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–Stimulated Group IIA Phospholipase A2 Expression by Peroxisome Proliferator–Activated Receptor Activators in Rat Mesangial Cells
Pharmazentrum Frankfurt, Klinikum der Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany.
Correspondence to Dr. Marietta Kaszkin, Pharmazentrum Frankfurt, Klinikum der Johann Wolfgang Goethe-Universität, Theodor-Stern-Kai-7, D-60590 Frankfurt am Main, Germany. Phone: 49-69-6301-6955; Fax: 49-69-6301-7942; E-mail: kaszkin{at}em.uni-frankfurt.de
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Abstract |
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activators have an effect on sPLA2-IIA mRNA expression in rat mesangial cells, thus constituting a feedback modulation of sPLA2-IIA transcription. In the presence of tumor necrosis factor– (TNF-), the PPAR agonists WY14643 and LY171883 as well as the lipid-lowering compound clofibrate potentiated expression, secretion, and activity of group IIA sPLA2 in mesangial cells. MK886, known as a noncompetitive inhibitor of PPAR, completely abolished the potentiation of sPLA2-IIA secretion and activity by WY14643, thus indicating that the effect of WY14643 is specifically mediated by PPAR. When cells were transfected with different constructs of the rat sPLA2-IIA promoter fused to a luciferase reporter gene, a stimulation with TNF- in the presence of the PPAR activators caused an enhanced promoter activity compared with that induced by TNF- alone. Site-directed mutagenesis of a putative PPRE site in the sPLA2-IIA promoter abolished the potentiating effect of PPAR agonists, thus strongly indicating its contribution to the enhanced promoter activity. In summary, this study shows that the rat sPLA2-IIA promoter is sensitive to PPAR agonists, which act synergistically with cytokines, resulting in an enhanced expression of sPLA2-IIA in rat mesangial cells. |
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(TNF-) or interleukin-1ß (1). sPLA2-IIA secreted by glomerular mesangial cells is thought to participate in the initiation and progression of proinflammatory reactions, eventually leading to glomerulosclerosis. sPLA2-IIA may act as an important mediator in promoting the action of the cytosolic phospholipase A2 (cPLA2) (2) by stimulating arachidonic acid release and, subsequently, increasing the amount of free fatty acids and prostaglandins (3). Moreover, in stimulated cells, cPLA2 and sPLA2-IIA may catalyze the release of a broad spectrum of free fatty acids. These lipid metabolites may themselves act as positive feedback modulators by activating the transcription of sPLA2-IIA gene expression. In human embryonic kidney 293 cells that overexpress sPLA2-IIA, it has been shown that polyunsaturated fatty acids potentiate cytokine-stimulated arachidonic acid release and subsequent prostaglandin production (4).
One possible and attractive mechanism of action of these lipid metabolites is via the family of peroxisome proliferator-activated receptors (PPAR). They belong to the family of nuclear receptors, which bind diverse endogenous saturated and unsaturated fatty acids and their metabolites (5) and which are key regulators of the expression of many genes involved in lipid uptake and catabolism (6,7). Three PPAR subtypes have been identified and are denoted as , ß/
, and 
. PPAR is mainly involved in the catabolism of fatty acids in the liver, whereas PPAR modulates the storage of fatty acids in adipose tissue. PPAR ß/ is ubiquitously expressed; however, its physiologic function has yet to be fully defined. As natural ligands, saturated and unsaturated fatty acids as well as eicosanoids such as leukotriene B4 and 15-deoxy-
12,14-prostaglandin J2 (15-dPGJ2) have been identified (5). Furthermore, therapeutic drugs such as the lipid-lowering fibrates and the antidiabetic thiazolidinediones are ligands for PPAR and PPAR, respectively (5).
In several studies, PPAR have been characterized as important mediators of lipid second messenger–induced transcription of genes that may negatively regulate inflammatory responses (8). Particularly in the liver, leukotriene B4 was found to activate PPAR, resulting in an increase in the expression of enzymes, which are involved in the catabolism of leukotrienes, thus proposing an antiinflammatory role of PPAR (9). Moreover, PPAR negatively regulates the vascular inflammatory response by interfering with the activation of nuclear factor–
B and activator protein–1 (AP-1) in smooth-muscle cells (10).
