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15-Deoxy-Δ12,14-Prostaglandin J2 Upregulates the Expression of LPS-Induced IL-8/CXCL8 mRNA in Vascular Smooth Muscle Cel s from Spontaneously Hypertensive RatsJung Hae Kim and Hee Sun Kim*Department of Microbiology, College of Medicine, and Aging-associated Vascular Disease Research Center, Yeungnam University, Background: 15d-PGJ2 has been known to act as an anti-in- flammatory agent and has anti-hypertensive effects. As a re-sult of these properties, we examined the effect of 15d-PGJ2 Infiltration of inflammatory cells and oxidative stresses in vas- on the LPS-induced IL-8/CXCL8 mRNA expression in cular walls have been shown to contribute to the patho- VSMCs from SHR. Methods: Effect and action mechanism genesis of hypertension (1-4), and monocytes/macrophages of 15d-PGJ2 on the expression of LPS-induced IL-8/CXCL8 infiltration and the proliferation of VSMCs and endothelial mRNA in VSMCs from SHR and WKY were examined by us-ing real-time polymerase chain reaction, electrophoretic mo- cells in arterial walls are mediated by chemokines (5,6). bility shift assay for NF-κB avtivity, Western blotting analy- Chemokine IL-8/CXCL8 is known to play an important role sis for ERK and p38 phosphorylation and flow cytometry for in the migration of monocytes into the subendothelial space NAD(P)H oxidase activity. Results: 15d-PGJ in the early phase of atherosclerosis, and along with expression of LPS-induced IL-8/CXCL8 mRNA in WKY MCP-1/CCL2, plays an important role in the pathogenesis of VSMCs, but increased the expression of LPS-induced atherosclerosis (7). In addition, elevated levels of IL-8/CXCL8 IL-8/CXCL8 mRNA in SHR VSMCs. The upregulatory effect are associated with an increased risk of future coronary artery of 15d-PGJ2 in SHR VSMCs was mediated through PPARγ, disease (8). IL-8/CXCL8 may directly enhance membrane per- and dependent on NF-κB activation and ERK pho- meability to Ca2＋; thus, inducing vasoconstriction in the sphorylation. However, inhibition of the p38 signaling path- smooth muscle cells (9). Moreover, we have previously way augmented the upregulatory effect of 15d-PGJ2 on shown that expression of IL-8/CXCL8 in SHR VSMCs is stron- LPS-induced IL-8/CXCL8 mRNA. A NAD(P)H oxidase in- ger than in WKY VSMCs (10). Therefore, it has been sug- hibitor inhibited the upregulatory effect of 15d-PGJ2 on LPS- induced IL-8/CXCL8 mRNA expression in SHR VSMCs, and gested that IL-8/CXCL8 is also involved in the pathogenesis an increase in NAD(P)H oxidase activity was detected in and maintenance of hypertensive vascular wall formation in SHR VSMCs treated with 15d-PGJ 2/LPS. Conclusion: Our results indicate that the upregulatory effect of 15d-PGJ PPAR ligands have been known to reduce systemic blood LPS-induced IL-8/CXCL8 expression in SHR VSMCs is pressure (11-13), increase production of a potent endogenous mediated through the PPARγ and ERK pathway, and may be vasodilator, NO and act as an anti-inflammatory agent (14,15). related to NAD(P)H oxidase activity. However, p38 in- 15d-PGJ2, a metabolite of prostaglandin PGD2, is a natural activation may also play an important role in 15d-PGJ2/ and high affinity ligand for PPARγ (16). It is known to pro- LPS-induced IL-8/CXCL8 expression in SHR VSMCs.
duce anti-hypertensive effects, such as inhibiting cell migra- [Immune Network 2009;9(2):64-73] tion and proliferation in rat and human VSMCs, and stimulat-ing HO-1 expression in rat VSMCs (17-19). 15d-PGJ2 also in-hibits the production of inflammatory mediators (TNF-α, Received on January 28, 2009. Revised on February 23, 2009. Accepted on March 4, 2009.
*Corresponding Author. Tel: 82-53-620-4363; Fax: 82-53-653-6628; E-mail: [email protected] Keywords: 15d-PGJ2, IL-8/CXCL8, vascular smooth muscle cells IMMUNE NETWORK http://www.ksimm.or.kr Volume 9 Number 2 April 2009 15d-PGJ2 Upregulates IL-8/CXCL8 Expression in SHR VSMCs Jung Hae Kim and Hee Sun Kim IL-6, IL-1β, IL-2, IP-10, MCP-1, gelatinase B, and cyclo- Experimental animal oxygenase-2) and reduces the expression of iNOS (15,20-22). Specific pathogen-free male inbred WKY and SHR, 20 to 30 As a result of these effects, 15d-PGJ2 has been suggested to weeks of age, were purchased from Japan SLC Inc. be a potential therapeutic compound for use as an anti-in- (Shizuoka, Japan). All experimental animals received auto- flammatory agent. However, there are also evidences that claved food and bedding to minimize exposure to viral or 15d-PGJ2 can promote inflammation (23-26). Thus, the role microbial pathogens. The rats were cared for in accordance and effects of 15d-PGJ2 on inflammation is complex and re- with the Guide for the Care and Use of Experimental Animals mains controversial. Moreover, the precise role of 15d-PGJ2 of Yeungnam Medical Center.
