Konstruktion und Charakterisierung eines konstitutiv aktiven Zytokin Rezeptors: gp130
keine dt.-spr. Zusammenfassung vh.
Cytokines are soluble proteins of low molecular weight which exert important functions via the regulation of intercellular communication in the immune and nervous system. The transmembrane receptor gp130 is the common subunit of all IL-6 type cytokines. Ligand binding induces the dimerization of receptor subunits which leads to the transduction of signals. The glykoprotein 130 transduces signals which result in the activation of the signal transducers and activators of transcription proteins STAT1 and STAT3. The constitutive activation of STAT3 has been reported to be associated with a variety of human cancers. We investigated the consequences of a constitutive active gp130 molecule. To achieve this, two chimeric gp130 receptors have been constructed and expressed. The truncation of the extracellular part of the wildtype receptor lead to construct ƒ´¡Vgp130. For the protein L-gp130 the extracellular region of the wildtype receptor has been replaced by the leucine zipper region of the human c-jun protein. Both proteins were expressed but only L-gp130 was properly processed by the cell and could be detected in the plasmamembrane of transfected cells. L-gp130 was phosphorylated and therefore active. Transfection experiments with eukaryotic cells showed that the expression of L-gp130 results in a ligand-independent phosphorylation of STAT3. Resulting active STAT3 molecules are biologically active which could be shown by binding studies with specific DNA sequences. Additionally, these transcription factors were able to induce promoter activity in a reporter gene assay. To be independent from endogenous signals we established a stable BAF/3 cell line expressing the active L-gp130 molecule. We obtained stable cells with the help of a retroviral transfection system. Concerning survival and proliferation, BAF/3 cells are vitally dependent upon the cytokine IL-3. IL-3 signalling activates the transcription factor STAT5 instead. IL-6/sIL-6R as well as Hyper-IL-6 lead to the activation of STAT3. Hyper-IL-6 is a designercytokine consisting of IL-6 and the soluble IL-6R. Thus the transfection of BAF/3 cells with the wildtype gp130 receptor results in a switch concerning the dependence from IL-3 to IL-6/sIL-6R or HIL-6. Successfully L-gp130 transfected BAF/3 cells grew and proliferated independently from any cytokine. In L-gp130-BAF/3 cells the permanent activation of the gp130 transduced signal leads to the activation of STAT3 proteins. With the constitutively active L-gp130 we now for the first time have the opportunity to directly investigate the consequences of disregulated permanent gp130 transduced signals in cells. The advantage of this model lies in the independence from a ligand-induced stimulation. The model can help to answer a series of scientific questions. The effects of a constitutively active gp130 signal upon adult or embryonic stem cells can be investigated. From the biotechnological point of view, L-gp130 could possibly be used to cultivate cytokine dependent cells like hybridomas which are being used to produce monoclonal antibodies. The hypothetical oncogenic potential of a permanent gp130 signal is not only of a scientific but with respect to the search for new therapeutic targets also of a considerable medical interest.