On the other hand, only little evidence has been presented concerning the induction of proinflammatory processes by PPAR activation. The only study known so far in this respect was performed by Hill et al. (11), who have shown that fibrates increase the plasma levels of TNF- in mice via PPAR.
All PPAR subtypes are also expressed in the kidney at distinct locations in the nephron, which implicates different roles of PPAR subtypes in renal functions (12). A recent review described the involvement of PPAR in physiologic processes in the kidney (13). The regulation of fatty acid ß oxidation by PPAR represents an important mechanism for keeping the balance of energy production and use in the kidney. Besides metabolic functions, PPAR seems to have an important role in cytoprotection, given that PPAR null mice subjected to ischemia/reperfusion injury exhibited worse kidney function compared with wild-type controls (14). However, little is known about the role of the different PPAR and their endogenous ligands or synthetic activators in pathophysiological processes in the kidney.
The thiazolidinediones known to act as PPAR activators have been described as having direct beneficial effects in diabetic nephropathy (15). In renal mesangial cells, however, only a model system for studying proinflammatory responses covering the role of PPAR has been published. An involvement of the PPAR agonist 15-dPGJ2 has been shown in the inhibition of inducible nitric oxide synthase expression (16,17), in the modulation of proliferation and differentiation (18), and in the regulation of extracellular matrix expression (19).
Because sPLA2-IIA is thought to exert proinflammatory functions in mesangial cells at early phases of renal dysfunction by modulation of fatty acid release and eicosanoid formation, we investigated the effects of different PPAR activators on the regulation of this enzyme. Moreover, we performed transfection experiments with different deletion and point mutants of the rat sPLA2-IIA promoter, to identify the functionality of a putative PPAR binding site. We demonstrate that PPAR agonists significantly enhanced the cytokine-stimulated sPLA2-IIA expression and secretion through a PPAR-sensitive site in the sPLA2-IIA promoter.
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was a generous gift from Knoll AG (Ludwigshafen, Germany). [1-14C]oleic acid and [32P]dCTP (110 TBq mmol-1) were from Amersham-Pharmacia (Freiburg, Germany). Immobilone-PVDF membranes were purchased from Millipore (Eschborn, Germany), and Nylon membranes (Gene Screen) were purchased from NEN Life Science (Köln, Germany). All cell culture media and nutrients were from Life Technologies BRL (Eggenstein, Germany), and all other chemicals used were from either Sigma (Munich, Germany), Calbiochem (Bad Soden, Germany), or Biomol (Hamburg, Germany).
Cell Culture
Rat renal mesangial cells were cultured and characterized as described elsewhere (20). Cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum, penicillin (100 U/ml), streptomycin (100 µg/ml), and bovine insulin (0.66 units/ml); 24 h before stimulation and during the experiments, cells were incubated in Dulbecco’s modified essential medium that contained 0.1 mg/ml fatty acid–free bovine serum albumin (BSA).
Reverse Transcription–PCR
Expression of mRNA for the different PPAR receptor subtypes was analyzed by reverse transcription (RT)–PCR that used a total of 5 µg RNA. First-strand cDNA was transcribed with superscript II RNase H–RT obtained from Life Technologies BRL and Oligo d 15-Primer (Promega). PCR was performed on a Perkin Elmer Thermal Cycler with specific primers for each subtype as follows: PPAR: sense, 5'-ctgcagacctcaaatctctgg-3' and antisense, 5'-ggtgatgaagccattgcc-3', amplified product 422 bp; PPARß: sense, 5'-agatcagcgtgcatgtgttc-3' and antisense, 5'-gaagaggtactggctgtcgg-3', amplified product 470 bp; PPAR: sense, 5'-ccgagaaggagaagctgttg-3' and antisense, 5'-ttattcatcagggaggccag-3', amplified product 445 bp.
The different cDNA probes were amplified in a prepared Mastermix that contained dNTPs, specific primers, and Red Taq polymerase (Sigma) in the corresponding PCR buffer. For the PCR reactions, the following sequences were performed: PPAR, 94°C for 4 min (1 cycle) followed immediately by 94°C for 1 min, 60°C for 1 min 30 s, and 72°C for 3 min (30 cycles) and a final extension phase at 72°C for 10 min; PPARß, 94°C for 1 min (1 cycle) followed immediately by 94°C for 1 min, 60°C for 1 min 30 s, and 72°C for 3 min (31 cycles) and a final extension phase at 72°C for 10 min; and PPAR, 94°C for 1 min (1 cycle) followed immediately by 94°C for 1 min, 61°C for 1 min 30 s, and 72°C for 3 min (36 cycles) and a final extension phase at 72°C for 10 min. Amplified PCR products were separated on 1.4% agarose gels that contained 0.5 µg/ml ethidium bromide.
