| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2003: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 2002: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
We performed a yeast two-hybrid screening and identified the mouse sphingosine kinase 2 (SPHK2) as a molecule associating with mouse IL-12Rβ1 cytoplasmic region. Analyses on various mutants of each molecule revealed that the region including proline-rich domain in SPHK2 is likely to be responsible for the binding to IL-12Rβ1, while the regions including the carboxyl terminus and Box II in IL-12Rβ1 cytoplasmic region appear to be involved in the binding to SPHK2. Transient expression of wild-type SPHK2 in T cell hybridoma augmented IL-12-induced STAT4-mediated transcriptional activation. Ectopic expression of dominant-negative SPHK2 in Th1 cell clone significantly reduced IL-12-induced IFN-γ production, while that of wild-type SPHK2 enhanced it. In contrast, the expression minimally affected IL-12-induced proliferation. Similar decrease in IL-12-induced IFN-γ production was observed when dominant-negative SPHK2 was expressed in activated primary T cells by using a retroviral expression system. These results suggest that SPHK2 associates with IL-12Rβ1 cytoplasmic region and is likely to play a role in modulating IL-12 signaling. We also investigated the JAK/STAT signaling molecules activated by IL-27 and the role of STAT1 in IL-27-mediated responses using STAT1-deficient mice. We found that IL-27 activated JAK1, -2 TYK2, STAT1,-2,-3, and -5 in naive CD4+ T cells. Comparable proliferative response to IL-27 was observed between STAT1-deficient and wild-type naive CD4+ T cells. In contrast, IL-27 failed to induce T-bet arid IL-12Rβ2 expression, and synergistic IFN-γ production by IL-27 and IL-12 was also impaired in STAT1-deficient naive CD4+ T cells. These results suggest that IL-27 activates JAK1,-2, TYK2, STAT1, -2,-3, and -5 in naive CD4+ T cells and that STAT1 plays an indispensable role in IL-27-induced T-bet and IL-12Rβ2 expression but not proliferation.
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