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NDT Advance Access originally published online on July 2, 2008
Nephrology Dialysis Transplantation 2008 23(12):3776-3785; doi:10.1093/ndt/gfn361
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© The Author [2008]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org


15-Deoxy-{Delta}12,14-prostaglandin J2 inhibits INF-{gamma}-induced JAK/STAT1 signalling pathway activation and IP-10/CXCL10 expression in mesangial cells

Ulf Panzer1,*, Gunther Zahner1,*, Ulrike Wienberg1, Oliver M. Steinmetz1, Anett Peters1, Jan-Eric Turner1, Hans-Joachim Paust1, Gunter Wolf2, Rolf A. K. Stahl1 and André Schneider1

1 Medizinische Klinik III, Universitätsklinikum Hamburg Eppendorf, Germany 2 Klinik für Innere Medizin III, Klinikum der Friedrich-Schiller-Universität, Jena, Germany

Correspondence and offprint requests to: Ulf Panzer, Medizinische Klinik III, University of Hamburg, Martinistr 52, 20246 Hamburg, Germany. Tel: +49-40-42803-3908; Fax: +49-40-42803-5186; E-mail: panzer{at}uke.uni-hamburg.de



   Abstract
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 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Background. Activators of the peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}), originally found to be implicated in lipid metabolism and glucose homeostasis, have been shown to modulate inflammatory responses through interference with cytokine and chemokine production. Given the central role of mesangial cell-derived chemokines in glomerular leukocyte recruitment in human and experimental glomerulonephritis, we studied the influence of natural and synthetic PPAR{gamma} activators on INF-{gamma}-induced expression of the T cell-attracting chemokines IP-10/CXCL10, Mig/CXCL9 and I-TAC/CXCL11 in mouse mesangial cells.

Methods. INF-{gamma}-treated mesangial cells were cultured in the presence or absence of either the naturally occurring PPAR{gamma} ligand 15-deoxy-{Delta}12,14-prostaglandin J2 (15d-PGJ2) or synthetic PPAR{gamma} activators of the glitazone group. Chemokine mRNA and protein expression and activation of the JAK/STAT signalling pathway were analysed.

Results. The 15d-PGJ2, but not synthetic PPAR{gamma} ligands, dose-dependently inhibited INF-{gamma}-induced chemokine gene (mRNA and protein) expression. Combined results from EMSA and western blot analysis revealed the inhibitory ability of 15d-PGJ2, but not of synthetic PPAR{gamma} ligands, on IFN-{gamma}-induced tyrosine phosphorylation of JAK1, JAK2, STAT1 and nuclear STAT1 translocation and DNA binding.

Conclusions. Our results demonstrate that 15d-PGJ2 inhibits INF-{gamma}-induced chemokine expression in mesangial cells by targeting the JAK/STAT signalling pathway. This effect is independent of an interference with PPAR{gamma}.

Keywords: chemokines; CXCR3; glomerulonephritis; mesangial cells; T cells



   Introduction
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 Introduction
Methods
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The recruitment of leukocytes into the glomerulus and into the renal tubulointerstitium is a hallmark of almost any human and experimental form of glomerulonephritis. Chemokines are considered to be the main regulators of directional leukocyte trafficking under physiological and inflammatory conditions [1]. Chemokines are produced by resident tissue cells and inflammatory cells and exert their effects locally in a paracrine or autocrine fashion. The majority of chemokines are induced under pathological conditions by pro-inflammatory stimuli (e.g., LPS, INF-{gamma} or TNF{alpha}). All of the ~50 chemokines act by activating heptahelical G protein-coupled chemokine receptors predominantly located on the leukocyte plasma membrane [1].

