15-Deoxy-Δ 12,14 -Prostaglandin J 2 Modulates Lipopolysaccharide-Induced Chemokine Expression by...

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Original Paper

Nephron Exp Nephrol 2013;123:1–10 DOI: 10.1159/000353232

15-Deoxy-Δ 12,14 -Prostaglandin J 2 Modulates Lipopolysaccharide-Induced Chemokine Expressionby Blocking Nuclear Factor-κB Activation via Peroxisome Proliferator Activated Receptor-γ-IndependentMechanism in Renal Tubular Epithelial Cells

Ying Lu Qiao Zhou Fang Zhong Shanmai Guo Xu Hao Cong Li

Weiming Wang Nan Chen

Department of Nephrology, Ruijin Hospital, Shanghai Jiao Tong University, Shanghai , PR China

sults showed that application of LPS significantly increased IL-8 and MCP-1 production. Phosphorylation of IκBα, IKK and nucleus translocation of NF-κB significantly increased in LPS-stimulated HK-2 cells. 15d-PGJ 2 downregulated LPS-induced IL-8 and MCP-1 production. Interestingly, in PPAR-γ-deficient HK-2 cells, 15d-PGJ 2 was still capable of inhibiting chemo-kines expression and attenuating phosphorylation of IκBα and nucleus translocation of NF-κB. Conclusion: Collective-ly, these results suggest that 15d-PGJ 2 exerts anti-inflam-matory actions on HK-2 cells by attenuating chemokinesexpression. 15d-PGJ 2 inhibits chemokines expression via a PPAR-γ-independent way, which is related to block NF-κB pathway. Since NF-κB is an important regulator of the re-sponse of HPTECs to injury, PPAR-γ agonists may represent a key pharmacological target for ameliorating inflammation-associated tubulointerstitial fibrosis.

© 2013 S. Karger AG, Basel

Introduction

Renal fibrosis is the final common pathway for most forms of progressive renal disease and involves interstitial fibrosis and/or glomerular and vascular sclerosis. Tubu-

Key Words

Peroxisome proliferator activated receptor-γ · 15d-PGJ 2 · Human proximal tubular cells · Chemokine · Nuclear factor-κB

Abstract

Background/Aims: Inflammation is an unavoidable milieu for renal tubular cells during the development of renal tubu-lointerstitial fibrosis. It has been demonstrated that chemo-kines including monocyte chemoattractant protein-1 (MCP-1) and IL-8 are related to tubulointerstitial lesions. 15d-PGJ 2 may modulate renal tubulointerstitial fibrosis progression via anti-inflammatory effects. However, no information is known about the effects of 15d-PGJ 2 on chemokine expres-sion in human proximal renal tubular cells (HPTECs) under inflammation. Methods: In the present study, HPTECs (HK-2 cells) were stimulated with lipopolysaccharide (LPS) only, or preincubated with 15d-PGJ 2 . IL-8 and MCP-1 expressions were determined by real-time PCR and ELISA. Nuclear factor-κB (NF-κB) location was detected by immunofluorescence analysis. The p-IKK, p-IκBα and p65/p50 were analyzedby immunoblotting. To investigate the mechanism of in-hibitory effects of 15d-PGJ 2 , the PPAR-γ gene was effectively silenced in HK-2 cells using specific siRNA. Results: The re-

Received: November 20, 2012 Accepted : May 22, 2013 Published online: July 25, 2013

Weiming Wang Department of Nephrology, Ruijin HospitalShanghai Jiao Tong University, No. 197 Ruijin Er Rd. Shanghai 200025 (PR China) E-Mail wweiming @ medmail.com.cn

© 2013 S. Karger AG, Basel1660–2129/13/1232–0001$38.00/0

www.karger.com/nee

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http://dx.doi.org/10.1159%2F000353232

Lu/Zhou/Zhong/Guo/Hao/Li/Wang/Chen


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lointerstitial inflammation plays an important role in the progression of renal fibrosis and is closely associated with the prognosis of chronic kidney diseases. Many lines of evidence suggest that chemokines including monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8) are related to tubulointerstitial lesions [1, 2] . In both experimental diabetic and nondiabetic kidney dis-ease, chemokines are overexpressed [1, 2] . Recent studies have suggested that renal tubular epithelial cells are the key cells which secreted such chemokines [3, 4] . These findings suggest that intervention strategies to prevent re-nal tubular epithelial cells secreting chemokines facilitate a reduction in tubulointerstitial inflammation lesion.

