5 polyclonal antibody detects the tyrosine-phosphorylated forms of both STAT5a and STAT5b. Immunoreactive proteins were detected with the ECL system as described above.

Assay of PKG Activity

Cells were treated with agents or AGE as described above. After rinsing the monolayer with PBS, cells were harvested and homogenized with cold buffer consisting of 20 mM sodium phosphate (pH 6.8), 2 mM EDTA, 15 mM 2-mercaptoethanol, 150 mM NaCl, 2 mM benzamidine, 0.1 mM phenylmethylsulfonyl fluoride, 10 μg/ml pepstatin A, and 10 μg/ml leupeptin. The suspension was centrifuged for 10 min at 14,000 × g to obtain cell extract. Aliquots of extract were analyzed for PKG activity and also for Western blotting with an affinity-purified polyclonal rabbit anti-PKG-I antibody (1:500). The anti-PKG-I polyclonal antibody detects the activation forms of both PKG-Iα and PKG-Iβ. PKG activity was assayed as described in Suhasini et al. (33) and Soh et al. (34) with a peptide substrate (RKISASEFDRPL) selective for PKG. The difference in the phosphorylation of substrate in the presence and absence of cGMP was taken as PKG activity.

Cdk4 Kinase Assay

For immunoprecipitation of cdk4, cells were washed twice with ice-cold PBS, harvested in cdk4 lysis buffer [50 mM HEPES (pH 7.5), 10% glycerol, 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 1 mM dithiothreitol, and 0.1% Tween 20, supplemented with the phosphatase and protease inhibitors 5 mM NaF, 0.1 mM sodium orthovanadate, 5 μg/ml leupeptin, 10 μg/ml aprotinin, 50 μg/ml phenylmethylsulfonyl fluoride, and 5 μg/ml pepstatin A], and lysed by repeated passages through a 25-gauge needle. Cellular debris was removed from soluble extracts by centrifugation at 16,000 × g for 10 min at 4°C. After normalization of protein content, lysates were precleared by incubation with protein A/G-agarose beads and preimmune rabbit serum for 30 min at 4°C. Endogenous cdk4-containing complexes were immunoprecipitated for 3 h at 4°C, using a rabbit polyclonal anti-human cdk4 antibody. Immunoprecipitates were washed twice with cdk4 lysis buffer and four times with glutathione S-transferase-RB (GST-RB) kinase buffer [50 mM HEPES (pH 7.5), 10 mM MgCl₂, 1 mM dithiothreitol, 2.5 mM EGTA, 10 mM β-glycerophosphate, 0.1 mM orthovanadate, 1 mM NaF] and then resuspended in 50 μl of GST-RB kinase buffer.

Kinase activity associated with anti-cdk4 immunocomplexes was assayed in 50 μl of GST-RB kinase buffer containing 2 μg of GST-RB substrate, and in each case supplemented with 2 mM EGTA and 2 μCi of [γ-³²P]ATP. Reactions were carried out for 30 min at room temperature; cold ATP (final concentration, 30 μM) was then added to each reaction mixture to reduce background signal. Reactions were stopped by addition of Laemmli sample buffer, and the reaction products were electrophoresed in 12% SDS-PAGE, whereupon the gels were dried, visualized by autoradiography, and quantitated with a scanning densitometer.

Synthesis of Oligodeoxynucleotides

The sequences of phosphorothioate double-stranded oligodeoxynucleotides (ODN) used in this study were synthesized using a DNA/RNA synthesizer (Applied Biosystems, Foster City, CA). An ODN containing the STAT5 binding, γ-IFN–activated sequence (GAS)-like element from the β-casein promoter (35), was used as a probe. The sequences of STAT5 ODN were 5′-TGCTTCTTGGAATT-3′, 3′-ACGAAGAACCTTAA-5′. The single-stranded ODN were annealed for 2 h as temperature descended from 80°C to 25°C.