Cytokines and chemokines expressed and secreted by activated mesangial cells are uniquely positioned to play an important role in the process of glomerular inflammation and leukocyte infiltration [2]. IP-10/CXCL10 is among the chemokines expressed by mesangial cells under inflammatory conditions [3]. IP-10/CXCL10 and the closely related chemokines Mig/CXCL9 and I-TAC/CXCL11 are induced upon stimulation with the Th1 cytokine INF-{gamma} in a variety of cells and bind to the chemokine receptor CXCR3, which is predominantly expressed on activated T cells of the Th1 phenotype [4]. Since crescentic glomerulonephritis is considered a Th1 type inflammation [5], the glomerular attraction of CXCR3-positive T cells by locally produced IP-10/CXCL10, Mig/CXCL9 and I-TAC/CXCL11 might play an important pathophysiological role. Indeed, blocking of IP-10/CXCL10 in a rat model of renal endothelial microvascular injury led to a reduction in renal T cell recruitment and improvement of renal function [6].

A naturally occurring metabolite of the prostaglandin D2 derivate of prostaglandin J2, 15-deoxy-{Delta}12,14-prostaglandin J2, is produced in a variety of tissues and cells during inflammatory processes [7]. It has been shown that 15d-PGJ2 acts via interaction with several intracellular targets. In particular, 15d-PGJ2 is recognized as a high-affinity ligand for the peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}), a member of the nuclear hormone receptor superfamily of ligand-activated transcription factors [8]. Activated PPARs regulate gene expression by heterodimerizing with the 9-cis retinoic acid receptor RXR and by binding to peroxisome proliferator response elements (PPRE) located in the promoter region of target genes [9].

Although originally thought to be mainly involved in lipid metabolism and glucose homeostasis, PPAR{gamma} agonists such as 15d-PGJ2 and the synthetic activators thiazolidinediones have recently been demonstrated to affect the immune response in vitro and in vivo by modulating the expression of inflammatory mediators, including cytokines, adhesion molecules and chemokines in leukocytes and tissue cells [10–13]. PPAR{gamma} activation is responsible for many of the 15d-PGJ2 anti-inflammatory functions. However, recent studies have demonstrated that 15d-PGJ2 can also mediate PPAR{gamma}-independent anti-inflammatory effects. Some of these effects may be mediated through covalent binding of 15d-PGJ2 to proteins involved in pro-inflammatory signalling pathways [14].

We hypothesized that PPAR{gamma} activators might modulate the regulation of INF-{gamma}-induced chemokine expression in mesangial cells, and therefore studied the effects of 15d-PGJ2 and the synthetic PPAR{gamma} activators troglitazone and ciglitazone on the expression of IP-10/CXCL10, Mig/CXCL9 and I-TAC/ CXCL11.



   Methods
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Results
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Reagents
Recombinant mouse INF-{gamma} was obtained from R&D Systems (Wiesbaden, Germany), and 15d-PGJ2 was from Merck Biosciences (Schwalbach, Germany). Ciglitazone and GW9662 were purchased from Alexis Biochemical (San Diego, CA, USA). Rabbit polyclonal antibodies against phospho- and total STAT1{alpha}, JAK1 and JAK2 were all purchased from Cell Signaling (Danvers, MA, USA). The goat polyclonal anti-IP-10/CXCL10 antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Troglitazone was kindly provided by M. Breyer (Vanderbilt University, Nashville, USA).

Cell culture
Mouse mesangial cells (MMCs) [15] were cultured in DMEM (Gibco-BRL/Invitrogen (Karlsruhe, Germany)) containing 10% FCS, 100 units/ml penicillin, 100 µg/ml streptomycin (Gibco-BRL) at 37°C in 5% CO2. Prior to stimulation, confluent cells were incubated in serum free DMEM for 24 h. The 15d-PGJ2, troglitazone and ciglitazone were solved in absolute ethanol or DMSO in accordance with the supplier's recommendations. Influence of solvent and of all stimulation conditions on cell viability was tested using an LDH-based cytotoxicity assay (CytoTox 96®, Promega, Madison, WI, USA) to rule out unspecific toxic effects.