The PCR products were purified with the QIAquick PCR purification kit (Qiagen, Hilden, Germany) for sequencing by use of a kit based on the dye terminator technology (Perkin Elmer Applied Biosystems, Weiterstadt, Germany) in combination with the automated sequence analyzer A310 (Perkin Elmer Applied Biosystems).
sPLA2 Assay
sPLA2 activity in the supernatant of mesangial cell cultures was determined with [1-14C]-oleate–labeled Escherichia coli as substrate, as described elsewhere (21). Briefly, assay mixtures (1 ml) contained 100 mM Tris/HCl (pH 7.0), 1 mM CaCl2, [1-14C]-oleate–labeled E. coli (
5000 cpm), and 5 µl of the enzyme-containing supernatants of the cell cultures, which is sufficient to produce <5% substrate hydrolysis to be in a linear range. Reaction mixtures were incubated for 30 min at 37°C in a thermomixer. The extraction of the lipids was performed by the Dole method exactly as described elsewhere (3). Free [1-14C]-oleate was measured in a ß-counter.
Northern Blot Analysis
Confluent mesangial cells were cultured in 100-mm diameter culture dishes. After stimulation, cells were washed twice with phosphate-buffered saline (PBS) and harvested with a rubber policeman. Total cellular RNA was extracted from the cell pellet by use of the guanidinium isothiocyanate/phenol/chloroform method; 20 µg total RNA was separated on a 1.4% agarose/formaldehyde gel, transferred to a gene screen membrane, and hybridized to the radiolabeled cDNA fragments for sPLA2-IIA or glyceraldehyde-3-phosphate dehydrogenase (GAPDH). For quantification, the signals of the filters were scanned densitometrically. The signal density of each of the RNA samples hybridized to the sPLA2-IIA probe was divided by that hybridized to the GAPDH probe.
Western Blot Analysis
sPLA2-IIA protein secretion by the cells was assayed by precipitating 500 µl of the culture supernatant with 200 µl of 20% TCA. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis that used a 15% polyacrylamide gel was performed under nonreducing conditions. The proteins were transferred to Immobilone-PVDF membranes for 30 min at 2 mA/cm. Nonspecific binding was blocked with 2% BSA in PBS for 1 hr at room temperature, followed by incubation with primary antibody at a 1:100 dilution in 0.01% milk powder in PBS (the monoclonal antibody against rat sPLA2-IIA was a gift from Prof. Henk van den Bosch, Utrecht). The blot was incubated with horseradish peroxidase–conjugated goat anti-mouse Ig G (Amersham Pharmacia Biotech) at a 1:15000 dilution in blocking buffer. The washing steps were performed in 0.05% Tween 20 in PBS. After the washing step, peroxidase activity was detected by developing the blots according to the enhanced chemiluminescence method (Amersham Pharmacia Biotech).
Construction of Reporter Gene Fusions
A fusion of a rat 2.67-kb sPLA2-IIA promoter fragment (accession number AF375595) to the luciferase gene was generated by cloning a BamHI/KpnI fragment to the respective sites in the polylinker of the pGL3 basic vector (Promega). Construction of unidirectional nested deletion sets were performed with the Erase-a-Base System (Promega).
Site-Directed Mutagenesis
Mutations within the putative PPAR binding site -909 to -888 of the rat sPLA2-IIA promoter were introduced by PCR-based site-directed mutagenesis according to the manufacturer’s instructions (Stratagene). The gene-specific primers used in the PCR reactions for mutation of this site in the sPLA2-IIA promoter are shown in Table 1.
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–treated cells were set as 1.