Peroxisome proliferator-activated receptors (PPARs) are a nuclear hormone receptor superfamily of ligand-de-pendent transcription factors. Three different subtypes designated PPAR-α, PPAR-β/δ and PPAR-γ exist, charac-terized by distinct expression patterns, different ligand-binding specificity, and biological functions. These PPARs form heterodimers with the 9-cis-retinoic acid receptor, RXR-α, bind to characteristic DNA sequences termed per-oxisome proliferator response elements located in the pro-moter region of target genes, and exert specific biological actions. PPAR-γ plays a pivotal role in the regulation of adipogenesis and insulin sensitivity, whereas PPAR-α and PPAR-β/δ are implicated primarily in lipid metabolism and in the control of cell proliferation and differentiation [5] . The most potent natural agonist for PPAR-γ is the en-dogenous prostaglandin D 2 metabolite, 15-deoxy-Δ 12,14 -prostaglandin J 2 (15d-PGJ 2 ) containing a cyclopentenone ring [6, 7] . Synthetic agonists, antidiabetic thiazolidine-diones, including pioglitazone, activate PPAR-γ by bind-ing to the receptor with high affinity. Recently, accumulat-ing evidence has indicated that 15d-PGJ 2 and thiazolidine-diones not only improve insulin sensitivity and glucose metabolism but also possess anti-inflammatory potential that results in a reduction of pro-inflammatory cytokines and chemokines productions [8] . As for 15d-PGJ 2 , its anti-inflammatory action was shown to be both dependent and independent of PPAR-γ [9–13] . However, it has so far re-mained to be clarified how 15d-PGJ 2 regulates chemokine expression in renal tubular cells under inflammatory con-dition. Therefore, it seems more intriguing to investigate especially 15d-PGJ 2 , on the enhanced chemokine produc-tion in renal tubular cells stimulated with inflammatory factors, corresponding to cellular conditions in vivo dur-ing the progression of kidney disease.

Lipopolysaccharide (LPS), an important inflamma-tion stimulator, can induce endotoxemia, accelerate kid-ney injury of lupus nephritis and IgA nephropathy in an-

imal models. Nuclear factor-κB (NF-κB) is an LPS-sensi-tive transcription factor that most commonly exists as a p50/p65 heterodimer. NF-κB is a multiunit transcription factor that regulates the expression of pro-inflammatory cytokines, chemokines, enzymes that generate mediators of inflammation, immune receptors, and adhesion mol-ecules, all of which play a key role in the initiation of in-flammatory reactions. In resting cells, NF-κB is seques-tered in the cytoplasm by association with an inhibitory protein IκB. In response to signaling by inflammatory cy-tokines, IκB kinase (IKK) is activated and phosphorylates IκB on two serine residues. IκB is then ubiquitinated and degraded by the proteasome, freeing NF-κB to migrate into the nucleus and activate gene expression [14, 15] . Many lines of evidence suggest that IL-8 and MCP-1 pro-moters contain NF-κB-binding sites.

The aim of this study was to investigate whether 15d-PGJ 2 ameliorates tubulointerstitial inflammation through inhibiting tubular epithelial cells secreting chemokines. To further understand the molecular mechanism of 15d-PGJ 2 activity in tubular epithelial cells, we investigated the effects of 15d-PGJ 2 on the activation of the transcrip-tion factor NF-κB. Moreover, we examined whether or not the chemokine-modulating effect of 15d-PGJ 2 is PPAR-γ dependent.

Materials and Methods

Cell Cultures The HK-2 cell line derived from normal kidney was purchased

from ATCC (Manassas, Va., USA), and was grown in keratinocyte serum-free media (Gibco, USA) supplemented with bovine pitu-itary extract (Gibco) and epidermal growth factor (Gibco). HK-2 cells were rendered quiescent in serum-free medium for 24 h and all the following experiments were performed in quiescent HK-2 cells.