RNA preparation and real-time PCR analysis
Total renal RNA was prepared as described previously [6]. Real-time PCR was performed for 40 cycles (initial denaturation: 95°C, 10 min; denaturation: 95°C, 15 s; primer annealing and elongation: 60°C, 1 min) with 1.5 µl of cDNA samples in the presence of 2.5 µl (0.9 µM) specific murine primers (primer sequences are available upon request) and 12.5 µl of 2x Platinum® SYBR Green® qPCR Supermix (Invitrogen) in an AbiPrism Sequence Detection System 7000 (Applied Biosystems, Foster City, CA, USA). To account for small RNA and cDNA variability, an 18S rRNA PCR was run in parallel. All samples were run in duplicate and normalized to 18S rRNA as recently described [6].

Nuclear extracts
For preparation of nuclear extracts, cells were collected and incubated in buffer A (10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 M PMSF, 10 mg/ml aprotinin and leupeptin, respectively) for 10 min on ice. After adding Nonidet-P40 to a final concentration of 0.5%, cells were gently vortexed and the nuclei were collected by centrifugation. The pellet was resuspended in buffer B (20 mM HEPES (pH 7.9), 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, 10 mg/ml aprotinin and leupeptin) by gentle vortexing, and nuclear proteins were obtained by centrifugation. The protein concentration was measured using a Bio-Rad protein assay.

Electrophoretic mobility shift assay (EMSA)
Equal amounts of nuclear extract proteins were incubated with 1 x 105 cpm of a 32P-labeled STAT1{alpha} double-strand consensus oligomer (FW: 5' ACG CTT TGG AAA GTG AAA CCT ACC TCA 3') at room temperature for 30 min. Reactions were carried out in binding buffer (18 mM HEPES (pH 7.9), 0.2 mM EDTA, 0.1 mM EGTA, 40 mM NaCl, 0.2 mM DTT, 0.1 mM PMSF, 3% glycerol) containing 2 µg poly(dI·dC) (Amersham, Pharmacia Biotech, Germany), as previously described [16].

Chemokine ELISA
IP-10/CXCL10 and Mig/CXCL9 release of MMCs was measured using a mouse-specific IP-10/CXCL10 and Mig/CXCL9 ELISA (R&D Systems) according to the manufacturer's recommendations. MMCs (1 x 106 cells/well) were plated in six-well plates. After replacement of culture medium by DMEM without FCS for 24 h, cells were stimulated for 24 h and cell culture supernatants were harvested.

Western blotting
Equal amounts of proteins (20–50 µg) were loaded onto denaturing SDS polyacrylamide gels, electrophoresed and transferred to PVDF membranes (Hybond-P, Amersham, Freiburg, Germany). The membranes were blocked in 5% nonfat dry milk in PBS containing 0.1% Tween 20 and incubated with the different primary antibodies (all 1:1.000) in a blocking buffer, followed by 1 h of incubation with 1:3.500 diluted anti-rabbit IgG or anti-goat IgG antibody conjugated to HRP (Southern Biotechnology, Birmingham, AL, USA) [17]. Chemoluminescence was detected with the ECL detection system (Amersham).

Chemotaxis experiments
The migration of murine pre-B cells (300–19) stably transfected with human CXCR3 (accession number NM_001504 [GenBank] ) in response to recombinant IP-10/CXCL10 and supernatants of INF-{gamma} stimulated MMCs for 24 h was analysed using 48-well Boyden chambers (Neuro Probe, Gaithersburg, MD, USA) with 5-µm pore size polycarbonate membranes, as previously described [18]. Migration was allowed to proceed for 60 min at 37°C in 5% CO2. The membrane was then removed, washed on the upper side with PBS, fixed and stained. All assays were performed in triplicate, and the migrated cells were counted in five randomly selected high power fields (HPF) at 1000-fold magnification. Results are expressed as chemotactic index.

Statistical analysis
Results are expressed as mean ± SD. Differences between individual experimental groups were compared by Kruskal Wallis test with post hoc analysis by the Mann–Whitney test.