in hypertensive VSMCs is not yet fully understood. Therefore, the aim of this study is to investigate the action VSMCs preparation mechanism of 15d-PGJ2 on LPS-induced IL-8/CXCL8 expre- VSMCs were obtained from the thoracic aortas of 20- to ssion in VSMCs from SHR. 30-week-old male WKY and SHR as described previously (25). VSMCs were cultured in Dulbecco's modified Eagle's MATERIALS AND METHODS 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 Trizol reagent, lipofectamine 2000 and rat p38 siRNA oligom- a density of 105 cells per ml. All experiments were conducted ers were purchased from Invitrogen (Carlsbad, CA). Dulbec- at cell passage 3 to 7. Prior to stimulation, 95% confluent co's phosphate-buffered saline (PBS), Dulbecco's modified VSMCs were serum-starved overnight by incubating in DMEM Eagle's medium (DMEM), penicillin-streptomycin and fetal with 0.1% FBS. Cell cultures were incubated in a humidified bovine serum (FBS) were purchased from Gibco/BRL (Life incubator at 37oC and 5% CO2 in the presence or absence Technologies, Gaithersburg, MD). 15d-PGJ2 and GW9662 of stimuli for the indicated times.
were purchased from Biomol (Plymouth Meeting, PA). Escherichia coli LPS (O111:B4), diphenyleneiodonium chlor- Preparation of total RNA and real-time polymerase ide (DPI), dithiothreitol (DTT), phenylmethylsulfonyl fluoride chain reaction (real-time PCR) (PMSF), pepstatin, leupeptin, autipain, and aprotinin were ob- Total RNA was extracted using a Trizol reagent in accordance tained from Sigma Chemical Co. (St. Louis, MO). MAPK in- with the manufacturer's instructions. The quantity of total hibitors, 2'-amino-3'methoxyflavone (PD98059), 4-(4-fluorophenyl)- RNA obtained was determined by measuring optical density 2-(4-nitrophenyl)-5-(4-pyridyl)-1H-imidazole (PD169316), and (OD) at 260 and 280 nm. Real-time PCR for the amplification of IL-8/CXCL8 and PPARγ in VSMCs was performed using nitrile (Bay 11-7082) were purchased from Calbiochem (San a LightCycler (Roche). RNA was reverse transcribed to cDNA Diego, CA). Dichlorofluorescein diacetate (DCF-DA) was ob- from 1 μg of total RNA, and then subjected to real-time PCR. tained from Molecular probes (Eugene, OR). Nitrocellulose PCR was performed in triplicate. The total PCR volume was transfer membranes were obtained from Schleicher & Schuell 20 μl and contained the LightCycler FastStart DNA SYBR Bioscience (Dassel, Germany). [α-32P]dCTP was purchased Green I mix (Roche), appropriate primer and 2 μl of cDNA. from Dupont-New England Nuclear (Boston, MA). Oligonu- Prior to PCR amplification, the mixture was incubated at 95oC cleotide primers for polymerase chain reaction (PCR) of for 10 min, and the amplification step consisted of 45 cycles IL-8/CXCL8, PPAR and β-actin were synthesized by Bionics of denaturation (10 s at 95oC), annealing (5 s at the pri- (Seoul, Korea). The LightCycler FastStart DNA SYBR Green mer-appropriate temperature), and extension (10 s at 72oC) I Mix was obtained from Roche (Mannheim, Germany). with fluorescence detection at 72oC after each cycle. After the Anti-NF-κB, Phospho-ERK and phospho-p38 antibodies were final cycle, melting point analyses of all samples were per- obtained from Cell Signaling Technology (Danvers, MA). The formed over the range of 65 to 95oC with continuous fluo- γ-tubulin antibody was obtained from Sigma Chemical Co. rescence detection. β-actin expression levels were used for (St Louis, MO). All other reagents were from pure-grade com- 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 IMMUNE NETWORK http://www.ksimm.or.kr Volume 9 Number 2 April 2009 15d-PGJ2 Upregulates IL-8/CXCL8 Expression in SHR VSMCs Jung Hae Kim and Hee Sun Kim bp) sense, 5'-gaagatagattgcaccga-3'; antisense, 5'-catagcctctca- Seoul, Korea). Protein concentrations were determined by a cacatttc-3', for PPARγ (359 bp): sense, 5'-tgaggagaagtca- Bradford assay using bovine serum albumin as a standard. cactctg-3'; antisense, 5'-tgggtcagctcttgtgaatg-3' and for β-actin Thirty-micrograms of the protein samples were separated on (101 bp): sense, 5'-tactgccctggctcctagca-3'; antisense, 5'-tgga- 10% SDS-polyacrylamide gels, and then transferred to nitro- cagtgaggccaggatag-3'. The level of IL-8/CXCL8 mRNA was de- cellulose membranes. The membranes were soaked in 5% termined by comparing experimental levels to the standard nonfat dried milk in TBST (10 mmol/l Tris-HCl pH 7.5, 150 curves and was expressed as the fold of relative expression.