Cytotoxic Assay To determine the cytotoxicity of 15d-PGJ 2 , we detected lactate

dehydrogenase (LDH) activity released in the media 24 h after 15d-PGJ 2 exposure using a standard lactate dehydrogenase release as-say (Promega, USA) and quantitated by measuring wavelength ab-sorbance at 490 nm. In brief, 2 × 10 4 HK-2 cells were seeded into a 96-well plate containing 15d-PGJ 2 (0, 0.5, 1, 2.5, 5 or 10 μ M ) in a final volume of 100 μl. For maximum release, the plates were incu-bated for 23 h and 15 min at 37 ° C, and then 10 μl of 10× lysis buf-fer was added into the wells, and incubation continued for 45 min. Data were corrected by baseline LDH released from vehicle-treat-ed cells exposed to buffer only. 50 μl of supernatant was transferred to a fresh ELISA plate and 50 μl substrate was added to each well and mixed, and the plate was sealed. The mixture was incubated for 30 min at room temperature in the dark, and 50 μl of stop solu-tion was added to each well. The plate was read at 490 nm and the

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15d-PGJ 2 Inhibits LPS-Induced Chemokine Expression


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specific lysis was calculated by the following formula: % cytotoxic-ity = 100% × (experiment-target spontaneous)/(target maximum-target spontaneous). All experiments were performed in triplicate, and the data were expressed as mean inhibitory rate ± SD from three independent experiments.

Real-Time PCR Assay HK-2 cells were stimulated with LPS only, or preincubated with

15d-PGJ 2 (5 μ M ) for 2 h followed by stimulating with LPS 1 μg/ml (Sigma, USA) for 4 h. After treatment, cells were harvested and total RNA was extracted using TRIzol reagent (Invitrogen, USA) according to the manufacturer’s protocol. Real-time PCR amplifi-cation was performed using the SYBR Green master mix (Toyobo, Japan) and the Opticon 3 Real-Time PCR Detection System (Bio-Rad). Real-time RT-PCR was performed for 10 min at 95 ° C fol-lowed by 44 cycles (denaturation for 15 s at 95 ° C, annealing with extension for 30 s at 60 ° C). Relative amounts of mRNA were nor-malized by GAPDH. The sequences are shown in table 1 .

Enzyme-Linked Immunosorbent Assay HK-2 cells were stimulated with LPS only, or preincubated with

15d-PGJ 2 (5 μ M ) for 2 h followed by stimulating with LPS 1 μg/ml (Sigma, USA) for 24 h. Cellular supernatants from all experimental conditions were collected, centrifuged to remove cell debris, and stored at –80 ° C for analysis. IL-8 and MCP-1 protein concentra-tion were measured by commercial ELISA kits (Biosource, USA) according to the manufacture’s protocol. Results were corrected for cell numbers.

Transfection of siRNA HK-2 cells were seeded into 6-well plates containing 2 ml of

growth medium for 24 h. siRNA was transfected into HK-2 cells using Lipofectamine TM 2000 according to the manufacturer’s pro-tocol (Invitrogen, USA). Briefly, diluting 100 pmol scrambled RNA (siControl) or siRNA for PPAR-γ in 250 μl Opti-MEM (In-vitrogen, USA) (final concentration of RNA when added to the cells is 30 n M ). Then diluting 5 μl Lipofectamine 2000 in 250 μl Opti-MEM, incubating for 5 min at room temperature. After the 5-min incubation, combine the diluted oligomer with the diluted Lipofectamine 2000. After the incubation for 20 min, adding the oligomer-Lipofectamine 2000 complexes to each well replacing the growth medium.

Transfection and Reporter Gene Assay The κB-luciferase activity was measured after transfection with

a reporter plasmid which contains the NF-κB-binding site in the promoter region of the reporter luciferase gene [16] . The cotrans-fection with κB-luciferase plasmid and β-galactosidase expression vector was performed using the Amaxa Nucleofector II (Amaxa, Cologne, Germany) [17] . Two micrograms of κB-luciferase plas-mid and 1 μg β-galactosidase expression vector were mixed with 0.1 ml cell suspension containing 5 × 10 5 HK-2 cells and trans-ferred to an electroporation cuvette and then transfected by elec-troporation using the A24 pulsing program. After electroporation, transfected HK-2 cells were immediately transferred to 6-well plates containing 2 ml of growth medium for 24 h. The medium was replaced with serum-free medium and incubated with test substances for 30 min and LPS was then added for 4 h. After wash-es, 100 μl of reporter lysis buffer (Promega, USA) was added and the cells were scraped from 6-well plates. The supernatant was col-

lected after centrifugation at 12,000 g at 4 ° C for 2 min. Aliquots of 20 μl of cell lysates containing equal amounts of protein (20–30 μg) were added into wells of an opaque black 96-well plate and 100 μl of luciferase substrate was then added to each sample. The lumi-nescence was measured in an Orion II microplate luminometer (Berthold Pforzheim, Germany). The luciferase activity value was normalized to the activity of cotransfected β-galactosidase.