   Results
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 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
INF-{gamma}-induced expression of the chemokines IP-10/ CXCL10, Mig/CXCL9 and ITAC/CXCL11 mRNA in MMCs
To assess the mRNA expression of CXCR3 ligands IP-10/CXCL10, Mig/CXCL9 and ITAC/CXCL11 in MMCs, cells were stimulated with INF-{gamma} (100 ng/ml) for 0.5–24 h. Isolated total RNA was subjected to real-time RT-PCR. Unstimulated MMCs produced small amounts of CXCR3 chemokine ligand mRNA, whereas stimulation with INF-{gamma} led to an upregulation of IP-10/CXCL10 (up to 110-fold), Mig/CXCL9 (up to 5730-fold) and I-TAC/CXCL11 (up to 141-fold) (Figure 1). Enhanced mRNA expression was detectable after 0.5 h, with a maximum at 6 h (Figure 1).


Figure 1
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  		Fig. 1 INF-{gamma} induces mRNA expression of CXCR3 ligands IP-10/CXCL10, Mig/CXCL9 and ITAC/CXCL11 in mouse mesangial cells. Mesangial cells were incubated with INF-{gamma} (100 ng/ml) for the indicated time periods. Isolated total RNA was subjected to real-time RT-PCR using specific primers for IP-10/CXCL10 or Mig/CXCL9 or I-TAC/CXCL11. Data are expressed as x-fold of control mRNA expression (n = 4).

 
Effect of 15d-PGJ2 and synthetic PPAR{gamma} activators and inhibitors on IP-10/CXCL10, Mig/CXCL9 and ITAC/CXCL11 mRNA expression
To analyse whether PPAR{gamma} activators may modulate IP-10/CXCL10, Mig/CXCL9 and ITAC/CXCL11 mRNA expression, we stimulated MMCs with INF-{gamma} (100 ng/ml) together with the naturally occurring PPAR{gamma} ligand 15d-PGJ2 (10 µM) or the synthetic PPAR{gamma} activators troglitazone (10 µM) or ciglitazone (10 µM). Real-time RT-PCR analysis of isolated RNA revealed that IP-10/CXCL10, Mig/CXCL9 and ITAC/CXCL11 mRNA expression was significantly reduced by 15d-PGJ2 (10 µM) (P < 0.01 compared with INF-{gamma} stimulated cells) (Figure 2A). In contrast, various doses of the synthetic PPAR{gamma} activators troglitazone and ciglitazone (up to 10 µM) did not affect chemokine mRNA expression (Figure 2A). Stimulation with 15d-PGJ2 (10 µM), troglitazone (10 µM) and ciglitazone (10 µM) alone had no effect on chemokine mRNA expression. To demonstrate that the action of 15-{Delta}PGJ2 on INF-{gamma}-induced IP10/CXCL10, Mig/CXCL9 and I-TAC/CXCL11 production is PPAR{gamma} independently, we performed additional experiments in which the PPAR{gamma} blockade was achieved by the pharmacological inhibitor GW9662. The results showed that GW9662 did not influence the effect of 15-{Delta}PGJ2 strongly arguing against a PPAR{gamma}-dependent mechanism (Figure 2B). 15d-PGJ2 led to a reduction in IP-10/CXCL10, Mig/CXCL9 and I-TAC/CXCL11 mRNA expression in a concentration-dependent manner. A significant reduction of chemokine mRNA expression was observed at 5 and 10 µM of 15d-PGJ2 (Figure 2C). Treatment of MMCs with INF-{gamma} or 15d-PGJ2 at the concentrations indicated (up to 20 µM) did not affect cell viability as assessed by the LDH-based non-radioactive cyctotoxic assay (Figure 2D).


Figure 2
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  		Fig. 2 The 15d-PGJ2, but not synthetic PPAR{gamma} activators, attenuates IP-10/CXCL10, Mig/CXCL9 and I-TAC/CXCL10 mRNA expression in a dose-dependent manner. (A) Real-time RT-PCR analysis of mesangial cells stimulated with INF-{gamma} (100 ng/ml) in the presence or absence of 15d-PGJ2 and synthetic PPAR{gamma} ligands that are added simultaneously to INF-{gamma} for 3 h at the concentrations shown (*P < 0.01 versus control, #P < 0.05 versus INF-{gamma}-stimulated cells) (n = 4). (B) Real-time RT-PCR analysis of INF-{gamma}-stimulated (100 ng/ml) mesangial cells in the presence or absence of 15d-PGJ2 and the synthetic PPAR{gamma} antagonist GW9662 (10 µM) for 3 h at the concentrations shown (*P < 0.01 versus control, #P < 0.05 versus INF-{gamma} -stimulated cells) (n = 4). (C) The 15d-PGJ2 dose-dependently reduced INF-{gamma}-induced chemokine expression) (#P < 0.01 versus INF-{gamma}-stimulated cells). Data are expressed as x-fold mRNA expression. Chemokine expression of INF-{gamma}-stimulated cells = 1 (n = 4). (D) Mesangial cell viability was tested using an LDH-based cytotoxicity assay. Data are expressed in percentage (n = 4).