mmol/l NaCl and 0.05% Tween-20) for 1 h and then in-cubated for 16∼18 h with primary antibodies against phos- Electrophoretic mobility shift assay (EMSA) pho-ERK, phospho-p38 and γ-tubulin at 4oC. Membranes Nuclear extracts were prepared as described previously (25). were washed three times with TBST for 10 min and then in- Cells were washed three times with cold PBS, then scraped cubated with horseradish peroxidase-conjugated secondary and harvested by centrifugation. Cell pellets were re- antibody for 1 h at 4oC. After incubation with the secondary suspended and incubated on ice for 15 min in 400 μl of antibody, the membranes were rinsed three times with TBST hypotonic buffer A (10 mmol/l HEPES, 10 mmol/l KCl, 1.5 for 10 min and antigen-antibody complex was detected using mmol/l MgCl2, 0.5 mmol/l DTT, 0.1 mmol/l PMSF, 10 μg/ml an enhanced chemiluminescence detection system (LAS-3000, pepstatin, 10 μg/ml leupeptin, 10 μg/ml autipain, and 10 Fujifilm, Tokyo, Japan).
μg/ml aprotinin). Nonidet P-40 was then added to a final concentration of 2.5%, and the cells were vortexed for 10 s. Small interfering RNA Nuclei were separated from the cytosol by centrifugation at To confirm whether the p38 pathway contributes to the in- 12,000 g for 15 s. Pel ets were resuspended in 40 μl of hypo- hibitory effect of 15d-PGJ2 on LPS-induced IL-8/CXCL8 ex- tonic buffer C (20 mmol/l HEPES, 25% glycerol, 0.4 mol/l pression, p38 expression was silenced using small interfering NaCl, 1 mmol/l EDTA, 1 mmol/l EGTA, 0.5 mmol/l DTT, 0.1 RNA (siRNA). VSMCs were plated on 24-well plates and mmol/l PMSF, 10 μg/ml pepstatin, 10 μg/ml leupeptin, 10 grown to 90% confluence. VSMCs were then transfected with μg/ml autipain, and 10 μg/ml aprotinin). Samples were so- p38 siRNA oligomers (20 nmol/l) using lipofectamine 2000 in nicated at level 3-4 for 2-3 s, followed by centrifugation for accordance with the manufacturer's instructions. After 24 h 10 min at 4oC. The nuclear protein concentration was de- of incubation, VSMCs were placed in growth medium for 24 termined using the Bradford assay (Bio-Rad, Richmond, CA). h before the experiments. Cells were then cultured in the A consensus sequence for the NF-κB DNA binding site (5'- presence or absence of stimuli for 4 h. Sense and antisense AGTTGAGGGGACTTTAGGC-3') (sc-2505; Santa Cruz Bio- oligonucleotides corresponding to the rat p38 siRNA se- technology, Santa Cruz, CA) was labeled with [α-32P]dCTP quence: sense, 5'-uacauuugcgaaguucaucuucggc-3'; antisense, using a random-primed DNA labeling kit (Roche). The mutant 5'-gccgaagaugaacuucgcaaaugua-3' was purchased from Invi- NF-κB binding sequence was identical to sc-2505 except for trogen (Carlsbad, CA). a "G" → "C" substitution in the NF-κB DNA binding motif (sc-2511; Santa Cruz Biotechnology). DNA The labeled DNA Flow cytometry for ROS generation was purified over an S-200 HR column (Pharmacia, Piscataway, ROS production was measured using flow cytometric analysis NJ) to remove unbound nucleotides. Nuclear protein extracts of DCF-DA-stained cells. In brief, VSMCs were grown to 70% were incubated at room temperature for 20 min with approx- confluence in serum-enriched DMEM at 37oC in 5% CO2. The imately 50,000 cpm of labeled oligonucleotides that were sus- medium was then replaced with serum-free DMEM and the pended in the binding buffer [200 mmol/l HEPES, 500 mmol/l cells were cultured in the presence or absence of stimuli for KCl, 10 mmol/l EDTA, 50% glycerol, 10 mmol/l DTT, 1 the indicated times. Cells were incubated in the dark with mg/ml BSA, 1 μg/μl poly (dI-dC)]. Following this in- DCF-DA (50 μmol/l) for 1 h at 37oC, scraped, and re- cubation, samples were resolved on 4% polyacrylamide gels suspended in PBS. Fluorescence was monitored using flow at 140 V and exposed to film.
cytometry (FACScan, Becton Dickison). Statistical analysis Total lysates were prepared in a PRO-PREP buffer (iNtRON, Results are expressed as means±SD from at least three or IMMUNE NETWORK http://www.ksimm.or.kr Volume 9 Number 2 April 2009 15d-PGJ2 Upregulates IL-8/CXCL8 Expression in SHR VSMCs Jung Hae Kim and Hee Sun Kim four independent experiments. For comparison between mul- nificantly greater than those in the cel s treated with LPS alone tiple groups, statistical significance was determined by the from the 4 h period (Fig. 1B). Mann-Whitney test using the SPSS version 12.0.
