Charakterisierung neuer Interaktionspartner der NFAT-vermittelten Transkription im Pankreaskarzinom

The nuclear factors of activated T-cells (NFATs) were mainly characterized by the regulation of the immune response, in which they play an essential role as transcription factors (Rao et al. 1997). In recent years it has been reported that expression of NFAT-protein is not only restricted to T-cells, but can also be found in a number of cells outside the immune system, in which they control the expression of genes that regulate differentiation and growth (Viola et al. 2004). Pancreatic cells are shown to express NFAT (Buchholz et al. 2006). NFAT-proteins are primarily located in the cytoplasm and only translocate to the nucleus after activation. There in place they interact with other binding partners on the DNA (Hogan et al. 2003). Those binding partners are transcription factors that co-operate with NFAT to influence the selection and regulation of NFAT-controlled genes. This study focuses on NFATc2, which is highly expressed in pancreatic carcinomas. However, its abilities to regulate DNA remains elusive and is not investigated in detail. The aim of this study is to identify possible binding partners of NFATc2 in pancreatic carcinoma cells. In the first part of this study the expression of NFAT and its potential binding partners is analyzed in a number of established pancreatic carcinoma cell-lines. To find potential binding partners of NFATc2, a literature search is performed for transcription factors that have already been shown to interact with NFATc2 in other cell types and tissues. In addition they have to be calcium-regulated and known for their oncogenic potential. Ionomycin, is a stimulant that guarantees the translocation of NFATc2 into the nucleus by activating the calcium-calcineurin-signaling pathway. However, Ionomycin exerts cytotoxic effects are making long term stimulations impossible. The process describe above is proven by using immunofluorescence studies as well as western blot analysis. In the following experiments, using immunoprecipitation, a binding of NFATc2 with the transcription factor Sp1 is observed. To verify and characterize this interaction additional experiments are performed. Using immunofluorescence labeling a colocalization of the oncogenic transcriptions factors Sp1 and NFATc2 can be seen in pancreatic carcinoma cell-lines in the presents of Ionomycin. In additional studies it is shown that they interact in the same complex at the NFAT-target sequence GGAAA. Sp1 increases the activity of its binding partner at the NFAT-responsive promoter construct. This interaction intensifies with the presence of Ionomycin in the early phase of stimulation (< 60 min). In the second part of this study the physical interaction of NFATc2 and Sp1 is characterized in more detail using mutants with Sp1-deletions. Here, it is shown on multiple levels using immunoprecipitation and luciferase-assays that the N-terminus of Sp1 interacts with NFATc2 to control the function of their target genes. Thru gene expression analysis some really interesting target genes of NFATc2 and Sp1, which are expressed thru Ionomycin treatment, are identified in the third part of the study. Among them, the proto-oncogene c-fos, tumor necrosis factor-alpha (TNF-alpha) and the adhesion molecule integrin-β-3 are found. The knock-down of Sp1 using siRNA, decreases the effects of Ionomycin on c-fos and TNF-alpha, indicating that the DNA-binding activity of Sp1 has relevancy in promoting the NFATc2 regulation of these genes. This observation and its relevance for pancreatic cancer is confirmed in independent experiments for c-fos using qRT-PCR and thymdine-proliferation-assay. A decreased cell proliferation is observed after 24 and 48 hours following knock-down of both transcription factors. In summary it can be presumed that NFATc2 and Sp1 interact in pancreatic carcinoma cells and form a complex that regulates target genes that control cell proliferation. The loss of either one of the two transcription factors inhibits the formation of this complex and its downstream regulation of cell cycle regulating genes.