 
15d-PGJ2 decreases INF-{gamma}-induced chemokine protein expression
Given the well-established importance of IP-10/CXCL10 and Mig/CXCL9 (in contrast to I-TAC/CXCL11) in T cell recruitment in inflammatory disease, we focused on the characterization of the underlying mechanisms of 15d-PGJ2 leading to reduced INF-{gamma}-induced IP-10/CXCL10 (and Mig/CXCL9) expression.

The IP-10/CXCL10 protein secretion of INF-{gamma}-stimulated MMCs was assessed using either ELISA or western blot analysis, and Mig/CXCL9 production was assessed by ELISA. Cells were stimulated with INF-{gamma} (100 ng/ml) in the presence or absence of 15d-PGJ2 for 24 h before harvesting the supernatants. Stimulation with INF-{gamma} led to a significantly increased secretion of IP-10/CXCL10 (P ≤ 0.02) and Mig/CXCL9 (P ≤ 0.01); both chemokines were reduced in a dose-dependent manner by 15d-PGJ2, with the highest reduction at 10 µM (P ≤ 0.02 compared with INF-{gamma}-stimulated cells) (Figure 3A). Western blot analysis of the cell culture supernatants with an anti-IP-10/CXCL10 antibody confirmed the IP-10/CXCL10 ELISA results (Figure 3B).


Figure 3
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  		Fig. 3 The 15d-PGJ2 reduces INF-{gamma}-induced, functionally active chemokine expression. (A) IP-10/CXCL10 and Mig/CXCL9 release of mouse mesangial cells was measured by ELISA. Before harvesting of supernatants, MMCs were stimulated with INF-{gamma} in the presence or absence of 15d-PGJ2 for 24 h at the concentrations indicated. (B) Representative IP-10/CXCL10 western blot analysis of supernatants of stimulated and unstimulated mesangial cells. Recombinant CXCL10 protein served as a positive control. (C) The chemotactic activity of supernatants of stimulated mouse mesangial cells was assessed in a chemotactic assay using CXCR3-transfected cells. Results are expressed as chemotactic index (basal = 1) (*P< 0.05 versus basal, #P < 0.05 versus INF-{gamma}-stimulated cells, n = 3).

 
To analyse the potential functional relevance of inhibition by 15d-PGJ2 of INF-{gamma}-induced IP-10/CXCL10 protein formation in MMCs, we performed in vitro chemotaxis assays using CXCR3-transfected pre-B mouse cells and supernatants from cultured MMCs. The results demonstrated that supernatants of INF-{gamma}-stimulated MMCs had high chemotactic potency on CXCR3-transfected cells. Most importantly, in the presence of 15d-PGJ2, the chemotactic activity of INF-{gamma}-stimulated supernatants was reduced by about 70%, indicating ameliorated IP-10/CXCL10 protein production (Figure 3C). Application of a neutralizing polyclonal anti-IP-10/CXCL10 antibody [6] to INF-{gamma}-stimulated supernatants showed that IP-10/CXCL10 was the main chemotactic stimulus for CXCR3-transfected cells.

INF-{gamma} activates the JAK/STAT signalling pathway in MMCs
Previous reports have indicated that the JAK/STAT signalling pathway, while activated in response to a large number of ligands, appears to be essential for INF-{gamma}-mediated gene regulation. We therefore analysed JAK/STAT signalling in INF-{gamma}-stimulated MMCs. INF-{gamma} stimulation rapidly induces (3 min) the phosphorylation of the tyrosine residues 1007/1008 of JAK2 and 1022/1023 of JAK1 and STAT1{alpha} at tyrosine 701 as assessed by western blotting using a phosphor-specific antibody (Figure 4A).