Action mechanisms of 15d-PGJ2 on LPS-induced IL-8/CXCL8 expression in SHR VSMCs It is widely accepted that 15d-PGJ2 exerts its effects on pro-in- Effect of 15d-PGJ2 on the LPS-induced IL-8/CXCL8 flammatory genes in cells through PPARγ dependent or expression in SHR VSMCs PPARγ independent mechanisms (27-29). Before evaluating We examined the differential effect of LPS on IL-8/CXCL8 whether the mechanism of the upregulatory action of mRNA expression in SHR VSMCs in comparison to WKY 15d-PGJ2 in SHR VSMCs was PPARγ-dependent, we de- VSMCs. From this experiment, we found that the expression termined the expression pattern of PPARγ mRNA in SHR of LPS-induced IL-8/CXCL8 mRNA was greater in SHR VSMCs VSMCs treated with 15d-PGJ2/LPS. There was no meaningful than WKY. Real-time PCR was performed on VSMCs after difference between the level of PPARγ expression induced they were untreated (NT) or treated with LPS (1 μg/ml), 15d- by 15d-PGJ2/LPS and that induced by 15d-PGJ2 alone (Fig. PGJ2 (10 μM) or LPS plus 15d-PGJ2 simultaneously (15d-PGJ2/ 2A). To evaluate whether the upregulatory effect of 15d-PGJ2 LPS) for 4 h. 15d-PGJ2 treatment had different effects on SHR in SHR VSMCs is mediated by PPARγ, the effect of GW9662, VSMCs relative to WKY VSMCs, where 15d-PGJ2 had upregu- a PPARγ antagonist, was tested in SHR VSMCs. GW9662 latory effect on LPS-induced IL-8/CXCL8 mRNA expression in blocked LPS-induced IL-8/CXCL8 mRNA expression at the SHR VSMCs and suppressive effect on LPS-induced IL-8/CXCL8 dose of 10 μM. And, although 10 μM of GW9662 did not mRNA expression in WKY VSMCs. 15d-PGJ2 alone did not block the upregulatory effect of 15d-PGJ2 on LPS-induced induce IL-8/CXCL8 mRNA expression in SHR VSMCs sig- IL-8/CXCL8 mRNA expression, the high doses (40 and 100 μM) nificantly (Fig. 1A). The time course of 15d-PGJ2/LPS-induced of GW9662 inhibited the upregulatory effect of 15d-PGJ2 on IL-8/CXCL8 mRNA expression was determined in SHR VSMCs LPS-induced IL-8/CXCL8 mRNA expression (Fig. 2B). over a 0 to 8 h time period. In this experiment, we found To further understand the nature of the upregulatory effect that the expression of IL-8/CXCL8 mRNA induced by 15d-PGJ2/ of 15d-PGJ2 on LPS-induced IL-8/CXCL8 expressions in SHR LPS was almost the same as that for cells treated with LPS VSMCs, the role of NF-κB activation was investigated. alone until 2 h after treatment. However, the expression lev- Bay11-7082 is known to selectively block the phosphorylation els of IL-8/CXCL8 mRNA induced by 15d-PGJ2/LPS were sig- of IκB-α; thus, preventing the activation and nuclear trans- Figure 1. Effect of 15d-PGJ2 on the expression of LPS-induced IL-8/CXCL8 mRNA in VSMCs from SHR and WKY, and the time course of
15d-PGJ2/LPS-induced IL-8/CXCL8 mRNA expression in SHR VSMCs. (A) VSMCs were untreated (NT) or treated with LPS (1 μg/ml) or/and 15d- PGJ2 (10 μM) for 4 h, and the total RNA was analyzed by real-time PCR. Bars represent means±SD from three independent experiments. *p＜0.05 vs. VSMCs treated with LPS alone. (B) SHR VSMCs were untreated (NT) or treated with LPS (1 μg/ml) or LPS plus 15d-PGJ2 (10 μM) simul- taneously (15d-PGJ2/LPS) for the indicated times and the total RNA was analyzed by real-time PCR. Bars represent means±SD from three IMMUNE NETWORK http://www.ksimm.or.kr Volume 9 Number 2 April 2009 15d-PGJ2 Upregulates IL-8/CXCL8 Expression in SHR VSMCs Jung Hae Kim and Hee Sun Kim Figure 3. Upregulatory effect of 15d-PGJ2 on LPS-induced IL-8/CXCL8
Figure 2. Upregulatory effect of 15d-PGJ2 on LPS-induced IL-8/CXCL8
mRNA expression is dependent on NF-κB activation in SHR VSMCs.