Western Blot Analysis HK-2 cells were seeded onto a 10-cm dish. After reaching con-

fluence, cells were incubated with test substances, and then washed with cold PBS and lysed for 30 min at 4 ° C with lysis buffer (150 mmol/l NaCl, 0.25% deoxycholate, 1% NP40, 10 mmol/l NaF,2 mmol/l EGTA, 1 mmol/l PMSF, 1 μg/ml leupeptin and 1 μg/ml aprotinin). Cytoplasmic extracts and nuclear extracts were sepa-rated by NE-PER (Thermo Scientific-Pierce, USA). Equal protein (20 μg) was applied per lane, and electrophoresis was performed under denaturing conditions on a 10% SDS gel and transferred to an immobilon-P membrane (Millipore). The membranes were blocked in 5% nonfat milk for 2 h at room temperature, followed by incubating with indicated antibodies against phosphor-IκBα, IκBα, phosphor-IKKα/β, IKKα/β, NF-κB p50, and NF-κB p65(1: 500) at 4 ° C overnight. After washing, horseradish peroxidase-conjugated secondary antibody was added and incubated for 1 h at room temperature. Protein bands were visualized by the en-hanced chemiluminescence method, and the band intensity was quantified by densitometry. Results were verified with β-actin for total protein extracts and Lamin B for nuclear protein extracts.

Immunofluorescence Staining To test the localization of NF-κB, monolayer cells were cul-

tured on glass coverslips in K-SFM. After pretreating with 15d-PGJ 2 for 2 h, cells were stimulated with LPS (1 μg/ml) for 30 min, then fixed with 4% paraformaldehyde in 1× sodium phosphate (PBS 1×) (pH 7.5) and permeabilized with 0.2% Triton X-100 for 5 min, the coverslips were incubated with polyclonal anti-p65antibody (1: 70 dilution; Santa Cruz) overnight at 4 ° C. They were then washed with PBS 1× (PH 7.5) and incubated with rhodamine-conjugated goat anti-rabbit IgG (KPL, USA) for 2 h at room tem-perature. After washing, coverslips with the stained cells were mounted in 80% glycerol in PBS and then photographed with a Nikon fluorescence photomicroscope.

Table 1. Primers used for real-time PCR analysis

Gene Primer sequence

GAPDH forwardreverse

5′-CAGGGCTGCTTTTAACTCTGGTAA-3′5′-GGGTGGAATCATATTGGAACATGT-3′

MCP-1 forwardreverse

5′-CAGCCAGATGCAATCAATGC-3′5′-GTGGTCCATGGAATCCTGAA-3′

IL-8 forwardreverse

5′-GAATTGAATGGGTTTGCTAGA-3′5′-CACTGTGAGGTAAGATGGTGG-3′

PPAR-γ forwardreverse

5′-GGGCCCTGGCAAAACATT-3′5′-AAGATCGCCCTCGCCTTT-3′

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Statistical Analysis All the results were expressed as means ± SD. Multiple group

comparison was done using one-way analysis of variance followed by the Bonferroni test. The difference is significant if the p value is <0.05.

Results

Effect of 15d-PGJ 2 on Cell Viability To evaluate the cytotoxicity of 15d-PGJ 2 , HK-2 cells

were exposed to 15d-PGJ 2 of various concentrations for 24 h. The cell viability was estimated by measuring LDH released into the media from dead or dying cells 24 h after 15d-PGJ 2 exposure. As shown in figure 1 , 15d-PGJ 2 (10 μ M ) had significant cytotoxicity, and less than 10 μ M con-centration did not produce cell death.