Figure 4
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  		Fig. 4 INF-{gamma}-induced JAK/STAT1 activation in mouse mesangial cells. (A) Western blot analysis using phosphorylated-specific JAK1, JAK2 and STAT1 antibodies shows INF-{gamma} induced phosphorylation within 3 min, but did not influence total JAK1, JAK2 and STAT1 protein level. In the lower panel the density ratio of phosphorylated to total protein is shown as mean ± SD of three independent experiments. (B) Mouse mesangial cells were stimulated with INF-{gamma}, and after 3 h nuclear proteins were isolated and EMSA with STAT1 consensus oligonucleotide was performed. (C) IP-10/CXCL10, Mig/CXCL9 and I-TAC/CXCL11 RNA expression assessed by RT-PCR after INF-{gamma} stimulation in the presence and absence of the JAK2 inhibitor AG 490 (*P < 0.01 versus control, #P < 0.05 versus INF-{gamma} -stimulated cells) (expressed as mean ± SD of three independent experiments).

 
The electrophoretic mobility shift assay (EMSA) demonstrated STAT1{alpha} nuclear translocation and DNA binding to STAT1{alpha} consensus oligonucleotides 3 h after INF-{gamma} stimulation (Figure 4B). Unlabeled specific STAT1 and unspecific competitors were used in 50-fold molar excess, demonstrating the specificity of the nuclear DNA binding. Supershift experiments using an anti-STAT1 antibody leads to a reduction in the density of the band indicating the presence of STAT1. In contrast, an anti-NF-{kappa}B p50 antibody did not affect the band density (Figure 4B). NF-{kappa}B and AP-1 were not induced by INF-{gamma} (data not shown) underscoring the unique role of JAK/STAT activation in IP-10/CXCL10, Mig/CXCL9 and I-TAC/CXCL11 expression after INF-{gamma} stimulation in MMCs.

To address the functional relevance of the JAK/STAT pathway in INF-{gamma}-induced IP-10/CXCL10, Mig/CXCL9 and I-TAC/CXCL11 expression, we used the selective pharmacological JAK2 inhibitor AG 490. Upregulated chemokine expression with INF-{gamma} was inhibited by AG 490 at a concentration of 100 µM (ca. 90% reduction for IP-10/CXCL10 and Mig/CXCL9 and ca. 80% for I-TAC/CXCL11) (Figure 4C). Treatment of MMCs with AG 490 at a concentration up to 300 µM did not affect cell viability (data not shown).

Inhibition of INF-{gamma}-induced JAK/STAT signalling in MMCs by 15d-PGJ2
In order to assess whether the JAK/STAT signalling pathways are targeted by 15d-PGJ2, we stimulated MMCs with INF-{gamma} in the presence or absence of 15d-PGJ2 and analysed JAK/STAT activation. As shown in Figure 5A, stimulation with INF-{gamma} in the presence of 10 µM 15d-PGJ2 for 15 min decreased JAK1 and STAT1{alpha} and to a lesser degree JAK2 phosphorylation. This 15d-PGJ2 effect was attenuated at later time points. To test the effects of the different PPAR{gamma} agonists on STAT1{alpha} phosphorylation, cells were stimulated with 100 ng/ml INF-{gamma} in the presence of troglitazone (10 µM) and 15d-PGJ2 (10 µM) for 15 min and compared to INF-{gamma}-stimulated cells. As depicted in Figure 5B, 15d-PGJ2 again led to a decrease in INF-{gamma}-induced JAK1, JAK2 and STAT1{alpha} phosphorylation. In contrast, application of troglitazone (Figure 5B) and ciglitazone (data not shown) had no effect on IFN-{gamma}-induced JAK/STAT1 signalling activation. The amount of total JAK1, JAK2 and STAT1{alpha} protein in MMCs was not affected by any treatment (Figure 5B). In some western blot experiments beta-actin was used as an internal loading control that confirmed that there was no change in total JAK and STAT protein levels (data not shown).


