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.Remodeling of extracellular matrix (ECM) is an important feature of normal growth and developmental processes. Consequently, an imbalance of ECM synthesis and degradation is associated with many diseases. Although changes in the synthesis of ECM may play a certain role in dysregulation of matrix turnover, recent studies have underlined the paramount role of ECM-degradative systems. The main proteases regulating physiological degradation of ECM are the matrix metalloproteinases (MMPs), a family of zinc-dependent enzymes, including the interstitial collagenases, stromelysins, elastases, membrane-type MMPs, and gelatinases (8,38,61,62). Tight regulation of most of these proteases is accomplished by different mechanisms, including the regulation of MMP gene expression, the processing and conversion of the inactive zymogens by other proteases, and, finally, the inhibition of active MMPs by endogenous inhibitors of MMPs, the tissue inhibitors of metalloproteinases (for a review, see reference 38).Cultured mesangial cells (MC) respond to proinflammatory cytokines such as tumor necrosis factor alpha (TNF-␣) or interleukin-1 beta (IL-1␤) with the production of several MMPs, including MMP-9 (gelatinase-B), mainly by an increase of gene expression (20,65). In addition to MMPs, MC exposed to cytokines produce high levels of NO through the expression of the inducible NO synthase (iNOS) gene (30,42). Whereas the early and rapid actions of NO signaling affect pos...

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Renal mesangial cells express high levels of matrix metalloproteinase 9 (MMP-9) in response to inflammatory cytokines such as interleukin (IL)-1␤. We demonstrate here that the stable ATP analog adenosine 5-O-(thiotriphosphate) (ATP␥S) 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 ATP␥S 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 ATP␥S 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 ATP␥S. 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 ATP␥S, but mutation of all three ARE motifs caused a loss of ATP-dependent increase in luciferase activity without affecting IL-1␤-inducibility. By confocal microscopy we demonstrate that ATP␥S 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.The matrix metalloproteinases (MMPs) 1 are members of a family of zinc-dependent endopeptidases which specifically degrade components of the extracellular matrix (ECM). Therefore, MMPs have mainly been implicated in various diseases accompanied with an altered turnover of ECM. Besides the altered synthesis of single ECM components the increased expression and/or activity of MMPs seems of paramount importance for pathological remodeling processes within the kidney such as acute proliferative glomerulonephritis (1, 2). Mainly the altered expression of MMP-2 and MMP-9, which are also denoted as gelatinases, is crucially involved in the progression of glomerular inflammatory processes (2, 3). In addition to various inflammatory cytokines, the expression of MMP-9 can be activated by many other stimuli such as mitogens, growth factors, and activators of the Ras oncogene (for review, see Refs. 4 and 5). Although most of these stimuli can modulate gelatinolytic activity by influencing MMP-9 gene expression, the regulation of MMP-9 activity is also achieved by the processing of the inactive proenzyme by the action of other proteases and by an inhibition of the active enzyme by its endogenous inhibitors, the tissue inhibitors of MMPs (4,5). Previously, we have demonstrated an additional mode of posttranscript...