Study on transcriptional regulation in CD40 signaling.

CD40信号在免疫反应中起着重要作用。CD40信号可激活多种信号分子，如转录因子NF-kB。然而，目前还不清楚这些信号分子是如何调控靶基因的。目前也不清楚目标基因的产物是如何参与免疫反应的。在此之前，我们已经证明CD40信号会降低细胞周期抑制因子kip1的mRNA水平。首先，我们探讨了CD40信号抑制kip1的分子机制。我们用3T3细胞用荧光法检测了kip1基因的启动子活性，证明了核因子-kB抑制了kip1基因的启动子。此外，kip1启动子中含有-581到-348的区域携带两个核因子-kB识别序列，并且该区域的启动子活性被核因子-kB抑制。这提示该区域可能在CD40介导的抑制kip1水平中发挥作用。然而，即使核因子-kB位点发生突变，核因子-kB介导的抑制仍会发生。因此，核因子-kB如何抑制该区域的启动子活性仍不清楚。接下来，我们评估了kip1抑制在CD40介导的B细胞增殖和存活中的作用。由于p27kip1抑制CDKs的细胞周期进程，因此kip1抑制可能在CD40信号诱导的B细胞增殖中起作用。CD40信号通过消除抗原受体介导的细胞凋亡诱导B细胞存活，包括B细胞系WEHI-231。当我们用逆转录病毒载体在WEHI-231细胞中表达kip1时，抗原受体连接的WEHI-231细胞的存活受到部分损害。这表明，通过CD40信号通路完全恢复抗原受体诱导的细胞凋亡需要抑制kip1。综上所述，kip1抑制似乎与核因子-kB有关，并在B细胞的增殖和存活中发挥作用。

CD40 signaling plays an essential role in immune responses. CD40 signaling activates various signaling molecules such as the transcription factor NF-kB. However, it is not yet elucidated how these signaling molecules regulate the target genes. It is also not yet clear how the products of the target genes are involved in immune responses. Previously, we demonstrated that CD40 signaling reduces the mRNA level of the cell cycle inhibitor kip1. First, we addressed the molecular mechanisms for the repression of kip1 by CD40 signaling. We assessed the promoter activity of the kip1 gene by luciferace assay using 3T3 cells, and demonstrated that NF-kB repress the kip1 promoter. Further the region containing -581 to -348 in the kip1 promoter carry two NF-kB recognition sequences and the promoter activity in this region is suppressed by NF-kB. This suggests that this region may play a role in CD40-mediated suppression of the kip1 level. However, NF-kB-mediated suppression still occurs even if the NF-kB site is mutated. Thus, how NF-kB represses the promoter activity in this region is still unclear. Next, we assessed the role of kip1 repression in CD40-mediated B cell proliferation and survival. Since p27kip1 inhibits CDKs essential fur cell cycle progression, kip1 repression may play a role in B cell proliferation induced by CD40 signaling. CD40 signaling induces survival of the B cells including the B cell line WEHI-231 by abrogating antigen receptor-mediated apoptosis. When we expressed kip1 in WEHI-231 cells using a retrovirus vector, survival of antigen receptor-ligated WEHI-231 cells is partially impaired. This indicates that kip1 suppression is required for fully restore antigen receptor-induced apoptosis by CD40 signaling. Taken together kip1 repression appears to involve NF-kB and play a role in proliferation and survival of B cells.