| Co-Investigator(Kenkyū-buntansha) |
INOUE Toru Dept.of Toxicology, National Inst.of Health Science, Chief Scientist, 毒性部, 部長 (50100110)
NAKAHATA Tatsutoshi The University of Tokyo Inst.of Medical Science, , Professor, 医科学研究所, 教授 (20110744)
MASAI Hisao The University of Tokyo Inst.of Medical Science, Associate Professor, 医科学研究所, 助教授 (40229349)
WATANABE Sumiko The University of Tokyo Inst.of Medical Science, , Research Associate, 医科学研究所, 助手 (60240735)
YOKOTA Takashi The University of Tokyo Inst.of Medical Science, , Professor, 医科学研究所, 客員教授 (50134622)
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| Research Abstract |
The purpose of this study is to regulate the immune responses in vitro and in vivo by the alteration of the function of the transcription factors NF-AT and STAT,which are involved in the regulation of immune responses.
We have isolated human NF-AT (NF-ATp/c/x) genes at the beginning of this research program, and during this study we isolated murine counterparts. Among the NF-AT family members, we focused on human and mouse NF-ATx for the analysis and alteration of its function. First, we performed domain mapping of human NF-ATx, and identified several distinct functional domains including the DNA binding domain, AP-1 interaction domain, transactivation domain CN-regulated inhibitory domain (CRI). For CRI sequence, specially deletion of this domain, resulted in nuclear translocation independent of calcium signaling. Next, we identified the calcineurin binding domain (CNBR) of mouse NF-ATx, and found that the over-expression of CNBR protein fragment inhibited NF-AT site calcineurin binding domain acted as a dominant negative mutant that prevents mNF-ATx-mediated gene activation.
STAT6 has been shown to be required for mejor IL-4 responses. We constructed a conditionally active form of STAT6 by fusing STAT6 to a modified form of the hormone binding domain of the mouse estrogen receptor. This protein activates a specific receptor construct in response to the bestradiol analog 4-Hydroxy-tamoxifen. We also succeeded in the regulation of GM-CSF-induced response of BAF/3 cells by using a dominant negative mutant of JAK2, which is known to act upstream of STAT proteins.
Mutant forms of NF-ATx and STAT6 derived from the above study will be useful fox the regulation of immune responses. We are now planning to generate transgenic mice expressing these mutants for the analysis of the effects of these mutants in vivo.
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