Studies on the signal transduction passways through IL-12 receptor

我们进行了酵母双杂交筛选，鉴定出小鼠鞘氨醇激酶2(SPHK2)是一个与小鼠IL-12Rβ1细胞质相关的分子。对每个分子的各种突变体的分析表明，SPHK2中的富含Pro结构域的区域可能与IL-12Rβ1结合，而IL-12Rβ1细胞质区域中的羧基末端和框II似乎参与了与SPHK2的结合。野生型SPHK2在T细胞杂交瘤中的瞬时表达增强了IL-12诱导的STAT4介导的转录激活。在Th1细胞克隆中异位表达显性负性SPHK2显著降低了IL-12诱导的干扰素-γ的产生，而野生型SPHK2的异位表达则增强了IL-12诱导的干扰素-DNA的产生。相反，该基因的表达对IL-12诱导的增殖影响最小。用逆转录病毒表达系统在激活的原代T细胞中表达显性阴性的SPHK2时，IL-12诱导的干扰素-γ的产生也有类似的下降。这些结果表明，SPHK2与IL-12Rβ1胞浆区相关，可能在调节IL-12信号转导中发挥作用。我们还研究了由IL-27激活的JAK/STAT信号分子，以及STAT1在IL-27介导的反应中的作用。我们发现IL-27在初始的CD4+T细胞中激活了JAK1、-2、TYK2、STAT1、-2、-3和-5。在STAT1缺陷和野生型初始CD4+T细胞之间也观察到了类似的对IL-27的增殖反应。IL-27不能诱导T-bet和IL-12Rβ-2的表达，IL-27和IL-12协同产生γ的作用也受到抑制。这些结果提示，IL-27能激活初始T细胞JAK1、-2、TYK2、STAT1、-2、-3和-5，STAT1在IL-27诱导的T-bet和IL-12Rβ-2的表达中起着不可缺少的作用，但不能促进增殖。

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.