15d-PGJ 2 Inhibits LPS-Induced IL-8 and MCP-1 Expression in HK-2 Cells HK-2 cells were stimulated with LPS only or preincu-

bated with 15d-PGJ 2 (5 μ M ) for 2 h followed by stimula-tion with LPS. Results show that treatment of LPS (1 μg/ml) for 4 h markedly increased the mRNA expression of IL-8 and MCP-1 in HK-2 cells about 6- to 7-fold, respec-tively ( fig. 2 a, c). After application of LPS (1 μg/ml) for24 h, IL-8 and MCP-1 protein in cell supernatants in-creased to 2.62 ± 0.12 ( fig. 2 b) and 3.25 ± 0.40-fold ( fig. 2 d), respectively. Pretreatment of HK-2 cells with 15d-PGJ 2 (5 μ M ) significantly inhibited LPS-induced IL-8 and MCP-1 mRNA expression (inhibition was up to 74.23 ± 10.12% and 79.42 ± 4.53%; fig. 2 a, c). 15d-PGJ 2 (5 μ M ) also exerted similar inhibitory effects on LPS- induced IL-8 and MCP-1 protein release into supernatant

(inhibition was up to 69.03 ± 11.82% and 49.44 ± 5.12%, fig. 2 b, d).

PPAR-γ Is Knocked Down by a Specific siRNA To examine whether PPAR-γ is involved in the pro-

duction of IL-8 and MCP-1 by LPS in HK-2 cells, a spe-cific siRNA of PPAR-γ was used in this study. A specific siRNA (30 nM ) of PPAR-γ decreased PPAR-γ mRNA ex-pression by 87.83 ± 4.13% ( fig. 3 a). PPAR-γ protein was also decreased by 82.26 ± 6.32% ( fig. 3 b). NonspecificsiRNA is used as a control to exclude any effect caused by PPAR-γ siRNA not specifically due to silencing of PPAR-γ.

15d-PGJ 2 Inhibits LPS-Induced IL-8 and MCP-1 Expression in a PPAR-γ-Independent Manner After 48 h of transfection of siRNA, HK-2 cells were

treated with 15d-PGJ 2 and LPS as described above. The results showed that PPAR-γ-deficient cells were still ca-pable of producing IL-8 and MCP-1 in response to LPS ( fig. 4 ). Importantly, silencing PPAR-γ by siRNA did not significantly antagonize the inhibitory effects of 15d-PGJ 2 on LPS-induced IL-8 and MCP-1 expression ( fig. 4 ). These results indicate that 15d-PGJ 2 inhibits the induc-tion of IL-8 and MCP-1 mainly through a PPAR-γ-independent manner, consistent with the idea that this compound can exert anti-inflammatory effects via a PPAR-γ-independent mechanism.

15d-PGJ 2 Inhibits LPS-Induced NF-κB Nuclear Translocation in a PPAR-γ-Independent Manner NF-κB plays a central role in the regulation of cyto-

kines and chemokines expression. To examine whether NF-κB is involved in the production of IL-8 and MCP-1 by LPS in HK-2 cells, we detected NF-κB nuclear translo-cation by western blot and immunofluorescence analysis. As shown in figure 5 a, treatment of LPS (1 μg/ml) for 30 min markedly increased the levels of p50 and p65 subunit in nuclear extract when compared to unstimulated cells. Pretreatment of 15d-PGJ 2 (5 μ M ) inhibited the transloca-tion of NF-κB subunits from cytosol into nucleus ( fig. 5 a). To further examine whether PPAR-γ signaling is in-volved in this action or not, a specific siRNA of PPAR-γ was used. Interestingly, in PPAR-γ-silenced cells, 15d-PGJ 2 was still able to attenuate LPS-induced the nuclear translocation of NF-κB subunits ( fig. 5 a). Immunofluo-rescent staining also showed that pretreatment of 15d-PGJ 2 markedly attenuated LPS-induced nuclear translo-cation of p65 ( fig. 5 b). In addition, the NF-κB activity was measured using κB-luciferase reporter assay. As shown in figure 6 , treatment of LPS for 4 h significantly increased

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Fig. 1. The toxicity of 15d-PGJ 2 in HK-2 cells. Cells were treated with 15d-PGJ 2 at the indicated concentrations for 24 h. 15d-PGJ 2 toxicity was evaluated by measuring LDH activity released in the media. Results are from three separate platings. * * * p < 0.001 vs.0 μ M group.

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Fig. 2. 15d-PGJ 2 inhibits LPS-induced IL-8 and MCP-1 expression in HK-2 cells. IL-8 and MCP-1 mRNA were quantified after incubation with 15d-PGJ 2 (5 μ M ) for 2 h followed by stimulation with LPS (1 μg/ml) for 4 h. IL-8 and MCP-1 protein in cell su-pernatants were measured after incubation with 15d-PGJ 2 (5 μ M ) for 2 h followed by stimulation with LPS (1 μg/ml) for 24 h. The results shown are representative of three separate experiments. a The expres-sion of IL-8 mRNA in different groups. b IL-8 protein in cell supernatants in differ-ent groups. c The expression of MCP-1 mRNA in different groups. d MCP-1 pro-tein in cell supernatants in different groups. ### p < 0.001 vs. control group; * * * p < 0.001 vs. LPS group.

