Regulation of matrix-metalloproteinase 9 (MMP-9) in glomerular mesangial cells

Remodeling of extracellular matrix (ECM) is an important physiologic feature of normal growth and development. In addition to this critical function in physiology many diseases have been associated with an imbalance of ECM synthesis and degradation. In the kidney, dysregulation of ECM turnover can lead to interstitial fibrosis, and glomerulosclerosis. The major physiologic regulators of ECM degradation in the glomerulus are the large family of zinc-dependent proteases, collectively refered to matrix metalloproteinases (MMPs). The 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 zymogen by other proteases such as serine proteases and finally the inhibition of active MMPs by endogenous inhibitors of MMPs, denoted as tissue inhibitors of metalloproteinases (TIMPs). Namely, the MMP-9 has been shown to be critically involved in the dysregulation of ECM turnover associated with severe pathologic conditions such as rheumatoid arthritis or fibrosis of lung, skin and kidney. In the present work I searched for a possible modulation of MMP-9 expression and/or activity in glomerular mesangial cells which are thought as key players of many inflammatory and non-inflammatory glomerular diseases. I found that various structurally different PPARalpha agonists such as WY-14,643, LY-171883 and fibrates potently suppress the cytokine-induced MMP-9 expression in renal MC. Furthermore, I demonstrate that the inhibition of MMP-9 expression by PPARalpha agonists was paralleled by a strong increase of cytokine-induced iNOS expression and subsequent NO formation, suggesting that PPARalpha-dependent effects on MMP-9 expression level primarily result from alterations in NO production which in turn reduces the MMP-9 mRNA half-life. Searching for the detailed mechanism of NO-dependent effects on MMP-9 mRNA stability, I found that NO either given from exogenous sources or endogenously produced increases the MMP-9 mRNA degradation by decreasing the expression of the mRNA stabilizing factor HuR. Furthermore, I demonstrate a reduction in the RNA-binding capacity of HuR containing complexes to MMP-9 ARE motifs in cells treated with NO. Since the reduction of HuR expression can be mimicked by the cGMP analog 8-Bromo-cGMP, I suggest that NO reduces in a cGMP-dependent manner the expression of HuR. Finally, I elucidated the modulatory effect of extracellular nucleotides, mainly ATP, on cytokine-triggered MMP-9 expression. Interestingly, I found that in contrast to NO, gamma-S-ATP the stable analog of ATP potently amplifies the IL-beta mediated MMP-9 expression. The increase in mRNA stability was paralleled by an increase in the nuclear-cytosolic shuttling of the mRNA stabilizing factor HuR. Furthermore, I demonstrate an increase in the RNA-binding capacity of HuR containing complexes to the 3'-UTR of MMP-9 by ATP. In summary, the data presented here may help to find new targets (posttranscriptional regulation) that could be used to manipulate or modulate the expression of not only MMP-9 but also other genes regulated on the level of mRNA stability.Umbauprozesse der Extrazellulären Matrix (ECM) spielen eine wichtige Rolle für normale Wachstums- und Entwicklungsprozesse. In der Niere kann der fehlerhafte Umsatz von ECM beispielsweise zur interstitiellen Fibrose und Glomerulosklerose führen. Zu den wichtigsten physiologischen Regulatoren des Abbaus von ECM im Glomerulus zählen die Zink-abhängigen Proteasen, die zur Familie der Matrixmetalloproteasen (MMPs) zusammengefasst werden. In der vorliegenden Arbeit untersuchte ich schwerpunktmässig nach Möglichkeiten die MMP-9 Expression und/oder MMP-Aktivität in glomerulären Mesangiumzellen zu verändern. Mesangiumzellen gelten als Hauptakteure von glomerulären Erkrankungen mit entzündlichen- als auch nicht entzündlichen Genese. Wie ich gezeigt habe, sind unterschiedliche PPARalpha Agonisten wie beispielsweise WY-14,643, LY-171883 und Fibrate in der Lage, die Zytokin-induzierte MMP-9 Expression in Mesangiumzellen potent zu hemmen. Weiterhin konnte von mir gezeigt werden, dass die Hemmung der MMP-9 Expression durch PPARalpha Aktivatoren mit einer Steigerung der iNOS Expression und der unmittelbaren Steigerung der NO Freisetzung einhergeht. Interessanterweise konvertieren die hemmenden Effekte der PPARalpha Aktivatoren in der Gegenwart eines iNOS Hemmstoffes zu einer massiven Verstärkung der Zytokin-induzierten MMP-9 Expression was darauf hinweist, dass die PPARalpha-vermittelten Effekte in erster Linie durch Veränderungen der NO Synthese hervorgerufen werden. Auf der Suche nach dem Mechanismus der NO-vermittelten Effekte auf die MMP-9 Expression konnte ich zeigen, dass sowohl exogen zugeführtes NO als auch über eine Induktion der iNOS entstandenes NO, in der Lage ist, den Abbau von MMP-9 mRNA durch eine Expressionshemmung des mRNA Stabilitätsfaktor HuR zu beschleunigen. In einem weiteren Projekt untersuchte ich, ob extrazellulären Nukleotide in der Lage sind, einen modulierenden Einfluss auf die Zytokin-induzierte MMP-9 Expression auszuüben. Ich konnte zeigen, dass das chemisch stabilisierte ATP-Analog gamma-S ATP im Unterschied zu NO in der Lage ist, den IL- 1beta vermittelten Anstieg der MMP-9 in potenter Weise zu verstärken. Der Anstieg der mRNA Stabilität korreliert mit einer Zunahme des Transports von HuR aus dem Zellkern in das Zytoplasma. Der verstärkte Export von HuR aus dem Zellkern war verbunden mit der verstärkten RNA Bindungsaffinität von HuR-haltigen Komplexen an AU-reiche Sequenzen innerhalb des 3'-untranslatierten Bereichs (3'-UTRs) des MMP-9 Gens. Zusammenfassend könnten die vorliegende Arbeit helfen, neue (posttranskriptionellen) Ansätzen zu finden, die eine spezifische Manipulation von MMP-9 und anderen auf Ebene der mRNA Stabilität regulierten Genen, ermöglichen