mRNA expression is dependent on the PPARγ pathway in SHR (A) VSMCs were untreated or treated with LPS (1 μg/ml) and/or VSMCs. (A) VSMCs were untreated (NT) or treated with LPS (1 μg/ml) 15d-PGJ2 (10 μM) in the absence or presence of Bay11-7082 (10 μ and/or 15d-PGJ2 (10 μM) for 4 h, and the total RNA was analyzed M) for 4 h. Bars represent means±SD from three independent by real-time PCR. Bars represent means±SD from three independent real-time PCR experiments. a: p＜0.05 vs. VSMCs treated with LPS experiments. (B) VSMCs were untreated or treated with LPS (1 μg/ml) alone. b: p＜0.05 vs. VSMCs treated with 15d-PGJ2/LPS. (B) Specific and/or 15d-PGJ2 (10 μM) in the absence or presence of GW9662 (10, binding activity of NF-κB from nuclear extracts was assessed by 40, and 100 μM) for 4 h. Bars represent means±SD from three EMSA. Aliquots of the nuclear extract were incubated with a 100-fold independent experiments. *p＜0.05 vs. VSMCs treated with 15d-PGJ2/ excess of the mutant probe (m) or with 2 μg of the anti NF-κB antibody before EMSA. Data shown are representative of four location of NF-κB. Real-time PCR and EMSA were performed on VSMCs after they were untreated or treated with LPS (1 PGJ2/LPS-induced IL-8/CXCL8 mRNA was decreased by the μg/ml) and/or 15d-PGJ2 (10 μM) in the absence or pres- ERK inhibitor PD98059. And the expression of LPS alone-in- ence of Bay11-7082 (10 μM) for 4 h. Bay11-7082 remarkably duced IL-8/CXCL8 mRNA was also decreased by PD98059 blocked the upregulatory effect of 15d-PGJ2 on LPS-induced (Fig. 4A). However, although ERK phosphorylation in cells IL-8/CXCL8 mRNA expression. And it also remarkably blocked treated 15d-PGJ2 alone was not detected, a remarkable in- LPS-induced IL-8/CXCL8 expression (Fig. 3A). However, in crease in ERK phosphorylation in VSMCs that were treated spite of negative NF-κB activity in cells treated 15d-PGJ2 with 15d-PGJ2/LPS relative to VSMCs that were treated with alone, NF-κB activity in SHR VSMCs treated with15d-PGJ2/ LPS alone was also detected (Fig. 4B). PD169316 increased LPS was remarkably increased compared to the activity in the IL-8/CXCL8 mRNA expression in VSMCs stimulated with cells treated with LPS alone (Fig. 3B). 15d-PGJ2/LPS, rather than inhibiting IL-8/CXCL8 expression Next we investigated whether the MAPK signaling path- (Fig. 5A). Moreover, 15d-PGJ2 decreased LPS-induced p38 ways are involved in the upregulatory effect of 15d-PGJ2 in phosphorylation (Fig. 5B). More specifically, blocking p38 SHR VSMCs. After VSMCs were untreated (NT) or pretreated phosphorylation caused an increase in 15d-PGJ2/LPS-induced with PD98059 (ERK inhibitor, 10 μM, 4A), or PD169316 (p38 IL-8/CXCL8 expression. To confirm this result, real-time PCR inhibitor, 10 μM, 5A) for 30 min, cells were untreated or using p38-directed small interfering RNA (siRNA) was treated with LPS (1 μg/ml) and/or 15d-PGJ2 (10 μM) for performed. In this experiment we found that while LPS in- 4 h. Real-time PCR was then performed on these treated cells. hibited the expression of IL-8/CXCL8 mRNA, 15d-PGJ2 in- In addition, these results were further confirmed by inves- duced IL-8/CXCL8 expression and 15d-PGJ2/LPS increased tigating the phosphorylation of MAP kinases in VSMCs that IL-8/CXCL8 mRNA expression in p38 siRNA transfected SHR had been treated with 15d-PGJ2/LPS. The expression of 15d- VSMCs (Fig. 5C). IMMUNE NETWORK http://www.ksimm.or.kr Volume 9 Number 2 April 2009 15d-PGJ2 Upregulates IL-8/CXCL8 Expression in SHR VSMCs Jung Hae Kim and Hee Sun Kim Figure 4. Upregulatory effect of 15d-PGJ2 on LPS-induced IL-8/CXCL8
expression is mediated through the ERK pathway in SHR VSMCs. (A) VSMCs were untreated (NT) or pretreated with PD98059 (ERK inhibitor, 10 μM) for 30 min, and then untreated or treated with LPS (1 μg/ml) and/or 15d-PGJ2 (10 μM) for 4 h. Real-time PCR was performed after total mRNAs were isolated. Bars represent means±SD from three independent experiments. a: p＜0.05 vs. VSMCs treated with LPS alone. b: p＜0.05 vs. VSMCs treated with 15d-PGJ2/LPS. (B) Figure 5. Blocking of p38 phosphorylation increased the expression of
Cell lysates were separated on 10% SDS-polyacrylamide gels and then 15d-PGJ2/LPS-induced IL-8/CXCL8 mRNA expression in SHR VSMCs. immunoblotted with the phospho-ERK antibody. The data shown are (A) VSMCs were untreated (NT) or pretreated with PD169316 (p38 representative of three independent experiments.
