Regulation of matrix degrading enzymes and their intrinsic inhibitors in renal inflammation and during tumorigenesis

Wolfgang Eberhardt (group leader), Roswitha Müller, A. Biyanee, A. Mohamed, U. Nasrullah, S. Kusch

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, most prominently rheumatoid arthritis, fibrosis and tumor metastasis. 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, which include the interstitial collagenases, stromelysins, elastases, membrane-type MMPs and gelatinases A 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 (TIMPs). We use cultured rat mesangial cells (MC) as a model system to investigate the mechanistical details of extracellular matrix remodeling. These cells highly respond to proinflammatory cytokines such as tumor necrosis factor a (TNFa) or interleukin-1b (IL-1b) with the transcriptional upregulation of different MMPs, including MMP-9 (gelatinase-B). In addition to MMPs, MC exposed to cytokines produce high levels of NO through the expression of the inducible NO synthase (iNOS) gene.

Mechanisms of intracellular shuttle of the mRNA regulating factor HuR
A main focus is the evaluation of posttranscriptional regulation of proteases and their intrinsic inhibitors by mRNA stability. A major candidate of our studies is the mRNA stabilizing protein HuA (HuR) a member of the embryonic lethal abnormal vision (ELAV) protein family. We could recently demonstrate that in MC, HuR-dependent mRNA stabilization is regulated by different mechanisms depending on which stimuli is experimentally used. These include a) a change in HuR expression as was observed by NO and cGMP donating compounds but similarly endogeneously via induction of the inducible nitric oxide synthase (iNOS). A further modulation of HuR-dependent mRNA decay is reached by changes in the nucleo cytoplasmic shuttling of HuR.
A current project deals with the elucidation of mechanisms involved in the stimuli-dependent nuclear shuttling processes of HuR. The main focus of this project lies in the identification of signaling pathways involved in the shuttling process. By use of different approaches including, pharmacological inhibitors, overexpression of activity-deficient kinases and by use of siRNA technology we try to identify the kinases functionally involved in the directed shuttling of HuR. Besides MMP-9, we try to indentify additional target genes posttranscriptionally regulated by HuR. The identification and functional role of putative phosphorylation sites within the HuR protein is an additional important aspect of this project.
In collaboration with other research groups we test whether our in vitro observations have an impact on renal pathologies. As animal model systems we use the Thy 1. model of acute glomerulonephritis (collaboration with L. Schäfer, Uniklinikum Münster).
This project is finacially supported by the Deutsche Forschungsgemeinschaft (EB 257/2-1)

Modulation of MMPs by Nitric oxide in breast cancer
The project deals with the elucidation of modulatory effects of NO on MMP expression in different breast cancer cell lines (e.g. MCF-7, MDA-231) and its consequences for tumor cell migration and invasion. NO has been described as a mediator exerting pro-tumorigenic but also anti-tumorigenic properties depending on which cancer cell model has been used. By use of exogeneous NO donors and by overexpresison of human iNOS we try to identify molecular mechanisms possibly involved in the NO-dependent modulation of MMP /expression and activity. Possible modulatory roles in addition to MMP transcription may involve posttranscriptional (e.g. the regulation of MMP mRNA stability) as well as posttranslational events, especially the regulation of MMP secretion. The functional consequences of the NO-dependent MMP regulation are finally monitored by use of different cellular assays including migration and invasion assays.

Full publication list of Wolfgang Eberhardt:

Ausgewählte Publikationen:

Huwiler A., Akool E.S., Aschrafi A., Pfeilschifter J., and Eberhardt W.
ATP potentiates IL-1b-induced MMP-9 expression in mesangial cells via recruitment of the ELAV-protein HuR.
J. Biol. Chem. (2003); 278 (51), 51758-51769

Akool E.S., Kleinert H., Hamada F.M.A., Abdelwahab M.H., Förstermann U., Pfeilschifter J, and Eberhardt W.Nitric oxide increases the decay of MMP-9 mRNA by inhibiting the expression of mRNA stabilizing factor HuR.
Mol. Cell. Biol. (2003); 23 (14), 4901-4916

Eberhardt W., Engels C., Müller R., and Pfeilschifter J.:
Mechanisms of dexamethasone-mediated inhibition of cAMP-induced tPA expression in rat
mesangial cells. Kidney Int. (2002); 62, 809-821

Eberhardt W., Akool E.S., Rebhan J., Frank S., Beck K.F., Franzen R., Hamada F.M.A., and Pfeilschifter J. Inhibition of cytokine-induced expression of MMP-9 expression by PPARa agonists is indirect and is due to a NO-mediated reduction of mRNA stability. J. Biol. Chem. (2002); 277 (6), 33518-33528

Eberhardt W., Schulze M., Engels C., Klasmeier E. and Pfeilschifter J.: Glucocorticoid-mediated suppression of cytokine-induced matrix metalloproteinase-9 expression in rat mesangial cells: involvement of NF-kB and Ets transcription factors.
Mol. Endocrinol. (2002); 16, 1752-1766

Eberhardt W.,Beck K.-F. , Pfeilschifter J.
Cytokine-induced expression of tissue plasminogen activator (tPA) is differentially modulated by NO and ROS in rat mesangial cells. Kidney Int. (2002); 61, 20-30

Eberhardt W., Beck K.-F., Huwiler A., Walpen S., Pfeilschifter J.
Amplification of interleukin-1b-triggered MMP-9 expression in rat mesangial cells by superoxide is mediated by increased activity of NF-kB and AP-1 and involves activation of the MAPKs pathways.
J. Immunol. (2000); 165, 5788-5797


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