Abstract
Rat renal mesangial cells express high levels of matrix metalloproteinase 9 (MMP-9) in response to inflammatory cytokines such as interleukin 1beta (IL-1beta). We tested whether ligands of the peroxisome proliferator-activated receptor (PPARalpha) could influence the cytokine-induced expression of MMP-9. Different PPARalpha agonists dose-dependently inhibited the IL-1beta-triggered increase in gelatinolytic activity mainly by decreasing the MMP-9 steady-state mRNA levels. PPARalpha agonists on their own had no effects on MMP-9 mRNA levels and gelatinolytic activity. Surprisingly, the reduction of MMP-9 mRNA levels by PPARalpha activators contrasted with an amplification of cytokine-mediated MMP-9 gene promoter activity and mRNA expression. The potentiation of MMP-9 promoter activity functionally depends on an upstream peroxisome proliferator-responsive element-like binding site, which displayed an increased DNA binding of a PPARalpha immunopositive complex. In contrast, the IL-1beta-induced DNA-binding of nuclear factor kappaB was significantly impaired by PPARalpha agonists. Most interestingly, in the presence of an inducible nitric-oxide synthase (iNOS) inhibitor, the PPARalpha-mediated suppression switched to a strong amplification of IL-1beta-triggered MMP-9 mRNA expression. Concomitantly, activators of PPARalpha potentiated the cytokine-induced iNOS expression. Using actinomycin D, we found that NO, but not PPARalpha activators, strongly reduced the stability of MMP-9 mRNA. In contrast, the stability of MMP-9 protein was not affected by PPARalpha activators. In summary, our data suggest that the inhibitory effects of PPARalpha agonists on cytokine-induced MMP-9 expression are indirect and primarily due to a superinduction of iNOS with high levels of NO reducing the half-life of MMP-9 mRNA.
Highlights
§ To whom correspondence should be addressed: Pharmazentrum Frankfurt, Klinikum der Johann Wolfgang Goethe-Universitat Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany
To test whether the inhibitory effects of the peroxisome proliferator-activated receptors (PPARs)␣ activators on the IL-1-induced matrix metalloproteinase 9 (MMP-9) mRNA levels depends on NO production, mesangial cells (MC) were treated with IL-1 and different PPAR␣ agonists in the presence or absence of the NOS inhibitor L-NMMA
We demonstrate that various structurally different PPAR␣ agonists such as WY-14,643, LY-171883, and fibrates potently suppress cytokine-induced matrix metalloproteases (MMPs)-9 expression in renal MC
Summary
Reagents—Human recombinant IL-1 was from Cell Concept (Umkirch, Germany). The PPAR␣ activators were WY-14,643 and LY171883, and the NO donors were DETA-NONOate and S-nitroso-Dpenicillamine. A glyceralaldehyd-3-phosphate dehydrogenase cDNA clone was generated using the internal primers of the coding sequence of rat glyceralaldehyd-3-phosphate dehydrogenase mRNA (accession number NM017008). Like site (GT to CA) to generate pGL-MMP-9-⌬PPRE-1 was performed using the forward primer 5Ј-ATG GAG ACT CAA GCA CAC CTA TGT GT-3Ј (corresponding to a region from Ϫ1763 to Ϫ1738). Cell-free Incubation Experiments—In these experiments we tested the effects of the PPAR␣ agonist WY-14,643 on MMP activities in the conditioned culture medium harvested from MC. Nuclear RNA and 100,000 cpm of the labeled T7-derived MMP-9 antisense transcript were co-precipitated by ethanol precipitation and hybridized at 42 °C overnight in 30 l of FAB hybridization buffer containing 80%, 1 mM EDTA, 40 mM PIPES (pH 6.4), and 400 mM NaCl. After hybridization, samples were digested with RNase A and T1 for 1 h at 30 °C. Statistical analysis was performed using Student’s t test and analysis of variance for significance. p values Ͻ 0.01 (** or ##) were considered significant
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