inhibitor, 10 μM) for 30 min, and then untreated or treated with LPS (1 μg/ml) and/or 15d-PGJ2 (10 μM) for 4 h. Real-time PCR was per- formed after total mRNAs were isolated. Bars represent means±SD from three independent experiments. *p＜0.05 vs. VSMCs treated with Effect of NAD(P)H oxidase activity on 15d-PGJ2/LPS- 15d-PGJ2/LPS. (B) Cell lysates were separated on 10% SDS-poly- acrylamide gels and then immunoblotted with the phospho-p38 anti- induced IL-8/CXCL8 expression in SHR VSMCs body. Data shown are representative of four independent experiments.
VSMCs generate reactive oxygen species (ROS), which play (C) VSMCs were plated on 24-well plates, grown to 90% confluence and then transfected with p38 siRNA oligomers (20 nmol/l). VSMCs an important role in the pathogenesis of hypertensive vas- were then untreated or treated with LPS (1 μg/ml) and/or 15d-PGJ2 cular injury. A major source of ROS is NAD(P)H oxidase. (10 μM) for 4 h. Bars represent means±SEM from three independent Therefore, we investigated whether NAD(P)H oxidase activity is related to the upregulatory effect of 15d-PGJ2 on LPS-in-duced IL-8/CXCL8 expressions in SHR VSMCs. From this analysis, SHR VSMCs treated with 15d-PGJ2/LPS was Real-time PCR was performed on VSMCs SHR after they shown to increase DCF-DA fluorescence slightly compared to were untreated or treated with LPS (1 μg/ml) and/or 15d- those treated with LPS alone (Fig. 6B).
PGJ2 (10 μM) in the absence or presence of flavin containing oxidase inhibitor, DPI (10 μM) for 4 h. DPI remarkably de- creased the expression of 15d-PGJ2/LPS-induced IL-8/CXCL8 mRNA; thus, blocking the upregulatory effects of 15d-PGJ2 on In relation to IL-8/CXCL8 expression, 15d-PGJ2 was shown to LPS-induced IL-8/CXCL8 expression (Fig. 6A). To support have pro-inflammatory effects in SHR VSMCs and anti-in- these results, the ability of 15d-PGJ2/LPS to induce NAD(P)H flammatory effects in WKY VSMCs. Wakino et al. (30) also oxidase activity was examined in SHR VSMCs. ROS gen- observed similar differential effects of PPARγ ligands on vas- eration in SHR VSMCs was measured by flow cytometric anal- cular tissues from SHR and WKY. They demonstrated that ysis of DCF-DA-stained VSMCs. DCF-DA fluorescence can be pioglitazone reduces the stimulated Rho-kinase activity in the also used as a measurement of NAD(P)H oxidase activity. vascular tissue from SHR, but not WKY. Troglitazone mark- IMMUNE NETWORK http://www.ksimm.or.kr Volume 9 Number 2 April 2009 15d-PGJ2 Upregulates IL-8/CXCL8 Expression in SHR VSMCs Jung Hae Kim and Hee Sun Kim In the EMSA, an increase in NF-κB activity in SHR VSMCs treated with 15d-PGJ2/LPS relative to VSMCs treated with LPS alone was detected, and the expression of 15d-PGJ2/LPS-in-duced IL-8/CXCL8 mRNA was decreased by the presence of Bay11-7082. These results indicate that the upregulatory effect of 15d-PGJ2 in SHR VSMCs is dependent on NF-κB acti-vation. It is widely accepted that 15d-PGJ2 exerts its effects on inflammatory mediated-genes in cells by either inhibiting or activating NF-κB signaling (24,26,33-35). The anti-in-flammatory activity of 15d-PGJ2 has been shown to be medi-ated mainly through the inhibition of NF-κB activation (33, 34), but 15d-PGJ2 has also been shown to upregulate IL-8/ CXCL8 and MIP-2/CXCL2 expression through NF-κB activa-tion (24,26).
