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%0 Thesis
%A Rauen, Thomas
%T Der Einfluss des Y-box-Proteins-1 (YB-1) auf den Notch-vermittelten Signaltransduktionsweg
%C Aachen
%I Publikationsserver der RWTH Aachen University
%M RWTH-CONV-112972
%P 133 S. : Ill., graph. Darst.
%D 2008
%Z Aachen, Techn. Hochsch., Diss., 2008
%X Recent studies indicate an active secretion of Y-box protein-1 (YB-1) by mesangial and inflammatory cells. GFP-tagged YB-1 could be detected in microvesicular structures adjacent to the cell membrane after challenge with PDGF-BB in indirect immunofluorescence. YB-1 is known to fulfill pleiotropic functions in the cytoplasmic and the nuclear compartment; extracellular functions have not been described thus far. A yeast two hybrid screen yielded, amongst other proteins, Notch-3 receptor as a direct interaction partner for YB-1. The human Notch receptor superfamily comprises four members of transmembrane receptors that are involved into cellular processes, such as differentiation, proliferation and apoptosis. Receptor activation is mediated by ligand binding to the extracellular receptor domains leading to consecutive cleavages within the receptor protein. Subsequently, intracellular receptor domains are translocated to the nucleus where they interact with DNA binding proteins, such as RBP-Jk, and influence gene transcription. In the present study Notch-3 expression was analyzed in various cell lines [human embryonic kidney cells (HEK293T), tubular cells (HK2) and human mesangial cells]. Notch-3 and YB-1 expression pattern was detected in the time course of a rodent model of mesangioproliferative glomerulonephritis (Anti-Thy1.1 nephritis) and yielded a concordant time and spatial up-regulation of both proteins within the glomerular compartment around day 7-9 after disease induction. Physical interaction of YB-1 and extracellular Notch-3 domains was corroborated in co-immunoprecipitation experiments. Confocal laser scanning microscopy showed a co-localisation of both proteins at the outer aspect of cell membranes of tubular cells. Co-culture of cells with Notch-3 overexpression and cells stably expressing Notch ligand Jagged1 leaded to a translocation of the intracellular receptor domains when recombinant YB-1 was added to culture medium, highly suggestive for receptor activation. Reporter assays with Notch downstream target genes HES1 and HES2 yielded a differential influence of secreted YB-1 on trans-regulation of the respective promoters. Whereas HES2 promoter was significantly activated in a YB-1 concentration-dependent manner, HES1 promoter was not regulated by extracellular YB-1. The effect on HES2 promoter is abrogated when Notch signalling is specifically inhibited by addition of a gamma-secretase inhibitor. In co-culture of Notch-3- and Jagged1-carrying cells extracellular YB-1 mediates a down-regulation of Jagged1-induced activation of the HES2 promoter. This underscores the context-specifity of Notch signalling. In summary, Notch-3 is identified as a receptor for secreted YB-1 protein and physiological relevance of YB-1 binding to Notch-3 might play a role in inflammatory diseases, e.g. mesangioproliferative glomerulonephritis. Reporter studies suggest a differential regulation of Notch target genes by extracellular YB-1.
%K Gen notch (SWD)
%K Signaltransduktion (SWD)
%K Signaling (SWD)
%K Glomerulonephritis (SWD)
%K Transkriptionsfaktor (SWD)
%F PUB:(DE-HGF)11
%9 Dissertation / PhD Thesis
%U https://publications.rwth-aachen.de/record/50426