Electrophoretic Mobility Shift Assay
The sequences of the double-stranded oligonucleotides used to detect the DNA binding activities of PPAR were chosen as described in Table 1. The complementary DNA strands were radioactively labeled by T4 polynucleotidase kinase by use of -32P-ATP (3000 Ci/mmol; Amersham-Pharmacia). Nuclear extracts from stimulated cells were isolated as described elsewhere (22), with the modification that buffer D was supplemented with 0.1% nooidet-40. Binding reactions were performed for 30 min at room temperature with 5 µg of total protein in 20 µl of 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1 mM ethylenediaminetetraacetic acid, 10% glycerol, 1 µg acetylated BSA, 2 µg poly(dI-dC), 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 20,000 d.p.m. of 32P-labeled oligonucleotides. DNA-protein complexes were separated from unbound DNA probe on native 10% polyacrylamide gels at 20 mA in 34 mM Tris-HCl (pH 7.5), 17 mM sodium acetate, and 0.5 mM ethylenediaminetetraacetic acid (pH 8.0). Gels were vacuum dried and analyzed with a phosphorimager.
Statistical Analyses
Data are presented as mean ± SD (n = 3; in transfection experiments, n = 6) and show one representative experiment out of three with similar results. Statistical analyses were performed by t test to determine significant differences between two groups. For comparison of different drug concentrations with the control group, statistical analyses were performed by repeated-measures ANOVA followed by Dunnett’s test. P < 0.05 was considered significant.
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, PPARß, and PPAR, RT-PCR was performed. The RT-PCR products are shown in Figure 1, and their identity was confirmed by sequence analysis. As shown in Figure 1, rat mesangial cells as well as extracts of whole rat kidneys do express PPAR, PPARß, and PPAR isotypes.
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Activators on TNF-–Induced Enzyme Secretion and Activity of sPLA2-IIA (1 nM) stimulated the secretion of sPLA2-IIA, as shown by Western blot analysis (Figure 2A) and measurement of enzyme activity (Figure 2B) in the cell culture supernatants. Treatment of the cells with the PPAR activator WY14643 alone up to 100 µM had no effects on enzyme secretion and activity. Higher concentrations of WY14643 caused cytotoxic effects, as monitored by the MTT test (data not shown). A combination of TNF- and WY14643 resulted in a potentiation of the cytokine-stimulated secretion of sPLA2-IIA, with a maximal amplification of twofold (±0.3) seen at 100 µM of WY14643 (average of three independent experiments).
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activator (5), also enhanced the TNA-–stimulated sPLA2-IIA secretion and activity in a dose-dependent manner to a similar degree as WY14643 (Figure 2). The concentrations of all compounds tested in this study are in the range known to specifically stimulate PPAR activation in cell culture.
In summary, these results suggest that PPAR ligands positively modulate TNF-–stimulated sPLA2-IIA secretion. A comparable potentiating effect by these compounds was also observed after stimulation of mesangial cells with interleukin-1ß or interferon- (data not shown).
Effect of PPAR Activators on TNF-–Induced sPLA2-IIA mRNA Steady-State Levels
To evaluate whether this enhanced sPLA2-IIA induction is due to an increase in mRNA steady-state levels, Northern blot analysis was performed. The data in Figure 3 show that sPLA2-IIA mRNA was present in TNF-–stimulated mesangial cells as a single band of 
0.9 kb (Figure 3) when cDNA specific for rat sPLA2-IIA was used. WY14643 and LY171883 increased TNF-–induced sPLA2-IIA mRNA steady-state levels in rat mesangial cells in a concentration-dependent manner, up to a maximum of twofold. The compounds alone had no inducing effect. These data indicate that coinduction of TNF-–stimulated sPLA2-IIA expression by PPAR activators occurs at the transcriptional level.
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activators on sPLA2-IIA induction is not restricted to the prototypic ligands WY14643 and LY171883, we investigated the effects of clofibrate, a therapeutically used hypolipidemic drug and activator of PPAR (24). The results in Figure 4 show that, in rat mesangial cells, clofibrate exerts a dose-dependent enhancing effect on sPLA2-IIA mRNA induction and activity comparable to that of the other PPAR activators.
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Antagonist MK-886 on TNF-–Stimulated sPLA2-IIA Protein Secretion and Activity with only minimal inhibitory effects on PPARß and PPAR (26). To confirm that the action of WY14643 on sPLA2-IIA induction was specifically mediated via PPAR activation, cells were pretreated for 30 min with different concentrations of MK-886 and then treated with TNF- in the absence or presence of WY14643. MK-886 alone up to 3 µM had no effect on basal sPLA2-IIA protein secretion and activity and did not inhibit TNF-–stimulated sPLA2-IIA induction. Higher concentrations of MK-886 were toxic to the cells. However, 3 µM of MK-886 reduced the potentiation of sPLA2-IIA secretion (Figure 5A) and activity (Figure 5B) by WY14643 back to the levels obtained with TNF- alone. These results support our hypothesis that the WY14643-stimulated effects on sPLA2-IIA induction are mediated via PPAR activation.