Electrophoretic Mobility Shift Assay

Electrophoretic mobility shift assay (EMSA) was performed as in our previous study (13). Briefly, ³²P-labeling of STAT5 ODN was carried out using T4-polynucleotide kinase (New England BioLabs) and [γ-³²P]ATP (3000 Ci/mmol). Labeled DNA was separated from the unincorporated radioactivity. Binding reactions were carried out by adding 5 μg of nuclear protein to 20 μl of binding buffer and [γ-³²P]ATP labeled STAT5 ODN probes. Where indicated, cold competitive oligonucleotides were included during the preincubation periods. Samples were incubated at room temperature for 25 min and fractionated by electrophoresis. After electrophoresis, gels were transferred to filter paper, dried, and exposed to x-ray Hyperfilm-MP at −70°C using an intensifying screen. The results were quantified by a scanning densitometer.

MTT Assay

MTT assays were performed to evaluate the proliferation of NRK-49F cells. Briefly, 100 μl of each cell type (1 × 10⁵ cells/ml) were plated and incubated for 24 h in wells of a 96-well plate. Then various concentrations of each drug were added to the wells. After another 24 h incubation, 10 μl of sterile MTT dye were added, and the cells were incubated for 6 h at 37°C. Then 100 μl of acidic isopropanol (0.04 M HCl in isopropanol) were added and thoroughly mixed. Spectrometric absorbance at 595 nm (for formazan dye) was measured with the absorbance at 655 nm for reference.

Flow-Cytometric Analysis of the Cell Cycle

For flow-cytometric experiments to determine DNA content and the cell cycle profile, cells were plated at a density of 1 × 10⁶ to 2 × 10⁶ cells per T-25 flask. At various time points, cells were harvested and fixed with ice-cold 100% ethanol with vortexing at low speed, and cells were then stored at −20°C for overnight. After fixation, cells were centrifuged and washed once with PBS containing 1% bovine serum albumin. For staining with DNA dye, cells were resuspended in 0.5 to 1 ml of propidium iodide (PI) solution containing RNase and incubated at 37°C for 30 min, and then they were incubated overnight at 4°C. Cell cycle profiles were obtained with a FACScan flow cytometer (Becton Dickinson, San Jose, CA) and data were analyzed with ModFit software (Verity Software House, Inc., Topsham, ME) for cell cycle analysis.

Statistical Analyses

The results were expressed as the mean ± standard errors of the mean (SEM). Unpaired t tests were used for the comparison between two groups. One-way ANOVA followed by unpaired t test was used for the comparison among more than three groups. P < 0.05 considered statistically significant.

Effects of AGE on NO Production, cGMP Synthesis, and PKG-I Activation in NRK-49F Cells

We previously reported that AGE induces NRK-49F cell proliferation via activation of the JAK-STAT pathway (13). However, the involvement of NO in the proliferative effects of AGE is unclear. Therefore, the goal of this study was to elucidate the mechanisms by which NO and its downstream signals mediate the AGE-induced proliferative response in NRK-49F cells. To investigate whether AGE could affect NO production in NRK-49F cells, we treated these cells with nonglycated BSA and AGE and endogenously analyzed NO production by the Griess method. We found that AGE dose-dependently decreased nitrite production at 8 and 12 h (Figure 1). However, nonglycated BSA (100 μg/ml) did not induce any changes at the same times. It is well established that the downstream target of the NO pathway is PKG, and that many of the biologic effects of NO are mediated through the important kinase PKG. We examined whether NO blockade prevented cGMP synthesis and PKG activation. We found that AGE (100 μg/ml) significantly decreased cGMP synthesis and PKG activity at 1, 1.5, and 2 h (Figure 2, A and B), while nonglycated BSA (100 μg/ml) did not affect control cGMP synthesis and PKG activity in NRK-49F cells. We also found that the synthesis of PKG-I was significantly reduced by AGE but not by nonglycated BSA at 0.5, 1, and 2 h (Figure 2C). The activation and protein synthesis of PKG in the absence of added test agents were similar to nonglycated BSA (100 μg/ml) (data not shown). These results indicate that the biologic effects of AGE in NRK-49F cells may be caused by inhibition of the NO-cGMP-PKG pathway.

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Figure 1.

