Publikationsserver der RWTH Aachen University
Untersuchungen zum crosstalk zwischen Interleukin-6 und Platelet-derived growth factor (PDGF) sowie zur Signaltransduktion von PDGF-Rezeptoren
Abstract
dc:descriptionThe investigation of the molecular mechanisms that lead to the degeneration and increased proliferation of cells and are thus part of oncogeny is of essential importance to the development of new cancer therapies. For a concerted intervention the signalling pathways involved have to be identified and their dysregulation has to be understood. There is evidence for IL-6 type cytokines, and for the Platelet Derived Growth Factor (PDGF) as well, to play a part in the pathogeny of different types of cancer (e.g., multiple myeloma, prostate cancer). Furthermore, IL-6 and PDGF have huge relevance for inflammation processes (e.g., rheumatoid arthritis, mesangioproliferative glomerulonephritis, atherosclerosis). In this work, a crosstalk-mechanism between IL-6 and PDGF is particularly investigated that leads to phosphorylation of the IL-6-induced feedback-inhibitor SOCS3. It was shown in several cell lines and primary cells that this crosstalk-mechanism leads to an enhanced STAT1 and STAT3 phosphorylation. An influence on ERK1/2-activation was not found. Several experiments, where multiple kinase inhibitors were used for instance, indicate that within the crosstalk SOCS3 phosphorylation could ensue directly through the kinase of the PDGF receptor and not through kinases further down the signalling pathway. Interaction between SOCS3 and the two PDGF receptors alpha and beta could be found in coimmunoprecipitations. For a more detailed investigation of this interaction, peptide precipitations with biotinylated phospho-peptides of PDGFR beta and SOCS3 were carried out. The results of these experiments were not conclusive. However, preferences for a binding of SOCS3 to the phosphotyrosine motives pY1009 and pY1021. The amino acid sequence V-L-pY-T-A-V-Q-P of the phosphotyrosine motive pY1009 matches the consensus sequence h-X-pY-h/S/T-XL/V-h-h predicted by Hörtner et al. for the binding of the SH2 domain of SOCS3. SOCS3 is known as a negative regulator of the IL-6 signalling pathway. However, in the frame of this work SOCS3 had no negative regulatory influence on the immediate signalling molecules of the PDGFR examined here. Physiological quantities of SOCS3 did not change the activation of the signalling molecules PLC, ERK1/2 and Akt after PDGF stimulation. Furthermore, in this work the migration and proliferation of cells as biological read out was examined in the crosstalk. Migration experiments in modified Boyden chambers showed that both IL-6 and PDGF initiate the migration of TUR cells and VSMC. Thereby, the VSMC migrated more strongly after PDGF stimulation than after IL-6 stimulation. In the crosstalk, the migration of the TUR cells was apparently reduced compared to PDGF stimulation. VSMC, however, showed a strong migration after PDGF stimulation and in the crosstalk as well. A potential difference was hard to evaluate here. Experiments with the thymidine analogon BrdU showed that both IL-6 and PDGF promote proliferation in NIH 3T3 and VSMC, with PDGF being more potent. When being simultaneously stimulated with IL-6 and PDGF proliferation of NIH 3T3 and VSMC was reduced compared to cells treated with PDGF alone. In order to analyse the gene expression in the crosstalk of IL-6 and PDGF in more detail and to gain clues on genes or gene products of interest in this context, a PIQOR Immunology Microarray was done. For this purpose, primary human dermal fibroblasts (HDFs) are used. The results of the Microarray show numerous genes that are regulated up or down in the crosstalk. Among those are many chemokines and transcription factors. The expression of some interesting genes was validated with the help of a real-time RT-PCR and the results of HDFs from different donors were compared with each other.
Degree
thesis:*- Grantor dc:publisher
- Publikationsserver der RWTH Aachen University
- Year dc:date
- 2009
Author and committee
dc:creator, dc:contributor.*- Author dc:creator
-
- Borggrebe, Kathrin
- Contributors dc:contributor
-
- Haan, Serge
Subjects
dc:subject × 9Rights
dc:rights- Statement dc:rights
-
- info:eu-repo/semantics/openAccess
- Language dc:language
- ger