Figure 5
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  		Fig. 5 The 15d-PGJ2 inhibited INF-{gamma}-induced activation of the JAK/STAT1 signalling pathway. (A) Time course of INF-{gamma}-induced JAK/STAT phosphorylation assessed by western blotting with and without 15d-PGJ2 (10 µM). In the lower panel the density ratio of phosphorylated to total protein is shown as mean ± SD of three independent experiments. (B) Effect of different PPAR{gamma} agonists (all 10 µM) on INF-{gamma}-induced JAK1, JAK2 and STAT1 phosphorylation at 15 min. In the lower panel the density ratio of phosphorylated to total protein is shown as mean ± SD of three independent experiments. (C) Mouse mesangial cells were stimulated with INF-{gamma} in the presence or absence of 15d-PGJ2 (10 µM) and the synthetic PPAR{gamma} ligand troglitazone (10 µM). Three hours after stimulation, nuclear proteins were isolated and gel shift assays for STAT1 was performed (expressed as mean ± SD of three independent experiments).

 
Nuclear extracts from INF-{gamma}-stimulated MMCs (3 h) in the presence or absence of PPAR{gamma} ligands were isolated, and gel shift assays were performed with STAT1{alpha} consensus oligonucleotides. As shown in Figure 5C, 15d-PGJ2 markedly reduced INF-{gamma}-induced STAT1{alpha} DNA-binding activity, whereas the synthetic PPAR{gamma} ligand troglitazone had no effect.



   Discussion
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 Methods
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References
 
The molecular signalling pathways that initiate an inflammatory reaction have been extensively studied. However, the mechanisms that are important for an attenuation of the proinflammatory mediators—and thus potentially for a switch from the inflammatory process toward ‘resolution’ or healing—are less well characterized [19,20]. Studies by Lawrence and colleagues indicated that the resolution of inflammation can be regarded as an integral component of the programme of acute inflammatory reactions. They suggested that although cyclooxygenase 2 induces the onset of inflammation through formation of pro-inflammatory mediators (e.g., prostaglandin E2), it also plays a role in the inflammatory resolution process through production of anti-inflammatory mediators, such as 15d-PGJ2 [21]. The potential role of the PPAR{gamma} agonist 15d-PGJ2 as an endogenous anti-inflammatory mediator is a focus of ongoing research activities [14,22,23].

The kidney has been shown to express differentially all PPAR isoforms [24]. PPAR{alpha} is predominantly expressed in proximal tubules and medullary thick ascending limbs, while PPAR{gamma} is expressed in medullary collecting ducts and glomerular mesangial cells. PPARβ is expressed at low levels in all segments of the nephron. The availability of PPAR-selective agonists and antagonists provides an approach to modulate the renal response to diseases, including glomerulonephritis. Rovin and colleagues demonstrated that treatment of mesangial cells with the PPAR{gamma} activator 15d-PGJ2 at a concentration of 50 µM blocked IL1β-induced MCP-1/CCL2 mRNA expression and protein production by reducing the degradation of the NF-{kappa}B inhibitor I{kappa}B-alpha [13].

In this study we focussed on the influence of 15d-PGJ2 on the expression of IP-10/CXCL10, an INF-{gamma}-inducible chemokine whose transcriptional regulation is mainly dependent on JAK/STAT signalling pathway activation. Gomez-Chiarri et al. showed that INF-{gamma}-stimulated mesangial cells express IP-10/CXCL10 [3]. Furthermore, a recent study demonstrated a mesangial and podocyte-specific expression pattern of IP-10/CXCL10 in a rat model of mesangioproliferative glomerulonephritis [25], and Romagnani et al. demonstrated mesangial expression of IP-10/CXCL10 in patients with IgA nephropathy by immunohistochemistry [26].