Time course and dose-dependent effects of advanced glycation end-products (AGE) on nitric oxide (NO) production in normal rat kidney fibroblasts (NRK-49F) cells. Serum-deprived NRK-49F cells (1.2 × 10³ cells/well) were treated with serum-free (SF) medium, 5% FBS (control), AGE (50, 100, and 200 μg/ml) or BSA (100 μg/ml) for 0, 4, 8, and 12 h. NO production was determined by monitoring nitrite production by Griess assay as described in Materials and Methods. AGE (but not BSA) significantly decreased nitrite production at 8 and 12 h. Results were expressed as the mean ± SEM (n = 6). *P < 0.01 versus control.

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Figure 2.

Inhibition of cyclic guanosine 5′ monophosphate (cGMP) synthesis and cGMP-dependent protein kinase (PKG) activity by AGE in NRK-49F cells. Serum-deprived NRK-49F cells were treated with SF medium, BSA (100 μg/ml), or AGE (100 μg/ml) for 0, 0.5, 1, 1.5, and 2 h, and then assayed for cGMP synthesis (A) and PKG activity (B) as described in Materials and Methods. *P < 0.01 versus control. (C) Serum-deprived cells were treated with BSA (100 μg/ml) or AGE (100 μg/ml) for 0, 0.5, 1, and 2 h, and then total cell lysates were subjected to Western blot analysis for PKG-I. Results were expressed as the mean ± SEM (n = 4). *P < 0.01 versus BSA.

Effects of NO Donors, PKG Inhibitor, and PKG Activator on AGE-Inhibited NO-PKG Pathway

To investigate whether exogenously induced NO/PKG could affect AGE-inhibited NO-PKG pathway in NRK-49F cells, we treated these cells with NO donors, PKG inhibitor, and PKG activator and examined iNOS synthesis, nitrite production, and PKG activation in the presence of BSA (100 μg/ml) and AGE (100 μg/ml). Figure 3 illustrates that AGE significantly reduced iNOS synthesis and nitrite production at 8 h as compared with the control and BSA. Basal (control) nitrite production was not affected by BSA treatment. The NO donors SNAP (5 μM), SNP (5 μM), and the PKG activator 8-pCPT-cGMP (5 μM) markedly induced iNOS synthesis and nitrite production in the presence of AGE at 8 h. However, the inhibitor of PKG, KT5823, slightly enhanced AGE-inhibited nitric oxide production in NRK-49F cells. We also examined whether the NRK-49F cells express other NOS isoenzymes and found that the amount of endothelial NOS or neuronal NOS in the cells is very small (data not shown). Furthermore, the effects of NO donors, PKG inhibitor, and PKG activator on AGE-inhibited cGMP synthesis and PKG activation were also tested in NRK-49F cells and similar results were obtained (Figure 4). These data suggest that AGE can inhibit iNOS synthesis and NO-PKG pathway and NO donors or PKG activator can reverse these AGE-induced effects.

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Figure 3.

Effects of NO donors, PKG inhibitor, and PKG activator on AGE-blocked inducible NO synthase (iNOS) synthesis and NO production. Serum-deprived NRK-49F cells were treated with S-nitroso-N-acetylpenicillamine (SNAP) (5 μM), sodium nitroprusside (SNP) (5 μM), KT5823 (1 μM), 8-para-chlorophenylthio-cGMP (8-pCPT-cGMP) (5 μM), and DMSO for 8 h in the presence of BSA or AGE, and then assayed for iNOS synthesis by Western blotting (A). Cells treated with the same conditions (1.2 × 10³ cells/well) were assayed for nitrite production by Griess assay (B). SF and Control represent cells grown in the presence of 0.1% FBS and 5% FBS, respectively. DMSO was the internal control used to dissolve SNAP, SNP, KT5823, and 8-pCPT-cGMP. Results were expressed as the mean ± SEM (n = 6). *P < 0.01 versus AGE.

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Figure 4.

