Trizol reagent, lipofectamine 2000 and rat p38 siRNA oligomers were purchased from Invitrogen (Carlsbad, CA). Dulbecco's phosphate-buffered saline (PBS), Dulbecco's modified Eagle's medium (DMEM), penicillin-streptomycin and fetal bovine serum (FBS) were purchased from Gibco/BRL (Life Technologies, Gaithersburg, MD). 15d-PGJ₂ and GW9662 were purchased from Biomol (Plymouth Meeting, PA). Escherichia coli LPS (O111:B4), diphenyleneiodonium chloride (DPI), dithiothreitol (DTT), phenylmethylsulfonyl fluoride (PMSF), pepstatin, leupeptin, autipain, and aprotinin were obtained from Sigma Chemical Co. (St. Louis, MO). MAPK inhibitors, 2'-amino-3'methoxyflavone (PD98059), 4-(4-fluorophenyl)-2-(4-nitrophenyl)-5-(4-pyridyl)-1H-imidazole (PD169316), and NF-κB inhibitor, (E)3-[(4-Methylphenyl)sulfonyl]-2-propenenitrile (Bay 11-7082) were purchased from Calbiochem (San Diego, CA). Dichlorofluorescein diacetate (DCF-DA) was obtained from Molecular probes (Eugene, OR). Nitrocellulose transfer membranes were obtained from Schleicher & Schuell Bioscience (Dassel, Germany). [α-³²P]dCTP was purchased from Dupont-New England Nuclear (Boston, MA). Oligonucleotide primers for polymerase chain reaction (PCR) of IL-8/CXCL8, PPAR and β-actin were synthesized by Bionics (Seoul, Korea). The LightCycler FastStart DNA SYBR Green I Mix was obtained from Roche (Mannheim, Germany). Anti-NF-κB, Phospho-ERK and phospho-p38 antibodies were obtained from Cell Signaling Technology (Danvers, MA). The γ-tubulin antibody was obtained from Sigma Chemical Co. (St Louis, MO). All other reagents were from pure-grade commercial preparations.

Specific pathogen-free male inbred WKY and SHR, 20 to 30 weeks of age, were purchased from Japan SLC Inc. (Shizuoka, Japan). All experimental animals received autoclaved food and bedding to minimize exposure to viral or microbial pathogens. The rats were cared for in accordance with the Guide for the Care and Use of Experimental Animals of Yeungnam Medical Center.

VSMCs were obtained from the thoracic aortas of 20- to 30-week-old male WKY and SHR as described previously (25). VSMCs were cultured in Dulbecco's modified Eagle's medium (DMEM) that was supplemented with 10% FBS and 1% penicillin-streptomycin. Cells were detached using 0.25% trypsin/EDTA and seeded into 75-cm² tissue culture flasks at a density of 10⁵ cells per ml. All experiments were conducted at cell passage 3 to 7. Prior to stimulation, 95% confluent VSMCs were serum-starved overnight by incubating in DMEM with 0.1% FBS. Cell cultures were incubated in a humidified incubator at 37℃ and 5% CO₂ in the presence or absence of stimuli for the indicated times.

Total RNA was extracted using a Trizol reagent in accordance with the manufacturer's instructions. The quantity of total RNA obtained was determined by measuring optical density (OD) at 260 and 280 nm. Real-time PCR for the amplification of IL-8/CXCL8 and PPARγ in VSMCs was performed using a LightCycler (Roche). RNA was reverse transcribed to cDNA from 1 µg of total RNA, and then subjected to real-time PCR. PCR was performed in triplicate. The total PCR volume was 20 µl and contained the LightCycler FastStart DNA SYBR Green I mix (Roche), appropriate primer and 2 µl of cDNA. Prior to PCR amplification, the mixture was incubated at 95℃ for 10 min, and the amplification step consisted of 45 cycles of denaturation (10 s at 95℃), annealing (5 s at the primer-appropriate temperature), and extension (10 s at 72℃) with fluorescence detection at 72℃ after each cycle. After the final cycle, melting point analyses of all samples were performed over the range of 65 to 95℃ with continuous fluorescence detection. β-actin expression levels were used for sample normalization. Results for each gene were expressed as the relative expression level compared with β-actin. The primers used for PCR were as follows: for IL-8/CXCL8 (365 bp) sense, 5'-gaagatagattgcaccga-3'; antisense, 5'-catagcctctcacacatttc-3', for PPARγ (359 bp): sense, 5'-tgaggagaagtcacactctg-3'; antisense, 5'-tgggtcagctcttgtgaatg-3' and for β-actin (101 bp): sense, 5'-tactgccctggctcctagca-3'; antisense, 5'-tggacagtgaggccaggatag-3'. The level of IL-8/CXCL8 mRNA was determined by comparing experimental levels to the standard curves and was expressed as the fold of relative expression.