Interestingly, human and murine Th1 and Th2 cells display distinct patterns of chemokine receptor expression. Th1-polarized cells preferentially express CXCR3 and CCR5, while Th2 cells express higher amounts of CCR3, CCR4 and CCR8 [27]. It is assumed that the Th1 immune response is the dominant Th phenotype in some forms of glomerulonephritis (e.g., anti-GBM nephritis, ANCA-associated GNs) [5]. Expression of the three CXCR3 chemokine ligands IP-10/CXCL10, Mig/CXCL9 and I-TAC/CXCL11 by mesangial cells may therefore have a central role in the recruitment of CXCR3-positive Th1 cells into the glomerulus.

In a first step, we demonstrated that INF-{gamma}-induced RNA expression of IP-10/CXCL10, Mig/CXCL9 and I-TAC/CXCL11 in MMCs was dose-dependently reduced by the naturally occurring PPAR{gamma} ligand 15d-PGJ2 but not by the synthetic PPAR{gamma} activators troglitazone and ciglitazone. In addition, the PPAR{gamma} blockade by the pharmacological inhibitor GW9662 did not influence the effect of 15-{Delta}PGJ2 on INF-{gamma}-induced chemokine expression. Taken together these results strongly argue against a PPAR{gamma}-dependent mechanism. Chemotactic assay experiments using CXCR3-transfected cells demonstrated that the 15d-PGJ2-mediated reduction of IP-10/CXCL10 RNA and protein formation was of functional relevance.

INF-{gamma} induces a rapid tyrosin phosphorylation of JAK1, JAK2 and STAT1{alpha} that leads to nuclear translocation of STAT1{alpha} and DNA binding. Moreover, the blockade of JAK2 by the pharmacological inhibitor AG 490 significantly reduced INF-{gamma}-induced IP-10/CXCL10, Mig/CXCL9 and I-TAC/CXCL11 RNA expression in mesangial cells. Having established that JAK/STAT signalling is involved in INF-{gamma}-induced IP-10/CXCL10 expression we next examined the effect of 15d-PGJ2 on this signalling pathway. Treatment with INF-{gamma} in the presence of 15d-PGJ2 decreased the amount of JAK1, JAK2 and STAT1{alpha} phosphorylation and STAT1 translocation to the nucleus. These results indicate that the inhibitory effect of 15d-PGJ2 on INF-{gamma}-induced IP-10/CXCL10 expression is mediated by inhibition of the JAK/STAT1{alpha} signalling pathway. The results are in agreement with a recent study in murine macrophages, which showed that 15d-PGJ2 inhibits the INF-{gamma}-mediated nitric oxide synthase induction by blocking the upstream STAT1 signalling pathway independently of PPAR{gamma} activation [28].

The in vivo function of 15d-PGJ2 as an endogenous anti-inflammatory mediator has recently been challenged by some investigators mainly because of the low concentration of free15d-PGJ2 measured in diverse human tissues and cells [29,30]. Although there is an ongoing debate about the biological role of 15d-PGJ2, possible explanations for a function despite the low levels of concentration may be derived from newer studies showing covalent binding of 15d-PGJ2 to multiple proteins, thus making it difficult to detect 15d-PGJ2 and also showing covalent binding of 15d-PGJ2 to PPAR{gamma} with a possible accumulative effect through irreversible activation [31]. Further research is necessary to elucidate the in vivo importance of 15d-PGJ2 in more detail.

In summary, our data demonstrate that 15d-PGJ2 inhibits INF-{gamma}-induced chemokine expression in mesangial cells by targeting the JAK/STAT1 signalling pathway independently of PPAR{gamma} activation. These findings indicate that 15d-PGJ2 exerts anti-inflammatory effects by attenuating the formation of Th1 cell-specific chemokines. Enhancement of endogenous 15d-PGJ2 formation or exogenous application might be a new therapeutic option in the treatment of T cell-mediated glomerular inflammation.



   Acknowledgments
 
This work was supported by a grant from the Deutsche Forschungsgemeinschaft to U.P. (PA 754/6-3).

Conflict of interest statement. None declared.



   Notes
 
* These authors contributed equally to the work. Back



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 References
 

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Received for publication: 18.10.07
Accepted in revised form: 4. 6.08


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