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–Induced sPLA2-IIA Promoter Activity by PPAR Activators-induced enhancement in TNF-–mediated sPLA2-IIA mRNA steady-state levels is due to a potentiation of sPLA2-IIA gene transcription, we transiently transfected a 2.67-kb promoter fragment (accession number AF375595) that was fused to a luciferase reporter gene into mesangial cells. Luciferase activity was then measured in cells treated with TNF- or TNF- plus one of the PPAR ligands (WY14643, LY171883, or clofibrate) for 24 h after transfection. Figure 6 shows that TNF- significantly stimulated luciferase activity of the promoter construct by fourfold (P < 0.001). The degree of stimulation varied between twofold and fivefold in different experiments.
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ligands, when given alone, also stimulated luciferase activity that was at least as potent as TNF- alone. However, in contrast to TNF-, the increase in promoter activity by PPAR activators was not reflected by an appropriate mRNA and protein expression (Figures 2 and 3).
Coincubation of transfected mesangial cells with TNF- and the PPAR activators caused a significant amplification of the TNF-–stimulated luciferase activity. The degree of stimulation varied in several experiments between twofold and fourfold. These data indicate that the sPLA2-IIA promoter contains PPAR-sensitive regulatory elements, which act synergistically with TNF-–activated transcription.
Effect of the PPAR Activator WY14643 on Different Deletion Mutants of the sPLA2-IIA Promoter in TNF-–Stimulated Mesangial Cells
To identify the regions of the sPLA2-IIA promoter responsible for PPAR activator–triggered luciferase activity in TNF-–stimulated cells, the Eras-a-Base System (Promega) was used to get unidirectional nested deletion sets of the promoter. Four different lengths of the sPLA2-IIA promoter (2.2, 1.4, 0.4, and 0.177 kb) fused to the luciferase gene were transfected into rat mesangial cells and subsequently incubated with TNF- in the absence or presence of WY14643 (100 µM). The data in Figure 7A show that TNF- increased luciferase activity of all promoter constructs except the 0.177-kb construct, which was neither stimulated by TNF- alone nor by a coincubation of TNF- with WY14643 (Figure 7B).
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–stimulated luciferase activity by WY14643 was detectable only with the 2.2-kb and the 1.4-kb sPLA2-IIA promoter fragments. In contrast, the 0.4-kb promoter fragment, which is activatable by TNF-, was not modulated by WY14643 (Figure 7A). Comparable results were also obtained with other PPAR activators like LY171883 and clofibrate (data not shown). In summary, these data indicate that PPAR-sensitive regulatory elements reside between -1400 and -400 of the rat sPLA2-IIA promoter.
Point Mutation of the Putative PPAR Binding Site
Sequence analysis of the promoter region upstream of -400 revealed a putative PPAR binding site between -909 and -888 (Table 1). To investigate whether this region represents a functional PPAR binding site, a promoter construct comprising the 2.67-bp fragment with an exchange of 4 bp in this region was generated by site-directed mutagenesis denoted as the "PPRE mutant" (Table 1). Mesangial cells were transfected with this mutated construct stimulated with TNF- in the absence or presence of WY14643 (100 µM) or clofibrate (500 µM).
As shown in Figure 8, cells transfected with the PPRE mutant were still activated by TNF- to a similar degree as those transfected with the wild-type 2.67-kb promoter construct. However, the potentiation of luciferase activity by WY14643 or clofibrate was completely abolished with the PPRE mutant. This strongly indicates that this promoter sequence is essential for potentiation of cytokine-evoked sPLA2-IIA transcription by PPAR activators.
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to the putative PPRE in the sPLA2-IIA promoter, we performed electrophoretic mobility shift analysis using a radioactively labeled oligonucleotide comprising the PPRE site from -909 to -888 (Table 1). In unstimulated mesangial cells, no specific complex formation was observed. WY14643 as well as TNF- alone already stimulated the formation of a specific complex (Figure 9). Of importance, the TNF-–induced complex formation is strongly enhanced by WY14643. Similar results were obtained when an oligonucleotide that encompassed the PPAR consensus sequence (Figure 9) was used. Together, these results indicate that the identified PPRE site at -909 to -888 kb is functional and mediates the transcriptional activation of the sPLA2-IIA promoter by PPAR agonists.