Long Term Stabilization of Expanding Aortic Aneurysms by a Short Course of Cyclosporine A through Transforming Growth Factor-Beta Induction

Abdominal aortic aneurysms (AAAs) expand as a consequence of extracellular matrix destruction, and vascular smooth muscle cell (VSMC) depletion. Transforming growth factor (TGF)-beta 1 overexpression stabilizes expanding AAAs in rat. Cyclosporine A (CsA) promotes tissue accumulation and induces TGF -beta1 and, could thereby exert beneficial effects on AAA remodelling and expansion. In this study, we assessed whether a short administration of CsA could durably stabilize AAAs through TGF-beta induction. We showed that CsA induced TGF-beta1 and decreased MMP-9 expression dose-dependently in fragments of human AAAs in vitro, and in animal models of AAA in vivo. CsA prevented AAA formation at 14 days in the rat elastase (diameter increase: CsA: 131.9±44.2%; vehicle: 225.9±57.0%, P = 0.003) and calcium chloride mouse models (diameters: CsA: 0.72±0.14 mm; vehicle: 1.10±0.11 mm, P = .008), preserved elastic fiber network and VSMC content, and decreased inflammation. A seven day administration of CsA stabilized formed AAAs in rats seven weeks after drug withdrawal (diameter increase: CsA: 14.2±15.1%; vehicle: 45.2±13.7%, P = .017), down-regulated wall inflammation, and increased αSMA-positive cell content. Co-administration of a blocking anti-TGF-beta antibody abrogated CsA impact on inflammation, αSMA-positive cell accumulation and diameter control in expanding AAAs. Our study demonstrates that pharmacological induction of TGF-beta1 by a short course of CsA administration represents a new approach to induce aneurysm stabilization by shifting the degradation/repair balance towards healing

ATP potentiates interleukin-1β-induced MMP-9 expression in mesangial cells via recruitment of the ELAV protein HuR

Renal mesangial cells express high levels of matrix metalloproteinase 9 (MMP-9) in response to inflammatory cytokines such as interleukin (IL)-1β. We demonstrate here that the stable ATP analog adenosine 5′-O-(thiotriphosphate) (ATPγS) 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 ATPγS 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 ATPγS 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 ATPγS. 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 ATPγS, but mutation of all three ARE motifs caused a loss of ATP-dependent increase in luciferase activity without affecting IL-1β-inducibility. By confocal microscopy we demonstrate that ATPγS 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

Posttranslational Modification of the AU-Rich Element Binding Protein HuR by Protein Kinase Cδ Elicits Angiotensin II-Induced Stabilization and Nuclear Export of Cyclooxygenase 2 mRNA▿

The mRNA stabilizing factor HuR is involved in the posttranscriptional regulation of many genes, including that coding for cyclooxygenase 2 (COX-2). Employing RNA interference technology and actinomycin D experiments, we demonstrate that in human mesangial cells (hMC) the amplification of cytokine-induced COX-2 by angiotensin II (AngII) occurs via a HuR-mediated increase of mRNA stability. Using COX-2 promoter constructs with different portions of the 3′ untranslated region of COX-2, we found that the increase in COX-2 mRNA stability is attributable to a distal class III type of AU-rich element (ARE). Likewise, the RNA immunoprecipitation assay showed AngII-induced binding of HuR to this ARE. Using the RNA pulldown assay, we demonstrate that the AngII-caused HuR assembly with COX-2 mRNA is found in free and cytoskeleton-bound polysomes indicative of an active RNP complex. Mechanistically, the increased HuR binding to COX-2-ARE by AngII is accompanied by increased nucleocytoplasmic HuR shuttling and depends on protein kinase Cδ (PKCδ), which physically interacts with nuclear HuR, thereby promoting its phosphorylation. Mapping of phosphorylation sites identified serines 221 and 318 as critical target sites for PKCδ-triggered HuR phosphorylation and AngII-induced HuR export to the cytoplasm. Posttranslational modification of HuR by PKCδ represents an important novel mode of HuR activation implied in renal COX-2 regulation

Nitric oxide induces TIMP-1 expression by activating the transforming growth factor beta-Smad signaling pathway

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