Effects of PKG inhibitor, NO donors, and PKG activator on AGE-inhibited cGMP and PKG-I synthesis and PKG activity. Total cell lysates from NRK-49F cells treated with KT5823 (1 μM), SNAP (5 μM), SNP (5 μM), and 8-pCPT-cGMP (5 μM) in the presence of AGE (100 μg/ml) for 2 h were subjected to analysis for synthesis of cGMP (A) and PKG-I (B). Serum-deprived NRK-49F cells were treated with the same test agents as above for 2 h, and then assayed for PKG activity as described in Materials and Methods (C). The concentration of BSA (control) was 100 μg/ml. These are representative experiments, each performed at least four times. *P < 0.01 versus AGE.

NO Donors and PKG Activator Block AGE-Induced Proliferation and JAK2-STAT5 Activation, But Not p42/p44 MAPK Activation

To determine whether the NO-PKG pathway played a role in AGE-induced mitogenesis, NO donors and PKG activator were used to pretreat NRK-49F cells in proliferation and JAK2-STAT5 activities. Both NO donors and PKG activator interfered with AGE-induced mitogenesis at 3 d (Table 1). This finding suggests that the antiproliferative effect of NO on AGE-induced proliferation is associated with PKG activation. Previously, we have shown that AGE induced tyrosine phosphorylation of JAK2 (but not JAK1, JAK3 or TYK2) and STAT5 at 15 min (14). Therefore, we also tested the effects of SNAP, SNP, and 8-pCPT-cGMP on AGE-induced JAK2-STAT5 activation at the same time. By using antibodies specific for JAK2 or phospho-JAK2, Western blots revealed that NO donors and PKG activator markedly reduced phospho-JAK2 without affecting JAK2 protein levels in AGE-treated NRK-49F cells (Figure 5, A and B). Interestingly, the PKG inhibitor KT5823 moderately enhanced the effect of AGE on phosphorylation of JAK2. Additionally, the experiments on phosphorylation of STAT5 showed similar results when cells were treated with AGE. To test whether the inhibition seen at the phosphorylation of STAT5 were also observed on protein-DNA binding activities, EMSA was performed. As shown in Figure 5C, the binding protein complexes were characterized by incubation with a 20-fold molar excess of unlabeled consensus ODN (Figure 5C, lane Comp.). Consistent with the results from the phosphorylation assays, treatment with SNAP, SNP, and 8-pCPT-cGMP inhibited AGE-enhanced STAT5 protein-DNA binding activities. On the other hand, recent studies have indicated that activation of the MAPK pathway is associated with AGE-increased cell proliferation (36, 37). Therefore, we also examined whether NO-PKG regulates p42/p44 MAPK activity in NRK-49F cells treated with AGE. We measured p42/p44 MAPK activity after exposure of cultured NRK-49F cells to FBS (0.1%), FBS (5%), BSA (100 μg/ml), and AGE (100 μg/ml). We found that AGE (100 μg/ml) markedly increased p42/p44 MAPK phosphorylation at 30 min in comparison with FBS (5%) and BSA (100 μg/ml) (data not shown). However, we found that p42/p44 MAPK phosphorylation was unaffected by NO donors or PKG activator when cells were treated with AGE (100 μg/ml) for 30 min (Figure 6). These observations demonstrate that JAK2-STAT5 but not MAPK may play roles as important signal mediators in the antiproliferative effects of the NO-PKG pathway in AGE-treated NRK-49F cells.

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Figure 5.

Effects of PKG inhibitor, NO donors, and PKG activator on AGE-induced phosphorylation of Janus kinase 2 (JAK2) and signal transducers and activators of transcription 5 (STAT5) and STAT5 protein-DNA binding activity. (A) Total cell lysates from NRK-49F cells treated with KT5823 (1 μM), SNAP (5 μM), SNP (5 μM), and 8-pCPT-cGMP (5 μM) in the presence of AGE (100 μg/ml) for 15 min were immunoprecipitated (IP) with anti-JAK2 and anti-STAT5. Immune complexes were separated by a polyacrylamide gel and immunoblotted (IB) with anti-phospho-JAK2 (P-JAK2) and anti-phospho-STAT5 (P-STAT5) antibodies (upper panels) or antibodies corresponding to the above antibodies (lower panels). (B) Laser densitometry of the gels shown in (A) and two additional tyrosine phosphorylation experiments. *P < 0.01 versus AGE. (C) Effects of KT5823 (1 μM), SNAP (5 μM), SNP (5 μM), and 8-pCPT-cGMP (5 μM) on AGE-induced STAT5 protein-DNA binding activities in NRK-49F cells. Serum-deprived cells were treated with the same above test agents for 15 min. Nuclear extracts were prepared and assayed by electrophoretic mobility shift assay (EMSA), as described in Materials and Methods. It was obvious that 20-fold molar excess of unlabeled STAT5 probe (Comp.) markedly abolished the DNA-protein complexes. Effects of BSA (100 μg/ml) on JAK2 and STAT5 activation are used as control. This is a representative experiment independently performed four times.

