ATP Potentiates Interleukin-1β-induced MMP-9 Expression in Mesangial Cells via Recruitment of the ELAV Protein HuR

Abstract

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...

Increased globulin and its association with hemorrhagic transformation in patients receiving intra-arterial thrombolysis therapy

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Differential modulation of the cytokine-induced MMP-9/TIMP-1 protease–antiprotease system by the mTOR inhibitor rapamycin

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Expression of CD83 Is Regulated by HuR via a Novel cis-Active Coding Region RNA Element

“…Taking into account the data of the present study, this suggests that the RNA stabilization and nuclear export function of HuR can be separated from each other, depending on the RNA target sequence. According to this, the stabilization of mRNAs depends on the interaction of HuR with an ARE sequence and occurs in the cytoplasm (61,93,94). In contrast, the binding of HuR to the CD83 PRE does not affect the decay of transcripts bearing this RNA sequence.…”