Study on the Role of Deltex3 in T Cell Activation

• Main Authors: Chen-Jhe Wang, 王晨哲
• Other Authors: Ming-Zong Lai
• Format: Others
• Language: zh-TW
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/33kfgx

碩士 === 國立陽明大學 === 生命科學暨基因體科學研究所 === 97 === Deltex (DTX) is a regulator of Notch signals in Drosophila, but its action mechanism and physiological functions are poorly understood in mammals. There are four DTX homologues (DTX1, DTX2, DTX3 and DTX4) in mammals. DTX1 has been identified as a negative regulator of T cell activation through degradation of active MEKK1 protein. DTX3 is unique among the DTX family for the absence of WWE domains, and its inability to bind Notch. DTX1 with both WWE domains deleted retains the partial capacity to inhibit T cell activation, suggesting DTX3 may also involved in T cell activation. In this study, we aim to elucidate whether DTX3 participates in T cell activation by DTX3 overexpression and Dtx3 downregulation. We found that Dtx3 was expressed in T cell lines including DO11.10 and EL4, as well as in primary T cells. We established DO11.10 clones transduced with DTX1 or DTX3. Overexpression of DTX3 in DO11.10 cells partially suppressed T-cell receptor (TCR)-stimulated IL-2 production, JNK activation, and p38 MAPK phosphorylation. The protein level of C-terminal catalytic domain of MEKK1 [MEKK1(C)] was also partially downregulated by DTX3. The physiological role of DTX3 in T cells was further examined by Dtx3 knockdown. We found that TCR-stimulated production of IL-2 and activation of JNK, p38 MAPK, and ERK were enhanced when Dtx3 was downregulated. The protein level of MEKK1(C) was also enhanced when Dtx3 was downregulated. Together, these data suggest that DTX3 also act as a negative regulator of T cell activation. For analysis on the physiological functions of DTX3, we are in the progress to generate DTX3-specific antibodies, as well as to produce Dtx3-KO mice. We obtained 329 ES cell genomic DNA from the Transgenic Core Facility and screened it by different PCR primers. After the PCR genotyping, 5 positive clones were identidied and the insertion of targeting construct was also confirmed by Sothern blotting. Blastocyst microinjection has generated a number of chimeric mice that will be used to produce Dtx3-/- mice in near future.