Nuclear extracts were prepared as described previously (25). Cells were washed three times with cold PBS, then scraped and harvested by centrifugation. Cell pellets were resuspended and incubated on ice for 15 min in 400 µl of hypotonic buffer A (10 mmol/l HEPES, 10 mmol/l KCl, 1.5 mmol/l MgCl₂, 0.5 mmol/l DTT, 0.1 mmol/l PMSF, 10 µg/ml pepstatin, 10 µg/ml leupeptin, 10 µg/ml autipain, and 10 µg/ml aprotinin). Nonidet P-40 was then added to a final concentration of 2.5%, and the cells were vortexed for 10 s. Nuclei were separated from the cytosol by centrifugation at 12,000 g for 15 s. Pellets were resuspended in 40 µl of hypotonic buffer C (20 mmol/l HEPES, 25% glycerol, 0.4 mol/l NaCl, 1 mmol/l EDTA, 1 mmol/l EGTA, 0.5 mmol/l DTT, 0.1 mmol/l PMSF, 10 µg/ml pepstatin, 10 µg/ml leupeptin, 10 µg/ml autipain, and 10 µg/ml aprotinin). Samples were sonicated at level 3-4 for 2-3 s, followed by centrifugation for 10 min at 4℃. The nuclear protein concentration was determined using the Bradford assay (Bio-Rad, Richmond, CA). A consensus sequence for the NF-κB DNA binding site (5'-AGTTGAGGGGACTTTAGGC-3') (sc-2505; Santa Cruz Biotechnology, Santa Cruz, CA) was labeled with [α-³²P]dCTP using a random-primed DNA labeling kit (Roche). The mutant NF-κB binding sequence was identical to sc-2505 except for a "G" → "C" substitution in the NF-κB DNA binding motif (sc-2511; Santa Cruz Biotechnology). DNA The labeled DNA was purified over an S-200 HR column (Pharmacia, Piscataway, NJ) to remove unbound nucleotides. Nuclear protein extracts were incubated at room temperature for 20 min with approximately 50,000 cpm of labeled oligonucleotides that were suspended in the binding buffer [200 mmol/l HEPES, 500 mmol/l KCl, 10 mmol/l EDTA, 50% glycerol, 10 mmol/l DTT, 1 mg/ml BSA, 1 µg/µl poly (dI-dC)]. Following this incubation, samples were resolved on 4% polyacrylamide gels at 140 V and exposed to film.

Total lysates were prepared in a PRO-PREP buffer (iNtRON, Seoul, Korea). Protein concentrations were determined by a Bradford assay using bovine serum albumin as a standard. Thirty-micrograms of the protein samples were separated on 10% SDS-polyacrylamide gels, and then transferred to nitrocellulose membranes. The membranes were soaked in 5% nonfat dried milk in TBST (10 mmol/l Tris-HCl pH 7.5, 150 mmol/l NaCl and 0.05% Tween-20) for 1 h and then incubated for 16~18 h with primary antibodies against phospho-ERK, phospho-p38 and γ-tubulin at 4℃. Membranes were washed three times with TBST for 10 min and then incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at 4℃. After incubation with the secondary antibody, the membranes were rinsed three times with TBST for 10 min and antigen-antibody complex was detected using an enhanced chemiluminescence detection system (LAS-3000, Fujifilm, Tokyo, Japan).