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Fig. 3. PPAR-γ was knocked down by a specific siRNA. a The efficient knockdown of PPAR-γ mRNA was determined after 48 h of transfection with siRNA (30 n M ) by real-time PCR. b The efficient knockdown of PPAR-γ protein was confirmed after 72 h of transfection with siRNA (30 n M ) by immunoblotting. * * p < 0.01 vs. HK-2 cells; * * * p < 0.001 vs. HK-2 cells.

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Fig. 4. 15d-PGJ 2 inhibits LPS-induced IL-8 and MCP-1 expression in a PPAR-γ-independent manner. After 48 h of trans-fection with siRNA, HK-2 cells were treat-ed with 15d-PGJ 2 and LPS as described above. The results shown are representa-tive of three separate experiments. a IL-8 mRNA in different groups. b IL-8 protein in cell supernatants in different groups. c MCP-1 mRNA in different groups. d MCP-1 protein in cell supernatants in different groups. ### p < 0.001 vs. control group; * * * p < 0.001 vs. LPS group.

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Fig. 5. 15d-PGJ 2 inhibits LPS-induced NF-κB nuclear translocation in a PPAR-γ-independent manner. HK-2 cells were transfected with siRNA for 48 h and then treated with 15d-PGJ 2 (5 μ M ) for 2 h. LPS (1 μg/ml) was then added for another30 min. a Cytosolic and nuclear extracts were separated by NE-PER kit. p50 and p65 in nucleus were determined by immuno-blotting. 15d-PGJ 2 (5 μ M ) significantly in-hibited the nuclear translocation of NF-κB subunits of p50 and p65. Knockdown of PPAR-γ exerted no influence on this inhib-itory effect. ### p < 0.001 vs. control group; ## p < 0.01 vs. control group; * * p < 0.01 vs. LPS group; * p < 0.05 vs. LPS group. b Im-munofluorescent staining showed that NF-κB p65 translocated into the nucleus after treatment with LPS (1 μg/ml) for 30 min and pretreatment with 15d-PGJ 2 (5 μ M )antagonized p65 nuclear translocation. Original magnification. ×200.

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κB-luciferase activity and the addition of 15d-PGJ 2 also markedly inhibited LPS-induced κB-luciferase activity. These results indicate that inhibition of NF-κB activation is involved in the antagonism of 15d-PGJ 2 on LPS-in-duced IL-8 and MCP-1 expression.

15d-PGJ 2 Inhibits LPS-Induced IKK and IκBα Activation in a PPAR-γ-Independent Manner As mentioned above, 15d-PGJ 2 inhibited LPS-induced

NF-κB translocation in a PPAR-γ-independent manner. In order to further examine the mechanism of 15d-PGJ 2 on the inhibition of IKK and IκBα activation, phosphor-IKK and phosphor-IκBα in cytoplasm was assessed. We found that that treatment of LPS activated IKK, which phosphorylated IκBα to cause the ubiquitination of IκBα and degradation by proteasome ( fig. 7 a, b). 15d-PGJ 2 was capable of inhibiting LPS-induced phosphorylation of IKK and IκBα ( fig. 7 a, b). Silencing PPAR-γ by siRNAdid not antagonize these inhibitory actions of 15d-PGJ 2 ( fig. 7 a, b). These results indicate that 15d-PGJ 2 inhibits LPS-induced NF-κB activation by the inhibition of IKK activation via a PPAR-γ-independent pathway.

Discussion

This study is the first to verify that the naturally occur-ring PPAR-γ agonist, 15d-PGJ 2 , inhibits LPS-stimulated chemokine expression in tubular epithelial cells. Our

results indicate that the protective effect of 15d-PGJ 2 is associated with attenuation LPS-induced IκB phosphory-lation, subsequently blocking NF-κB translocation tonucleus. We also provide evidence that the effect of15d-PGJ 2 occurred independently of PPAR-γ. The inhib-itory effect of 15d-PGJ 2 on chemokine expression sug-gests a possible mechanism responsible for its anti-in-flammatory action and its potential use as a therapeutic agent for treating tubulointerstitial inflammation lesion.

