Dissociated glucocorticoids are synthetic ligands of the glucocorticoid receptor (GR) and which discriminate between transrepression and transactivation. These compounds were predicted to have large therapeutic benefits when compared to conventional glucocorticoids because of reduced side effects. In this study, we compared the transrepressive properties of different dissociated glucocorticoids on the interleukin-1β (IL-1β)-activated metalloproteinase-9 (MMP-9) and tissue plasminogen activator (tPA) expression in rat mesangial cells (MC). Both proteinases regulate the turnover of extracellular matrix (ECM). We demonstrate that the GR agonist RU 24858, equipotent to dexamethasone (DEX), exhibited strong suppressive effects on the IL-1β-induced MMP-9 and tPA mRNA levels concomitant with an inhibition of corresponding enzyme activities. In contrast, RU 24782 and RU 40066 exhibited weaker inhibitory activities on both proteinases. Mechanistically, the changes in MMP-9 expression level by different RUs were accompanied by an inhibition of cytokine-induced promoter activity indicating that the inhibition occurs on a transcriptional level. In parallel to the reduction in mRNA levels, we observed an attenuation of cytokine-induced DNA binding of nuclear factor kappa B (NF-κB) and reduced contents of the p65 subunit of NF-κB within cell nuclei. Along with these transrepressive activities RU 24858, RU 24782 and RU 40066 displayed similar transactivation potentials as indicated by induction of the glucocorticoid-inducible mouse mammary tumor virus (MMTV) reporter gene and by induced expression level of plasminogen activator inhibitor 1 (PAI-1). Interestingly, the different RUs affected the expression of cAMP-induced tPA and inducible NO synthase with the same potency as the IL-1β-induced protease expression thus indicating that these compounds equipotently modulate cytokine- and cAMP-driven gene expression.

Excessive accumulation of the extracellular matrix is a hallmark of many inflammatory and fibrotic diseases, including those of the kidney. This study addresses the question whether NO, in addition to inhibiting the expression of MMP-9, a prominent metalloprotease expressed by mesangial cells, additionally modulates expression of its endogenous inhibitor TIMP-1. We demonstrate that exogenous NO has no modulatory effect on the extracellular TIMP-1 content but strongly amplifies the early increase in cytokine-induced TIMP-1 mRNA and protein levels. We examined whether transforming growth factor beta (TGFbeta), a potent profibrotic cytokine, is involved in the regulation of NO-dependent TIMP-1 expression. Experiments utilizing a pan-specific neutralizing TGFbeta antibody demonstrate that the NO-induced amplification of TIMP-1 is mediated by extracellular TGFbeta. Mechanistically, NO causes a rapid increase in Smad-2 phosphorylation, which is abrogated by the addition of neutralizing TGFbeta antisera. Similarly, the NO-dependent increase in Smad-2 phosphorylation is prevented in the presence of an inhibitor of TGFbeta-RI kinase, indicating that the NO-dependent activation of Smad-2 occurs via the TGFbeta-type I receptor. Furthermore, activation of the Smad signaling cascade by NO is corroborated by the NO-dependent increase in nuclear Smad-4 level and is paralleled by increased DNA binding of Smad-2/3 containing complexes to a TIMP-1-specific Smad-binding element (SBE). Reporter gene assays revealed that NO activates a 0.6-kb TIMP-1 gene promoter fragment as well as a TGFbeta-inducible and SBE-driven control promoter. Chromatin immunoprecipitation analysis also demonstrated DNA binding activity of Smad-3 and Smad-4 proteins to the TIMP-1-specific SBE. Finally, by enzyme-linked immunosorbent assay, we demonstrated that NO causes a rapid increase in TGFbeta(1) levels in cell supernatants. Together, these experiments demonstrate that NO by induction of the Smad signaling pathway modulates TIMP-1 expression.

Dysregulation of extracellular matrix turnover is an important feature of many inflammatory processes. Rat renal mesangial cells express high levels of matrix metalloproteinase 9 (MMP-9) in response to inflammatory cytokines such as interleukin-1 beta. We demonstrate that NO does strongly destabilize MMP-9 mRNA, since different luciferase reporter gene constructs containing the MMP-9 3' untranslated region (UTR) displayed significant reduced luciferase activity in response to the presence of NO. Moreover, by use of an in vitro degradation assay we found that the cytoplasmic fractions of NO-treated cells contained a higher capacity to degrade MMP-9 transcripts than those obtained from control cells. An RNA electrophoretic mobility shift assay demonstrated that three of four putative AU-rich elements present in the 3' UTR of MMP-9 were constitutively occupied by the mRNA-stabilizing factor HuR and that the RNA binding was strongly attenuated by the presence of NO. The addition of recombinant glutathione transferase-HuR prevented the rapid decay of MMP-9 mRNA, whereas the addition of a neutralizing anti-HuR antibody caused an acceleration of MMP-9 mRNA degradation. Furthermore, the expression of HuR mRNA and protein was significantly reduced by exogenously and endogenously produced NO. These inhibitory effects were mimicked by the cGMP analog 8-bromo-cGMP and reversed by LY-83583, an inhibitor of soluble guanylyl cyclase. These results demonstrate that NO acts in a cGMP-dependent mechanism to inhibit the expression level of HuR, thereby reducing the stability of MMP-9 mRNA.

Renal mesangial cells express high levels of matrix metalloproteinase 9 (MMP-9) in response to inflammatory cytokines such as interleukin (IL)-1beta. We demonstrate here that the stable ATP analog adenosine 5'-O-(thiotriphosphate) (ATPgammaS) potently amplifies the cytokine-induced gelatinolytic content of mesangial cells mainly by an increase in the MMP-9 steady-state mRNA level. A Luciferase reporter gene containing 1.3 kb of the MMP-9 5'-promoter region showed weak responses to ATPgammaS but confered a strong ATP-dependent increase in Luciferase activity when under the additional control of the 3'-untranslated region of MMP-9. By in vitro degradation assay and actinomycin D experiments we found that ATPgammaS potently delayed the decay of MMP-9 mRNA. Gel-shift and supershift assays demonstrated that three AU-rich elements (AREs) present in the 3'-untranslated region of MMP-9 are constitutively bound by complexes containing the mRNA stabilizing factor HuR. The RNA binding of these complexes was markedly increased by ATPgammaS. Mutation of each ARE element strongly impaired the RNA binding of the HuR containing complexes. Reporter gene assays revealed that mutation of one ARE did not affect the stimulatory effects by ATPgammaS, but mutation of all three ARE motifs caused a loss of ATP-dependent increase in luciferase activity without affecting IL-1beta-inducibility. By confocal microscopy we demonstrate that ATPgammaS increased the nucleo cytoplasmic shuttling of HuR and caused an increase in the cytosolic HuR level as shown by cell fractionation experiments. Together, our results indicate that the amplification of MMP-9 expression by extracellular ATP is triggered through mechanisms that likely involve a HuR-dependent rise in MMP-9 mRNA stability.

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 PPARa 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 clement-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 PPARa potentiated the cytokine-induced iNOS expression. Using actinomycin D, we found that NO, but not PPARa activators, strongly reduced the stability of MMP-9 mRNA. In contrast, the stability of MMP-9 protein was not affected by PPARa activators. In summary, our data suggest that the inhibitory effects of PPARa 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.