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is commonly thought to mediate antiinflammatory effects during pathophysiological processes in different organs and tissues (9,24,27). In this study, we have shown that in rat renal mesangial cells structurally different PPAR agonists such as WY14643, LY171883, and clofibrate enhanced rather than inhibited cytokine-stimulated expression and secretion of proinflammatory sPLA2-IIA. MK-886, a noncompetitive inhibitor of PPAR (26), abolished the potentiating effects of WY14643 on sPLA2-IIA secretion and activity, which suggests that PPAR is involved in this process. Transfection of mesangial cells with deletion and point mutants of the sPLA2-IIA promoter and sequence analysis allowed the identification of a functional PPAR binding site between -888 and -909 bases upstream of the transcription starting point. When cells were transfected with a promoter construct that contained a point mutation in this site, the TNF-–stimulated luciferase activity was no longer potentiated by WY14643. Of interest, all PPAR agonists, when given alone, caused the activation of the sPLA2-IIA promoter; however, they had no significant effects on sPLA2-IIA mRNA steady-state and protein levels. This strongly indicates that a specific cytokine signal is required for efficient sPLA2-IIA expression and that PPAR agonists may modulate in a synergistic fashion cytokine-induced proinflammatory responses in mesangial cells. The relatively high concentrations of PPAR agonists that were required to obtain an effect in vitro raises the question regarding the in vivo relevance of these findings. However, the concentrations of all compounds tested are in the range known to specifically stimulate PPAR activation in intact cells (5–7,10).
Analysis of PPAR binding by electrophoretic mobility shift analysis showed that treatment with TNF- stimulated specific complex formation, indicative for PPAR binding to the putative PPRE identified in the rat sPLA2-IIA promoter. We suggest that, similarly to interleukin-1, which has been shown to cause a rapid activation of cPLA2 in rat mesangial cells (28), TNF- also stimulated an early activation of cPLA2 followed by the release of free fatty acids that then activate PPAR binding.
Surprisingly, activation of the sPLA2-IIA promoter by TNF- does not depend on the activation of the PPRE site, because point mutation of the sequence did not abolish promoter activation by TNF-, although the response to the PPAR ligands was completely lost. Obviously, PPAR agonists synergize with TNF- to optimize sPLA2-IIA expression.
Our results are in contrast to a recent report by Couturier et al. (29) that showed that, in rat vascular smooth-muscle cells, the sPLA2-IIA promoter is activated by PPAR but not PPAR agonists. They concluded that, in this cell system, cytokines use nuclear factor–B and PPAR for sPLA2-IIA gene expression.
Furthermore, in contrast to Couturier et al. (29), who described a functional low-affinity PPAR binding site between -160 and -133, we observed that promoter constructs <0.178 kb, i.e., without the nuclear factor–B binding site (30), were not activated by cytokines or PPAR agonists at all. We do not yet have an explanation for these discrepant findings. Our data clearly define the region between -909 and -888 upstream of the transcription start site as a functional PPRE site involved in the rat sPLA2-IIA promoter activation by cytokines and PPAR agonists.
An important question concerns the pathophysiological consequences of PPAR-mediated enhancement of cytokine-stimulated sPLA2-IIA induction in renal mesangial cells. We suggest that, at early stages of glomerular inflammatory processes, endogenous ligands of PPAR might support the expression of proinflammatory enzymes and mediators. Potent endogenous ligands for PPAR are fatty acids and eicosanoids (5), which may be produced in the early course of glomerulonephritis by invading inflammatory cells or by mesangial cells themselves. Indeed, in anti–Thy-1 nephritis, a rat model of mesangial cell injury, an invasion of neutrophils was observed 1 h after administration of anti–Thy-1 antibody followed by a marked increase in expression levels of cPLA2, COX-1, and COX-2 in the glomeruli, which produce a row of lipid metabolites as potential PPAR agonists (31). We have observed that, in rat mesangial cells, docosahexaenoic acid and the arachidonic acid analog ETYA, both known as PPAR activators (5), also potentiated sPLA2-IIA expression (data not shown). It is tempting to speculate that sPLA2-IIA itself may modulate its own expressional regulation by mediating eicosanoid biosynthesis either directly by mobilization of free fatty acids or indirectly by cross-communication and activation of cPLA2 (2). Thus, PPAR-activating eicosanoids in cooperation with proinflammatory cytokines may contribute to early effects during the development of renal dysfunction by enhancing the expression of enzymes such as sPLA2-IIA via PPAR.