The renal tubular epithelium cells play a central role in the development of chronic kidney structural deteriora-tion, especially by the generation of inflammatory and profibrogenic cytokines [18] . Recent studies suggested that chemokines, such as MCP-1 and IL-8, were closely associated with kidney diseases, and tubular epithelial cells are the predominant site secreting these chemokines [19] . MCP-1 and IL-8 can induce tubulointerstitial le-sions by recruiting target cells, which include macro-phages/monocytes, T cells, neutrophils, eosinophils and basophils, into the tubulointerstitium. In the current study, we confirmed that LPS significantly stimulated HK-2 cells to secrete IL-8 and MCP-1 (mRNA levels in-creased to nearly 7-fold; protein levels increased to 2-fold). Possible reasons to explain why the proteins were not as significantly changed as their corresponding mRNAs are as follows. A series of biological process exists between mRNA and its corresponding protein, and the cellular mRNA level does not surely represent the protein level. The second reason might be due to the specific mode of action of LPS. LPS-induced MCP-1 and IL-8 se-cretion was a slow and ever-increasing process, at least within the experimental time period, indicating the en-hancement or at least maintenance of the effect of LPS might only be effective on the early stage but become less powerful as incubation time increases.

In line with this notion, there is a great interest in identifying and developing drugs to prevent tubularepithelial cells secreting chemokines. The cyclopen-tenone prostaglandin 15d-PGJ 2 , a natural ligand for PPAR-γ, has recently been received considerable atten-tion because of its widespread biological effects, such as adipocyte differentiation and anti-inflammatory activi-ty. Recently, much attention has been focused on the role of 15d-PGJ 2 on the regulation of the inflammatory process. It was reported that 15d-PGJ 2 inhibited the ex-pression of a number of inflammatory response genes in activated macrophages, including the genes encoding the inducible nitric oxide synthase, tumor necrosis factor-α, gelatinase B, and cy clo-oxygenase-2 genes [20] . In the current study, we demonstrated that pretreatment

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Fig. 6. 15d-PGJ 2 inhibits LPS-induced NF-κB reporter activity. HK-2 cells were transfected with κB-luciferase expression vector by electroporation and then pretreated with 15d-PGJ 2 (5 μ M ) for2 h before incubation with LPS (1 μg/ml) for 4 h. Luciferase activity was then assayed. Note that 15d-PGJ 2 markedly inhibited the κB-luciferase activity. Data are presented as mean ± SEM. ### p < 0.001 vs. control group; * * p < 0.01 vs. LPS group.

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of HK-2 cells with 15d-PGJ 2 significantly inhibited LPS-induced IL-8 and MCP-1 expression. Ricote et al. [21] demonstrated that the activity of the inducible nitric ox-ide synthase and gelB promoters in RAW264.7 macro-phages was strongly inhibited by 15d-PGJ 2 , and that this inhibition was potentiated in the presence of PPAR-γ. Specifically, mutations in PPAR-γ that eliminate ligand-dependent interaction with the coactivators SRC-1 and CBP abolished both ligand-dependent repression. These results suggest that ligand-dependent transrepression is associated with competition with limiting amounts of coactivators.

There are other previous studies also suggested the ex-istence of PPAR-γ -independent mechanism(s) for re-pression of gene expression by 15d-PGJ 2 . First, 15d-PGJ 2 represses inducible nitric oxide synthase promoter activ-ity even in cells that do not express PPAR-γ, although the dose-response curve is shifted toward higher 15d-PGJ 2 concentrations. Second, high-affinity synthetic PPAR-γ ligands such as BRL49653 are less active than 15d-PGJ 2 in repressing gene expression or promoter activity. Since the synthetic ligands bind to PPAR-γ with affinities that equal or exceed the affinity of 15d-PGJ 2 , these results strongly suggest the existence of receptor-independent pathway(s) for gene repression by 15d-PGJ 2 . In the cur-rent study, we found that 15d-PGJ 2 significantly inhibited LPS-induced IL-8 and MCP-1 expression via a PPAR-γ-independent mechanism.

