24 research outputs found
Regulation of matrix-metalloproteinase 9 (MMP-9) in glomerular mesangial cells
No full textRemodeling 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
MOLECULAR MECHANISMS OF TGF-/SMAD SIGNALING CASCADE INDUCED BY CYCLOSPORIN A IN A549 LUNG EPITHELIAL CELLS
No full textMolecular Mechanisms of the Protective Role of Wheat Germ Oil Against Oxidative Stress–Induced Liver Disease
No full textPosttranslational modification of the AU-rich element binding protein HuR by protein kinase Cdelta elicits angiotensin II-induced stabilization and nuclear export of cyclooxygenase 2 mRNA
No full textThe 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 Cdelta (PKCdelta), which physically interacts with nuclear HuR, thereby promoting its phosphorylation. Mapping of phosphorylation sites identified serines 221 and 318 as critical target sites for PKCdelta-triggered HuR phosphorylation and AngII-induced HuR export to the cytoplasm. Posttranslational modification of HuR by PKCdelta represents an important novel mode of HuR activation implied in renal COX-2 regulation
Propolis alleviates concanavalin A-induced hepatitis by modulating cytokine secretion and inhibition of reactive oxygen species
No full text
The potential antifibrotic impact of apocynin and alpha-lipoic acid in concanavalin A-induced liver fibrosis in rats: Role of NADPH oxidases 1 and 4
No full textModulatory effect of metformin and its transporters on immune infiltration in tumor microenvironment: a bioinformatic study with experimental validation
No full textAbstract Metformin is a traditional antidiabetic drug for type 2 diabetes mellitus. However, it showed antitumor activity in many types of tumors, and it also has an influence on tumor metastasis in several types of tumors. It is transported through organic cationic transporters (OCTs), OCT1, OCT2, and OCT3, into the cells or into tumor microenvironment (TME). The complex interaction of metformin and its transporters on immune infiltration in TME of different types of tumors of The Cancer Genomic Atlas (TCGA) is not yet studied. The objective of this study is to identify the most suitable therapeutic target of tumors and immune infiltrates for metformin and its transporters in the TME. TIMER2.0, a bioinformatic tool, and other computational analysis were used to investigate this complex interaction; moreover, the identification of metformin target protein in TME is also investigated. The results revealed that the most suitable therapeutic target for metformin and OCTs among 32 types of TCGA data tumor types is Breast Invasive carcinoma (BRCA), and the most relevant immune infiltrate among 14 types of immune infiltrates that yields better prognosis and better therapeutical effect in TME is Macrophage M1. Furthermore, metformin showed a cytotoxic effect and an inhibitory effect on Urokinase Plasminogen Activator (uPA) gene expression in a concentration dependent fashion in MDA-MB-231 breast cancer cell line. This may suggest that metformin is a promising antitumor drug, stimulant for natural antitumor immune infiltrates, and inhibitor for metastasis in breast cancer