To confirm whether the p38 pathway contributes to the inhibitory effect of 15d-PGJ₂ on LPS-induced IL-8/CXCL8 expression, p38 expression was silenced using small interfering RNA (siRNA). VSMCs were plated on 24-well plates and grown to 90% confluence. VSMCs were then transfected with p38 siRNA oligomers (20 nmol/l) using lipofectamine 2000 in accordance with the manufacturer's instructions. After 24 h of incubation, VSMCs were placed in growth medium for 24 h before the experiments. Cells were then cultured in the presence or absence of stimuli for 4 h. Sense and antisense oligonucleotides corresponding to the rat p38 siRNA sequence: sense, 5'-uacauuugcgaaguucaucuucggc-3'; antisense, 5'-gccgaagaugaacuucgcaaaugua-3' was purchased from Invitrogen (Carlsbad, CA).

ROS production was measured using flow cytometric analysis of DCF-DA-stained cells. In brief, VSMCs were grown to 70% confluence in serum-enriched DMEM at 37℃ in 5% CO₂. The medium was then replaced with serum-free DMEM and the cells were cultured in the presence or absence of stimuli for the indicated times. Cells were incubated in the dark with DCF-DA (50 µmol/l) for 1 h at 37℃, scraped, and resuspended in PBS. Fluorescence was monitored using flow cytometry (FACScan, Becton Dickison).

Results are expressed as means±SD from at least three or four independent experiments. For comparison between multiple groups, statistical significance was determined by the Mann-Whitney test using the SPSS version 12.0.

We examined the differential effect of LPS on IL-8/CXCL8 mRNA expression in SHR VSMCs in comparison to WKY VSMCs. From this experiment, we found that the expression of LPS-induced IL-8/CXCL8 mRNA was greater in SHR VSMCs than WKY. Real-time PCR was performed on VSMCs after they were untreated (NT) or treated with LPS (1 µg/ml), 15d-PGJ₂ (10 µM) or LPS plus 15d-PGJ₂ simultaneously (15d-PGJ₂/LPS) for 4 h. 15d-PGJ₂ treatment had different effects on SHR VSMCs relative to WKY VSMCs, where 15d-PGJ₂ had upregulatory effect on LPS-induced IL-8/CXCL8 mRNA expression in SHR VSMCs and suppressive effect on LPS-induced IL-8/CXCL8 mRNA expression in WKY VSMCs. 15d-PGJ₂ alone did not induce IL-8/CXCL8 mRNA expression in SHR VSMCs significantly (Fig. 1A). The time course of 15d-PGJ₂/LPS-induced IL-8/CXCL8 mRNA expression was determined in SHR VSMCs over a 0 to 8 h time period. In this experiment, we found that the expression of IL-8/CXCL8 mRNA induced by 15d-PGJ₂/LPS was almost the same as that for cells treated with LPS alone until 2 h after treatment. However, the expression levels of IL-8/CXCL8 mRNA induced by 15d-PGJ₂/LPS were significantly greater than those in the cells treated with LPS alone from the 4 h period (Fig. 1B).

It is widely accepted that 15d-PGJ₂ exerts its effects on pro-inflammatory genes in cells through PPARγ dependent or PPARγ independent mechanisms (27-29). Before evaluating whether the mechanism of the upregulatory action of 15d-PGJ₂ in SHR VSMCs was PPARγ-dependent, we determined the expression pattern of PPARγ mRNA in SHR VSMCs treated with 15d-PGJ₂/LPS. There was no meaningful difference between the level of PPARγ expression induced by 15d-PGJ₂/LPS and that induced by 15d-PGJ₂ alone (Fig. 2A). To evaluate whether the upregulatory effect of 15d-PGJ₂ in SHR VSMCs is mediated by PPARγ, the effect of GW9662, a PPARγ antagonist, was tested in SHR VSMCs. GW9662 blocked LPS-induced IL-8/CXCL8 mRNA expression at the dose of 10 µM. And, although 10 µM of GW9662 did not block the upregulatory effect of 15d-PGJ₂ on LPS-induced IL-8/CXCL8 mRNA expression, the high doses (40 and 100 µM) of GW9662 inhibited the upregulatory effect of 15d-PGJ₂ on LPS-induced IL-8/CXCL8 mRNA expression (Fig. 2B).