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Figure 6.

Effects of PKG inhibitor, NO donors, and PKG activator on AGE-induced phosphorylation of p42/p44 mitogen activated protein kinase (MAPK). Total cell lysates from NRK-49F cells treated with KT5823 (1 μM), SNAP (5 μM), SNP (5 μM), and 8-pCPT-cGMP (5 μM) in the presence of AGE (100 μg/ml) for 30 min were immunoprecipitated (IP) with anti-p42/p44 MAPK. Immune complexes were separated by a polyacrylamide gel and immunoblotted (IB) with anti-phospho-p42/p44 MAPK antibody (upper panel) or antibodies corresponding to the above antibodies (lower panel). This is a representative experiment independently performed four times.

NO Donors and PKG Activator Block the Effects of AGE on Cell Cycle Progression, Cyclin D1/cdk4 Activation, and p21^Waf1/Cip1 Expression

To gain further insight into the mechanism exerted by NO-PKG, we next wished to determine whether activation of the NO-PKG pathway is responsible for inhibition of AGE-mediated cell cycle progression in NRK-49F cells. AGE-incubated cells were treated for the indicated times with KT5823, SNAP, SNP, and 8-pCPT-cGMP, and then examined by flow-cytometric analysis. We found that a significant proportion of the cells entered S phase after the start of AGE treatment, whereas control cells largely remained arrested in the G₁ phase (Table 2). Clearly, SNAP, SNP, and 8-pCPT-cGMP caused potent G₁ arrest in AGE-treated NRK-49F cells. No significant difference in percentage of S phase was observed between AGE and KT5823-treated cells. These results demonstrate that AGE can induce S phase entry, and activation of the NO-PKG pathway may also have the ability to induce cell cycle arrest from AGE-treated cells. To investigate the possible role of NO-PKG in cell cycle progression, we examined the effects of SNAP, SNP, and 8-pCPT-cGMP on AGE-regulated expression of cell cycle regulatory molecules and cdk4 kinase activation. SNAP, SNP, and 8-pCPT-cGMP can prevent synthesis of cyclin D1 but not cdk4 (Figure 7A). However, cdk4 kinase analysis reveals that both NO donors and PKG activators reduced cdk4 kinase activation in AGE-treated cells (Figure 7B). Furthermore, NO donors and PKG activator significantly reversed AGE-inhibited expression of p21^Waf1/Cip1 (Figure 7A). These results indicated that suppression of cyclin D1/cdk4 activation and induction of p21^Waf1/Cip1 are the underlying mechanisms by NO donors and PKG activator to promote inhibition of cell cycle progression.

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Figure 7.

Effects of PKG inhibitor, NO donors, and PKG activator on AGE-regulated expression of cell cycle regulatory molecules and cdk4 kinase activation. (A) Total cell lysates from NRK-49F cells treated with KT5823 (1 μM), SNAP (5 μM), SNP (5 μM), and 8-pCPT-cGMP (5 μM) in the presence of AGE (100 μg/ml) for 4 h were subjected to Western blot analysis for cyclin D1, cdk4, and p21^Waf1/Cip1. (B) Serum-deprived NRK-49F cells were treated with the same above test agents for 4 h, and then assayed for cdk4 kinase activation was described in Materials and Methods. The concentration of BSA (control) was 100 μg/ml. These are representative experiments, each performed at least four times. *P < 0.01 versus AGE.