Studies on the signal transduction passways through IL-12 receptor

通过酵母双杂交技术筛选出小鼠鞘氨醇激酶2（SPHK 2），并鉴定其为与小鼠IL-12 R β1胞浆区相关的分子。对SPHK 2的各种突变体的分析表明，SPHK 2中富含脯氨酸的区域可能与IL-12 R β1的结合有关，而IL-12 R β1胞浆区的羧基端和Box II区域可能与SPHK 2的结合有关。野生型SPHK 2在T细胞杂交瘤中的瞬时表达增强了IL-12诱导的STAT 4介导的转录激活。显性阴性SPHK 2在Th 1细胞克隆中的异位表达可显著降低IL-12诱导的IFN-γ产生，而野生型SPHK 2则可增强IFN-γ产生，而对IL-12诱导的细胞增殖影响甚微。当使用逆转录病毒表达系统在活化的原代T细胞中表达显性阴性SPHK 2时，观察到IL-12诱导的IFN-γ产生的类似减少。这些结果表明SPHK 2与IL-12 R β1胞浆区结合，可能在调节IL-12信号转导中发挥作用。我们还研究了JAK/STAT信号分子激活的IL-27和STAT 1在IL-27介导的反应中的作用，使用STAT 1缺陷小鼠。我们发现IL-27激活了初始CD 4 + T细胞中的JAK 1、-2 TYK 2、STAT 1、-2、-3和-5。在STAT 1缺陷型和野生型幼稚CD 4 + T细胞之间观察到对IL-27的相当的增殖反应。相比之下，IL-27不能诱导T-bet和IL-12 R β2的表达，并且IL-27和IL-12的协同IFN-γ产生也在STAT 1缺陷的幼稚CD 4 + T细胞中受损。这些结果表明，IL-27激活初始CD 4 + T细胞中的JAK 1，-2，TYK 2，STAT 1，-2，-3和-5，并且STAT 1在IL-27诱导的T-bet和IL-12 R β2表达中起不可或缺的作用，但不参与增殖。

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.