To further understand the nature of the upregulatory effect of 15d-PGJ₂ on LPS-induced IL-8/CXCL8 expressions in SHR VSMCs, the role of NF-κB activation was investigated. Bay11-7082 is known to selectively block the phosphorylation of IκB-α; thus, preventing the activation and nuclear translocation of NF-κB. Real-time PCR and EMSA were performed on VSMCs after they were untreated or treated with LPS (1 µg/ml) and/or 15d-PGJ₂ (10 µM) in the absence or presence of Bay11-7082 (10 µM) for 4 h. Bay11-7082 remarkably blocked the upregulatory effect of 15d-PGJ₂ on LPS-induced IL-8/CXCL8 mRNA expression. And it also remarkably blocked LPS-induced IL-8/CXCL8 expression (Fig. 3A). However, in spite of negative NF-κB activity in cells treated 15d-PGJ₂ alone, NF-κB activity in SHR VSMCs treated with15d-PGJ₂/LPS was remarkably increased compared to the activity in cells treated with LPS alone (Fig. 3B).

Next we investigated whether the MAPK signaling pathways are involved in the upregulatory effect of 15d-PGJ₂ in SHR VSMCs. After VSMCs were untreated (NT) or pretreated with PD98059 (ERK inhibitor, 10 µM, 4A), or PD169316 (p38 inhibitor, 10 µM, 5A) for 30 min, cells were untreated or treated with LPS (1 µg/ml) and/or 15d-PGJ₂ (10 µM) for 4 h. Real-time PCR was then performed on these treated cells. In addition, these results were further confirmed by investigating the phosphorylation of MAP kinases in VSMCs that had been treated with 15d-PGJ₂/LPS. The expression of 15d-PGJ₂/LPS-induced IL-8/CXCL8 mRNA was decreased by the ERK inhibitor PD98059. And the expression of LPS alone-induced IL-8/CXCL8 mRNA was also decreased by PD98059 (Fig. 4A). However, although ERK phosphorylation in cells treated 15d-PGJ₂ alone was not detected, a remarkable increase in ERK phosphorylation in VSMCs that were treated with 15d-PGJ₂/LPS relative to VSMCs that were treated with LPS alone was also detected (Fig. 4B). PD169316 increased the IL-8/CXCL8 mRNA expression in VSMCs stimulated with 15d-PGJ₂/LPS, rather than inhibiting IL-8/CXCL8 expression (Fig. 5A). Moreover, 15d-PGJ₂ decreased LPS-induced p38 phosphorylation (Fig. 5B). More specifically, blocking p38 phosphorylation caused an increase in 15d-PGJ₂/LPS-induced IL-8/CXCL8 expression. To confirm this result, real-time PCR using p38-directed small interfering RNA (siRNA) was performed. In this experiment we found that while LPS inhibited the expression of IL-8/CXCL8 mRNA, 15d-PGJ₂ induced IL-8/CXCL8 expression and 15d-PGJ₂/LPS increased IL-8/CXCL8 mRNA expression in p38 siRNA transfected SHR VSMCs (Fig. 5C).

VSMCs generate reactive oxygen species (ROS), which play an important role in the pathogenesis of hypertensive vascular injury. A major source of ROS is NAD(P)H oxidase. Therefore, we investigated whether NAD(P)H oxidase activity is related to the upregulatory effect of 15d-PGJ₂ on LPS-induced IL-8/CXCL8 expressions in SHR VSMCs.

Real-time PCR was performed on VSMCs SHR after they were untreated or treated with LPS (1 µg/ml) and/or 15d-PGJ₂ (10 µM) in the absence or presence of flavin containing oxidase inhibitor, DPI (10 µM) for 4 h. DPI remarkably decreased the expression of 15d-PGJ₂/LPS-induced IL-8/CXCL8 mRNA; thus, blocking the upregulatory effects of 15d-PGJ₂ on LPS-induced IL-8/CXCL8 expression (Fig. 6A). To support these results, the ability of 15d-PGJ₂/LPS to induce NAD(P)H oxidase activity was examined in SHR VSMCs. ROS generation in SHR VSMCs was measured by flow cytometric analysis of DCF-DA-stained VSMCs. DCF-DA fluorescence can be also used as a measurement of NAD(P)H oxidase activity. From this analysis, SHR VSMCs treated with 15d-PGJ₂/LPS was shown to increase DCF-DA fluorescence slightly compared to those treated with LPS alone (Fig. 6B).