Study of the signaling pathway in TRAIL-induced T cell activation by yeast-two-hybrid system

TRAIL 是屬於第二型細胞膜蛋白質，並且是屬於 TNF 家族裡的一員，透過跟死亡受體的結合可以引起細胞的凋亡。TRAIL 主要是以膜結合型的形式存在，而 T 細胞在受到抗 CD3 的抗體或第一型干擾素的刺激之下可以引發 TRAIL 的表現。有越來越多的證據顯示 TNF 家族的成員，像是 CD40L，CD30L，FasL 和 TRANCE，在跟受體結合後都可以傳遞訊息。我們之前的研究顯示，同時給予 T 細胞抗 CD3 抗體和 DR4 的刺激，可以引起 T 細胞的活化和誘導干擾素-γ的產生。這樣的結果指出，TRAIL 在跟死亡受體結合後，除了會導致細胞的凋亡，也會傳遞反向的訊息導致 T 細胞的活化。然而這樣由 TRAIL 所引起的反向訊息傳導途徑仍然不甚清楚。我們之前的研究發現，在 TRAIL 所引起 T 細胞的活化當中可以觀察到 p38 MAPK 和 PI3K/Akt 的活化，顯示 p38 MAPK 和 PI3K/Akt 可能參與在 TRAIL 所引起的反向訊息傳導途徑之中。目前已知，NF-κB 訊息傳導途徑參與在 CD28 所引起的 T 細胞共活化之中，顯示 NF-κB 訊息傳導途徑對於 T 細胞的共活化相當重要。在這篇研究當中，我們利用酵母菌雙雜合系統來識別跟 TRAIL 的細胞質內區域相結合並且幫助 TRAIL 傳遞訊息的可能的分子。研究的結果顯示，JAK1 可能可以跟 TRAIL 的細胞質內區域相結合並且調節 TRAIL 所引起的訊息傳導而使得 T 細胞活化和產生誘導干擾素-γ。進一步的研究顯示，TRAIL 所引起的 T 細胞活化和誘導干擾素-γ的產生可以藉由加入 JAK 抑制劑而得到顯著的抑制，顯示 JAK-STAT 途徑可能參與在 TRAIL 所引起的 T 細胞活化當中。此外，我們也證明了 NF-κB 訊習傳導途徑在 TRAIL 所引起的 T 細胞共活化當中有被活化的現象。Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL), a type Ⅱ transmembrane protein, is one of several members of the TNF superfamily that induces apoptosis through engagement of death receptors. TRAIL exists mainly in membrane-bound form, and its expression on T cells is induced after T cell activation by anti-CD3 or type I interferon (IFN). There is growing evidence that ligands of the TNF family, such as CD40L; CD30L; FasL, and TRANCE, transduced signals after engagement with their receptors. Our previous study has demonstrated that TRAIL stimulated with immobilized DR4, in conjunction with suboptimal immobilized anti-CD3, induced T cell proliferation and enhanced IFN-γ production. This indicates that in addition to direct triggering apoptosis through death receptor, TRAIL can tranduce reverse signals to induce T cell activation. However, the reverse signaling pathways transduced by TRAIL is still unclear. In our previous study, we found that p38 MAPK and PI3K/Akt activation was detected after TRAIL-induced T cell activation, indicating that p38 MAPK and PI3K/Akt pathway is involved in TRAIL reverse signaling pathway. It has been demonstrated that the NF-κB signaling pathway was involved in CD28 costimulation signaling pathway, suggesting that NF-κB signaling pathway is critical in costimulation of T cells. In this study, we use yeast-two-hybrid system to identify the possible molecules associated with TRAIL intracytoplasmic domain to transduce signal. Our results showed that JAK1 may be an associated protein in the cytoplasmic region of TRAIL and modulate the signaling transduction of TRAIL-induced T cell proliferation and IFN-γ production. Furthermore, the effect of TRAIL-induced T cell proliferation and IFN-γ production can be significantly blocked by JAK inhibitor, indicating JAK-STAT pathway is critical in TRAIL-induced T cell activation. Moreover, we also demonstrated that NF-κB pathway is activated during TRAIL-induced costimulation of T cells.Chapter I. Introduction Part 1. T cell activation 1 Part 2. TRAIL and its receptors 2 Part 3. Reverse signal transduction in TNF superfamily 3 Part 4. JAK family 3 Part 5. NF-κB pathway 4 Part 6. Aims of the study 5 Chapter II. Materials and Methods Part 1. Experimental Materials 6 Part 2. Experimental Procedures 17 a. Human TRAIL cytoplasmic region cDNA construction 17 b. Yeast transformation 18 c. Lift assay 19 d. Isolation of positive plasmids 20 e. Human primary T cell purification 21 f. Human primary T cell proliferation assay 21 g. Human IFN-γ ELISA 22 h. Jurkat cell activation 22 i. Cytosolic and nuclear extract separation 23 j. Western blotting 23 k. Jurkat cell transfection and activation 24 l. Luciferase assay 25 Chapter III. Results Part 1. Identification of proteins associated with the cytoplasmic region of TRAIL 26 Part 2. CD2, JAK1, and Map2k3 as candidate proteins associated with the cytoplasmic region of TRAIL 27 Part 3. JAK1 is a possible regulator in TRAIL induced reverse signaling pathway 28 Part 4. Induction NF-κB luciferase activity by DR4-TRAIL engagement 29 Part 5. Western blotting of IκBα phosphorylation, IκBα degradation, and NF-κB nuclear translocation in TRAIL-induced reverse signaling pathway 30 Chapter IV. Discussion Part 1. CD2, JAK1, and Map2k3 as candidate proteins associated with the cytoplasmic region of TRAIL 32 Part 2. JAK1 is a regulator in TRAIL-induced reverse signaling pathway 34 Part 3. NF-κB pathway was involved in the signaling pathway of TRAIL-induced T cell activation 35 Part 4. Conclusion 37 Reference 39 Figures 43 Table1. Several encoding genes associate with the cytoplasmic region of TRAIL 43 Figure1. Three different plasmids used in the LexA yeast-two-hybrid system 45 Figure2. CD2, JAK1, and Map2k3 are associated with the cytoplasmic region of TRAIL in the LexA yeast-two-hybrid system 47 Figure3. TRAIL-induced human primary T cell proliferation could be blocked by JAK inhibitor 50 Figure4. TRAIL-induced IFN-γ production in human primary T cells could be blocked by JAK inhibitor 51 Figure5. DR4-TRAIL engagement induced NF-κB luciferase activity in EL4 cells 52 Figure6. DR4-TRAIL engagement induced IκBα phosphorylation and NF-κB nuclear translocation in Jurkat cells 53 Figure7. DR4-TRAIL engagement induced IκBα degradation and NF-κB nuclear translocation in Jurkat cells 5