Because of its ubiquitous role in the pathogenesis of inflammatory gene expression, NF-κB is a current target for the treatment of various diseases. It has been reported that 15d-PGJ 2 strongly inhibits NF-κB-dependent tran-scription by two distinct mechanisms [21, 22] . First, 15d-PGJ 2 can interrupt NF-κB-dependent gene expression through covalent modifications of critical cysteine resi-dues in IKK with subsequent prevention of IκB degrada-tion and nuclear entry of NF-κB. In this respect, the reac-tive α,β-unsaturated carbonyl group in the cyclopente-none ring is critical for IKK inactivation. 15d-PGJ 2 bearing the α,β-unsaturated carbonyl moiety can act as a Michael acceptor, and hence reacts with cellular thiols. The second mechanism by which 15d-PGJ 2 interferes with NF-κB ac-tivation involves direct inhibition of binding of NF-κB to target DNA without blocking IκB degradation and nucle-ar translocation of NF-κB. This is more likely to be achieved by alkylation of a conserved cysteine residue lo-cated in the DNA binding domain of Rel proteins (e.g. C38 in p65 and C62 in p50). Therefore, 15d-PGJ2 may act as a negative feedback regulator of pro-inflammatory en-zymes/molecules through NF-κB inactivation. Our results showed that 15d-PGJ 2 inhibited LPS-induced phosphory-lation of IKK, and attenuated the downstream steps of IKK, such as the phosphorylation and degradation of IκBα and the nuclear translocation of NF-κB p50 and p65 sub-units via a PPAR-γ-independent way. These results are consistent with the first mechanism.

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Fig. 7. 15d-PGJ 2 inhibits LPS-inducedIKK and IκB activation in a PPAR-γ-independent manner. HK-2 cells were pre-treated with 15d-PGJ 2 for 2 h, and LPS was then administered for another 30 min. a Immunoblotting showed that treatment of LPS enhanced the phosphorylation of IKK, and 15d-PGJ 2 inhibited LPS-induced phosphorylation of IKK. Knockdown of PPAR-γ did not antagonize the inhibitory action of 15d-PGJ 2 . b LPS enhanced the phosphorylation of IκB, and 15d-PGJ 2 in-hibited LPS-induced phosphorylation of IκB. Knockdown of PPAR-γ did not antag-onize the inhibitory action of 15d-PGJ 2 . Data are presented as mean ± SEM. ### p < 0.001 vs. control group; ## p < 0.01 vs. con-trol group; * * p < 0.01 vs. LPS group; * p < 0.05 vs. LPS group.

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Kang et al. [23] demonstrated that 15d-PGJ 2 (3 μ M ) induced opossum kidney (OK) cell death in a concentra-tion- and time-dependent manner. Additionally, death induction effects of 15d-PGJ 2 was associated with inter-ruption of NF-κB via a PPAR-γ-independent way. In the current study, 15d-PGJ 2 (10 μ M ) had significant cytotox-icity, and less than 10 μ M concentration had no significant cytotoxicity. 15d-PGJ 2 (10 μ M ) attenuated the ability of Helicobacter pylori -induced apoptosis in gastric epithelial cells by inhibiting the NF-κB activation [24] . Levonen et al. [25] had shown that 15d-PGJ 2 at low micromolar con-centrations caused a robust increase in cellular GSH which conferred resistance against oxidative death in hu-man endothelial cells. In contrast, a higher concentration of 15d-PGJ 2 induced endothelial cell apoptosis [25] . The results described above suggest that the proapoptotic and cytoprotective/antiapoptotic effects of 15d-PGJ 2 are de-pendent upon the dose and the type of target cells. It would be worthwhile elucidating the molecular mecha-nisms underlying differential effects of PPAR-γ ligands on cell survival/death.

Overall, our results delineate a PPAR-γ-independent inhibitory action of 15d-PGJ 2 on the expression of che-mokine genes in human renal tubular epithelial cells. Fur-thermore, the data presented provides evidence that the inhibition effect of 15d-PGJ 2 is mediated by blocking ac-tivation of NF-κB. These results suggest that 15d-PGJ 2 could be acting as a chemokine inhibitor in the inflam-matory kidney diseases. In this context, considerable in-terest would be focused on 15d-PGJ 2 as a potential thera-peutic agent for amelioration renal inflammation and re-nal injury in chronic kidney disease. Future studies will address this aspect in PPAR-γ-knockout mice in vivo.

Acknowledgements

This work was supported by grants from the National Nature Science Foundation of China (30270613 and 30771000), Leading Projects of Shanghai Science and Technology Commission (08dz1900502) and Shanghai Leading Academic Discipline Proj-ect (T0201), China.

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