Funktionsanalyse von Tec Kinasen in primären Mastzellen
Zusammenfassung der Projektergebnisse
In this project the role of the name giving member of the Tec kinase family, Tec, was investigated by analysing murine mast cell functions in vitro and in vivo. Until now the role of Tec in mast cells was not investigated in the field of mast cell biology. So far, only the role of the Tec kinase family member Btk was investigated in mast cells in more detail (nb: by now also several reports shed light into the function of the Tec kinase Itk in mast cells). The goal was to elucidate the functions of Tec in mast cells and to find out if Tec displays other functions than Btk in mast cells. Therefore, Tec-/- mice and mast cells derived thereof were compared to the respective wild-type, Btk-/- and Tec-/- // Btk-/- double-knock counterparts. As a first step, Tec was identified as a member of the FceRI signalosome since it was phosphorylated and demonstrated kinase activity after FceRI stimulation. Bone marrow cells from Tec-/- mice gave rise to much more mature mast cells after a 4-week culture than wild-type or Btk-/- bone marrow cells. In the double-knock the extend of the amplification was similar to the Tec-/- single knock-out. The reason for the increased expansion potential in all investigated knock-outs could not be clarified within this project and needs further investigation. The size and the morphology of bone marrow derived mast cells as well as cell surface marker expression was similar for all genotypes investigated. No differenes in cell numbers or morphology could be detected when skin mast cells of ears were investigated. Histamin release was not impaired in the Tec-/- background in vitro and in vivo while the reported decrease in a Btk-/- background could be confirmed which was impaired to a similar extend in the double knock-out. Interestingly, Tec appeared to regulate leukotriene release differentially since the release of LTC4 was impaired, but not the release of LTB4. Calcium flux was impaired only to a minor extend in Tec-/- mast cells, while it was severly impaired in Btk-/- and double-knockout cells. This data was in line with a dimished phosphorylation of PLCγ1 in Btk-/- and double-deficient cells while Tec-/- cells showed no impairment. Immunoblot analysis revealed that phosphorylation of p38 and jnk1/2 were impaired in Tec-/- cells in comparison to wild-type cells. Interestingly, phosphorylation of erk1/2 was enhanced in Tec-/- and double-deficent cells in comparison to wild-type and Btk-/- cells suggesting a negative role for Tec in this pathway. The secretion of IL-6 was normal Tec and Btk deficient cells, while only the double-knock out displayed a 2-fold reduction in IL-6 secretion. IL-13 was similary impaired for all genotypes. gm-csf secretion was only mildly impaired in Btk-/- cells, but was severly decreased in Tec-/- cells and even more severly impaired in the doubleknock out cells. TNFα was similarly dimished in Tec and Btk single knockouts and dramatically reduced in the double knock-out cells. Most interesting was the effect on IL-4. The secretion of IL-4 was heavily reduced in Tec-/- and double-knockout cells while it was enhanced in Btk-/- cells, both if stimulated via antigen or IgE. Taken together, the results obtained in this project demonstrate that Tec is a member of the FceRI signalosome and that its function is partially different to the role of the wellcharacterized family member Btk.
Projektbezogene Publikationen (Auswahl)
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(2006). Characterization, isolation and differentiation of murine skin cells expressing hematopoietic stem cell markers. Journal of Leukocyte Biology, 80, 816-826
S. Meindl, U. Schmidt, C. Vaculik and A. Elbe-Bürger
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(2007). Regulation of dendritic cell differentiation and subset distribution by the zinc finger protein CTCF. Immunological letters, 109, 165-74
C. Koesters, B. Unger, I. Bilic, U. Schmidt, S. Bluml, B. Lichtenberger, M. Schreiber, J. Stockl and W. Ellmeier
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(2007). The Btk tyrosine kinase is a major target of the Bcr-Abl inhibitor dasatinib. PNAS, 104 (33)13283-88
U. Schmidt, O. Hantschel, U. Rix, T. Bürckstümmer, M. Kneidinger, G. Schütze, K. Bennett, W. Ellmeier, P. Valent and G. Superti-Furga
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(2008). Dasatinib inhibits IgE receptor-dependent activation and histamine release in human blood basophils. Blood, 111(6) 3097-3107.
M. Kneidinger, U. Schmidt, U. Rix, K.V. Gleixner, A. Vales, C. Baumgartner, C. Lupinek, M. Weghofer, K.L. Bennett, H. Herrmann, A. Schebesta, W.R. Thomas, S. Vrtala, R. Valenta, F.Y. Lee, W. Ellmeier, G. Superti-Furga and P. Valent
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(2008). Essential roles for the Tec family kinases Tec and Btk in M-CSF receptor signaling pathways that regulate macrophage survival. JI, (180) 8048-8056
M. Melcher, B. Unger, U. Schmidt, I.A. Rajantie, K. Alitalo, and W. Ellmeier
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(2010). The protein tyrosine kinase Tec regulates a CD44highCD62L- Th17 subset. JI, Nov 1;(18) 5111-9
N. Boucheron, O. Sharif, A. Schebesta, A. Croxford, J. Raberger, U. Schmidt, B. Vigl, J. Bauer, R. Bankoti, H. Lassmann, M.M. Epstein, S. Knapp, A. Waisman and W. Ellmeier
