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1000
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Abstract/Summary
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Endothelin-1 (ET-1), first described as a vasoconstrictor peptide, is a powerful mitogen for a variety of tumour and non-tumour cells. Its signalling cascade, mainly analysed in tumor cells, induces the inflammatory NF-кB complex resulting in target gene expression. This thesis focuses on the ET-1 induced activation of the NF-кB transcription factor and the involvement of the two endothelin receptors (ETAR and ETBR) in tumor and renal proximal tubule cells. We demonstrate that NF-κB is up-regulated by two different time-dependent signal pathways in renal tubular epithelial cells. Protein microarray screening and different cell treatments with special receptor antagonists show activation of two independent cascades (ETAR and diacylglycerol kinase) leading to NF-кB induction. A co-upregulation of p38 was found and the role of this upregulation was subsequently analysed in HeLa cells. MAPK p38 has been regarded as potential phosphate donor for the p65 subunit of NF-кB. In ET-1 treated HeLa cells we have found that the NF-кB complex is activated via the ETAR as well and that the transcription factor complex contains not only p65 and p50 but also p38 (α and β). Using direct mutagenesis, we found that the nuclear location sequence of p65 is important for p38 nuclear location and that the complex formation already occurs in the cytoplasm. Several different treatments either for p38 up- or downregulation were performed to show the biologic effect and the expression levels of some known NF-κB target genes, identifying a distinct number of p38 dependent genes. The ET-1 treated cells were discovered to contain about twice the amount of p38 than TNFα treated cells. Analysing tumor cell lines of histogenetically different carcinomas and fresh tumor samples from renal cell tumors the observed p38/p65 transcription factor complex formation was found to be a general phenomenon. An unexpected finding was the high presence of this complex in benign kidney tumors (on
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