LIGHT經由其受體LTβR及HVEM訊息傳導之研究

Abstract

腫瘤壞死因子受體家族已知是免疫反應中的強效媒介物質，LIGHT屬於腫瘤壞死因子家族中的一員，其受體包括了LTβR、HVEM及DcR3。LIGHT可以經由與LTβR或HVEM的結合去引起細胞的死亡或NF-kB的活化。LTβR是二級淋巴組織發育及整合所必要的，而TRAF2、3及5會與LTβR結合去行使一連串的訊息傳遞而引起NF-kB的活化或程式性細胞凋亡。而HVEM參與了T細胞的活化，造成T細胞許多的反應。它可以與TRAF2及5結合去活化轉譯因子NF-kB及AP-1。 在第一部分的研究中，為了鑑定LT

Members of the tumor necrosis factor (TNF) superfamily are known to be potent mediators of immune responses. LIGHT is a member of the TNF superfamily, and its receptors have been identified as lymphotoxin β receptor (LTβR), herpes virus entry mediator (HVEM), and decoy receptor 3 (DcR3). LIGHT can induce either cell death and/or NF-κB activation via its interaction with LTβR and/or HVEM. LTβR is essential for the development and organization of secondary lymphoid tissue. TNF receptor factor (TRAF) 2,3 and 5 were associated with LTβR receptor and involved in LTβR receptor signaling that can induce NF-kB activation and/or cell death through apoptosis. HVEM is involved in T cell activation and can mediate a number of T cell responses. It binds to TRAF 2,5 and activates transcription factors NF-kB and AP1. In the part I of this study, LTβR was overexpressed in HEK293 cells in order to characterize the LTβR downstream signaling leading to chemokine IL-8 gene expression. We found overexpression of LTβR increases IL-8 promoter activity and leads to IL-8 release. LTβR-induced IL-8 gene expression requires NF-kB (-80 to –71) and AP-1 (-126 to –12) binding sites located in IL-8 promoter, and NF-kB is more crucial than AP-1 for IL-8 gene expression. Reporter assay with dominant-negative mutants of TRAFs reveals that TRAF 2, 3, and 5, as well as the downstream signal molecules, NIK, IKKa and IKKb are involved. LTbR-mediated IL-8 response was inhibited by the dominant-negative mutants of ASK1, MKK4, 7, and JNK, but not by those of MEKK1, TAK1, MEK, ERK and p38 MAPK. These data suggest that IL-8 induction by LTβR is via TRAFs-elicited signaling pathways, including NIK/IKK-dependent NF-kB activation and ASK/MKK/JNK-dependent AP-1 activation. In the part II of this study, we also investigated the inflammatory effects of LIGHT in human umbilical vein endothelial cells (HUVECs). We demonstrated that both LTβR and HVEM, but not DcR3, are present in HUVECs, and LIGHT can induce the secretion of chemokines (IL-8 and GRO-? cell surface expression of adhesion molecules (ICAM-1 and VCAM-1), PGI2 release, and COX-2 expression. However, the LIGHT mutein, LIGHT-R228E, which has been shown to exhibit binding specificity to LTβR, could not induce the secretion of GRO-? PGI2, or the expression of COX-2. These results indicate that both LTβR and HVEM can discriminatively mediate the expression of different genes in HUVECs, and suggest that LIGHT is a proinflammatory cytokine. Previous studies have indicated that LIGHT through LTβR signaling can induce cell death with features unlike classic apoptosis in the presence of IFN-g. In the part III of this study, we investigated the mechanism of LIGHT/IFN-g-induced cell death in HT29 cells, where the cell death was profoundly induced when sub-toxic concentrations of LIGHT and IFN-g were co-treated. LIGHT/IFN-g-induced cell death was accompanied by DNA fragmentation and slight LDH release. This effect was not affected by caspase, JNK nor cathepsin B inhibitors, but was partially prevented by p38 MAPK and PARP inhibitors, and abolished by aurintricarboxylic acid, which is an inhibitor of endonuclease and STATs signaling of IFN-g. LIGHT/IFN-g could induce p38 MAPK activity, Bak and Fas expression, but down-regulate Mcl-1. Besides, LIGHT/IFN-g could not activate caspase-3 and -9, but decreased mitochondrial membrane potential. Although LIGHT could not affect IFN-g-induced STAT1 phosphorylation and transactivation activity, which was required for the sensitization of cell death, survival NF-kB signaling of LIGHT was inhibited by IFN-g. These data suggest that co-presence of LIGHT and IFN-g can induce an integrated interaction in signaling pathways, which lead to mitochondrial dysfunction and mix-type cell death, not involving caspase activation.