Among the MAPK signaling pathways, the ERK pathway is known to be associated with the stimulatory activity of 15d- PGJ2 on the expression of some cytokine genes (23,24,36). 15d-PGJ2 induces the rapid activation of the ERK pathway in Figure 6. Activity of NAD(P)H oxidase mediates the upregulatory
VSMCs (37). We also detected an increase in ERK phosphor- effect of 15d-PGJ2 on the expression of LPS-induced IL-8/CXCL8 mRNA ylation in SHR VSMCs treated with 15d-PGJ2/LPS. Up-regu- in SHR VSMCs. (A) VSMCs were untreated or treated with LPS (1 μg/ ml) and/or 15d-PGJ lation of LPS-induced IL-8/CXCL8 expression by 15d-PGJ2 was 2 (10 μM) in the absence or presence of DPI (10 μM) for 4 h. Bars represent means±SD from three independent mediated through the ERK signaling pathway in SHR VSMCs. experiments. *p＜0.05 vs. VSMCs treated with 15d-PGJ2/LPS. (B) VSMCs were untreated or treated with LPS (1 Inhibition or activation by 15d-PGJ μg/ml) and/or 15d-PGJ 2 on p38 MAP kinase ap- (10 μM) for 4 h, stained with DCF-DA (50 μM) for ROS detection, pears to be target gene-, cell type- and stimulation con- and subjected to flow cytometry. Bars represent means±SD from four independent experiments. *p dition-dependent (19,38,39). Activation of the p38 MAP kin- ＜0.05 vs. VSMCs treated with LPS ase has been shown to be involved in 15d-PGJ2-induced HO-1 expression (19) and in IL-1β-induced IL-8/CXCL8 gene ex-pression in human VSMCs (38). However, 15d-PGJ2 is known edly decreased the expression of TGFβ-1, PDGF, or bFGF to inhibit IL-1-induced p38 MAP kinase expression in human mRNAs in SHR VSMCs, but not in WKY VSMCs (12). Therefore, astrocytes (39). In this study, blocking p38 phosphorylation these different effects of PPAR ligands on SHR and WKY increased 15d-PGJ2/LPS-induced IL-8/CXCL8 expression in VSMCs indicate that diverse, complex pathways mediate the SHR VSMCs. 15d-PGJ2 inhibited p38 phosphorylation in SHR action of PPARg ligands on hypertension. VSMCs treated with LPS. Furthermore, while the expression The upregulatory effect of 15d-PGJ2 on LPS-induced of LPS-induced IL-8/CXCL8 mRNA was abolished in SHR IL-8/CXCL8 expression was mediated through PPARg in SHR VSMCs that were transfected with p38 siRNA, 15d-PGJ2/LPS- VSMCs. 15d-PGJ2 and other cyclopentenone prostaglandin, induced IL-8/CXCL8 expression increased. Therefore, LPS-in- such as prostaglandin A1 (PGA1) are known to exert effects duced IL-8/CXCL8 mRNA expression appears to be related to on cytokine genes through PPARγ-dependent and PPARγ- p38 activation, but 15d-PGJ2 itself induces IL-8/CXCL8 mRNA independent mechanisms (25,26,28,29,31,32). In our previous expression without p38 activation. These combined results in- studies (25,26), the upregulatory effect of 15d-PGJ2 on LPS-in- dicate that the upregulatory effect of 15d-PGJ2 on LPS-induced duced MIP-2/CXCL2 and KC/CXCL1 gene expression was IL-8/CXCL8 expression is related to p38 inactivation. Although found to not mediated by the PPARγ pathway, but the effect the upregulatory effect of 15d-PGJ2 on LPS-induced IL-8/ of PGA1 on LPS-induced IL-10 expression was dependent on CXCL8 expression is mediated by the ERK pathway, an un- PPARγ in mouse peritoneal macrophages. In a separate VSMCs known mechanism via p38 inactivation may play an im- study, 15d-PGJ2-induced HO-1 expression was shown to be portant role in 15d-PGJ2/LPS-induced IL-8/CXCL8 expression independent of PPARγ (19).
in SHR VSMCs.