A further hint for a pathophysiological role of PPAR activators in the kidney is the observation, that fibrates, which are therapeutically used for systemic reduction of serum lipid levels in patients with hyperlipidemia, can cause renal dysfunction. In particular, patients with organ transplants (32) or cardiac surgery (33), who often also have elevated serum cholesterol levels, developed an impairment of renal function during fibrate therapy, as indicated by increased creatinine values (34). After discontinuation of fibrate therapy, renal function was restored in most patients.
It is tempting to speculate that during inflammatory processes the lipid-lowering effects of fibrates may lead to an accumulation of circulating endotoxins and proinflammatory cytokines that are no longer associated with lipid-binding proteins and that then may potentiate the activity of macrophages (11). Further support comes from the observation that hypolipidic animals were found to have higher LPS-induced TNF levels and higher mortality than the control groups (35). Elevated sPLA2-IIA levels may even contribute to the degradation of lipid-binding proteins. Pruzanski et al. (36) have found that lipoproteins and, in particular, high-density lipoprotein, are substrates for human sPLA2-IIA. By this sPLA2-mediated modification, high-density lipoprotein changes its function from an anti-inflammatory to a proinflammatory particle (37).
In summary, PPAR may be a candidate mediator responsible for positive feedback or crosstalk regulation of sPLA2-IIA expression by free fatty acids or their derivates generated by either cytosolic or secreted phospholipases A2 or other lipid-metabolizing enzymes. In this context, PPAR activation may have considerable relevance for the early progression of glomerular inflammatory diseases. On the other hand, it cannot be excluded that, as a long-term effect, products of the action of sPLA2-IIA exhibit negative feedback effects by mobilizing other lipid-metabolizing enzymes via PPAR activation, which are involved in degradation, uptake, lipid oxidation, or lipid transport and thus may counteract the proinflammatory effects of sPLA2-IIA. Further studies in animal models of renal diseases are needed to explore the functional role of sPLA2-IIA in inflammatory processes in the kidney and the regulation and function of PPAR in this respect.
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Y. J. Jiang, B. Lu, F. Y. Xu, J. Gartshore, W. A. Taylor, A. J. Halayko, F. J. Gonzalez, J. Takasaki, P. C. Choy, and G. M. Hatch Stimulation of cardiac cardiolipin biosynthesis by PPAR{alpha} activation J. Lipid Res., February 1, 2004; 45(2): 244 - 252. [Abstract] [Full Text] [PDF]
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S. Beck, G. Lambeau, K. Scholz-Pedretti, M. H. Gelb, M. J. W. Janssen, S. H. Edwards, D. C. Wilton, J. Pfeilschifter, and M. Kaszkin Potentiation of Tumor Necrosis Factor {alpha}-induced Secreted Phospholipase A2 (sPLA2)-IIA Expression in Mesangial Cells by an Autocrine Loop Involving sPLA2 and Peroxisome Proliferator-activated Receptor {alpha} Activation J. Biol. Chem., August 8, 2003; 278(32): 29799 - 29812. [Abstract] [Full Text] [PDF]
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E. Cernuda-Morollon, F. Rodriguez-Pascual, P. Klatt, S. Lamas, and D. Perez-Sala PPAR Agonists Amplify iNOS Expression While Inhibiting NF-{kappa}B: Implications for Mesangial Cell Activation by Cytokines J. Am. Soc. Nephrol., September 1, 2002; 13(9): 2223 - 2231. [Abstract] [Full Text] [PDF]
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W. Eberhardt, E.-S. Akool, J. Rebhan, S. Frank, K.-F. Beck, R. Franzen, F. M. A. Hamada, and J. Pfeilschifter Inhibition of Cytokine-induced Matrix Metalloproteinase 9 Expression by Peroxisome Proliferator-activated Receptor alpha Agonists Is Indirect and Due to a NO-mediated Reduction of mRNA Stability J. Biol. Chem., August 30, 2002; 277(36): 33518 - 33528. [Abstract] [Full Text] [PDF]
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