IMMUNE NETWORK http://www.ksimm.or.kr Volume 9 Number 2 April 2009 15d-PGJ2 Upregulates IL-8/CXCL8 Expression in SHR VSMCs Jung Hae Kim and Hee Sun Kim NAD(P)H oxidase is a known major source of reactive oxy- atherosclerosis. Oxidative stress and the mediation of arte- gen species (ROS). ROS are not only harmful cellular metabo- rial inflammatory response: a new perspective. Hyperten-sion 25;155-161, 1995 lites but are also essential molecules in cell signaling and reg- 2. Capers Q 4th, Alexander RW, Lou P, De Leon H, Wilcox ulation (40). Excessive ROS generation by NAD(P)H oxidase JN, Ishizaka N, Howard AB, Taylor WR: Monocyte chemo- is known to play an important role in the pathogenesis of attractant protein-1 expression in aortic tissues of hyper- hypertensive vascular injury and has also been implicated in tensive rats. Hypertension 30;1397-1402, 1997 3. Rodríguez-Iturbe B, Vaziri ND, Herrera-Acosta J, Johnson the pathogenesis of hypertension (1,41-44). Cruzado et al. RJ: Oxidative stress, renal infiltration of immune cells and (41) demonstrated that ROS generation was enhanced in SHR salt-sensitive hypertension: all for one and one for all. Am VSMCs during the development of hypertension. Therefore, J Physiol 286;F606-F616, 2004 4. Zhang Y, Griendling KK, Dikalova A, Owens GK, Talyor we examined the effect of NAD(P)H oxidase activity on IL-8/ WR: Vascular hypertrophy in angiotensin II-induced hyper- CXCL8 expression in SHR VSMCs treated with 15d-PGJ2/LPS. tension is mediated by vascular smooth muscle cell-derived An inhibitor of the flavin-containing oxidases, DPI, remark- H2O2. Hypertension 46;732-737, 2005 ably decreased the expression of 15d-PGJ 5. Gerszten RE: Pleiotropic effects of chemokines in vascular 2/LPS-induced IL-8/ lesion development. Artherioscler Thromb Vasc Biol 22; CXCL8 mRNA. Although a significant production of ROS by 15d-PGJ2 has been reported in Sprague-Dawley rat VSMCs 6. Luster AD: Chemokines - chemotactic cytokines that medi- (19) and ROS generation by LPS alone was detected in WKY ate inflammation. N Engl J Med 338;436-445, 1998 VSMCs (data not shown), ROS generation did not increase in 7. Gerszten RE, Garcia-Zepeda EA, Lim YC, Yoshida M, Ding HA, Gimbrone MA, Luster AD, Luscinskas FW, Rosenzweig SHR VSMCs after treatment with 15d-PGJ2 or LPS alone. A: MCP-1 and IL-8 trigger firm adhesion of monocytes to However, 15d-PGJ2/LPS did increase ROS generation in SHR vascular endothelium under flow conditions. Nature 398; VSMCs. These results suggest that the upregulatory effect of 8. Boekholdt S, Peters R, Hack CE, Day NE, Luben R, 15d-PGJ2 on LPS-induced IL-8/CXCL8 expression in SHR Bingham SA, Wareham NJ, Reitsma PH, Khaw K: IL-8 plas- VSMCs may be related to NAD(P)H oxidase activity.
ma concentrations and the risk of future coronary artery In conclusion, this is the first study to report on the upregu- disease in apparently healthy men and women: the latory effect of 15d-PGJ EPIC-Norfolk prospective population study. Arterioscler 2 on LPS-induced IL-8/CXCL8 gene ex- Thromb Vasc Biol 24;1503-1508, 2004 pression in SHR VSMCs and the inhibitory effect in WKY 9. Buemi M, Marino D, Floccari F, Ruello A, Nosto L, Aloisi VSMCs. In addition, we showed that the upregulatory effect C, Marino MT, Di Pasquale G, Corica F, Frisina M: Effect of interleukin 8 and ICAM-1 on calcium-dependent outflow 2 in SHR VSMCs is mediated through PPARγ path- of K+ in erythrocytes from subjects with essential hyper- way, NF-κB and ERK activation, and p38 inactivation may tension. Curr Med Res Opin 20;19-24, 2004 play an important role in 15d-PGJ2/LPS-induced IL-8/CXCL8 10. Kim HY, Kang YJ, Song IH, Choi HC, Kim HS: Upregu- expression. These results provide new insight into the poten- lation of Interleukin-8/CXCL8 in vascular smooth muscle tial diverse effects of 15d-PGJ cells from spontaneously hypertensive rats. Hypertens Res 2 on hypertensive vascular smooth muscle cells.
11. Dobrian AD, Schriver SD, Khraibi AA, Prewitt RL: Pioglita- zone prevents hypertension and reduces oxidative stress in diet-induced obesity. Hypertension 43;48-56, 2004 12. Fukuda N, Hu WY, Teng J, Chikara S, Nakayama K, Kanmatsuse K: Troglitazone inhibits growth and improves This work was supported by the Korean Science and insulin signaling by suppression of angiotensin II action in Engineering Foundation (KOSEF) grant funded by the Korean vascular smooth muscle cells from spontaneously hyper- government (MEST) (No. R13-2005-005-02002-0(2008)).
tensive rats. Atherosclerosis 163;229-239, 2002 13. Wakino S, Hayashi K, Tatematsu S, Hasegawa K, Takamatsu I, Kanda T, Homma K, Yoshioka K, Sugano N, Saruta T: CONFLICTS OF INTEREST Pioglitazone lowers systemic asymmetric dimethylarginine by inducing dimethylarginine dimethylaminohydrolase in The authors have no financial conflict of interest.
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Management of Patellofemoral PainTargeting Hip, Pelvis, and Trunk MuscleFunction: 2 Case Reports Catherine L. Mascal, PT, BSc1Robert Landel, DPT, OCS2Christopher Powers, PT, PhD3 Study Design: Case report.Objective: To describe an alternative treatment approach for patellofemoral pain.Background: Weakness of the hip, pelvis, and trunk musculature has been hypothesized toinfluence lower-limb alignment and contribute to patellofemoral pain. Two patients who had a