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IN VIVO AND INTRACELLULAR ROLES OF LEUKOTRIENE B4 RECEPTOR.

白三烯 B4 受体的体内和细胞内作用。

基本信息

批准号：
11670111
负责人：
YOKOMIZO Takehiko
金额：
$ 2.3万
依托单位：
UNIVERSITY OF TOKYO
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11670111/
关键词：
LEUKOTRIENE B4 BLT1 BLT2 G-PROTEIN-COUPLED RECEPTOR CLONING ロイコトリエン LTB4 受容体白血球遊走腎不全

项目摘要

To investigate the biological functions of leukotriene B4 receptor in vivo, we established a line of mice deficient in high-affinity leukotriene B4 receptor (BLT1). Embryonic stem (ES) cells were transfected with a targeting vector for BLT1, and several clones were isolated and confirmed for their homologous recombination by Southern blotting. These ES cells were microinjected to blastcysts, transferred to pseudopregnant female mice, and subsequently chimeric mice were obtained. These chimeric mice are crossed among them, and finally BLT1-deficient mice were born. Now these mice are under backcrossing to examine their phenotype.CHO cells stably expressing BLT1 showed robust chemotaxis toward LTB4 in vitro, so we tried to examine the chemotactic activities of these cells in vivo. Rat acute ischemic-reperfusion renal injury was initiated by 45 minute-clamping of the renal artery. After 24 hrs, cells were injected into the injured kidney, and were allowed to chemotax for 4 hrs. Only in ca … More se of ischemic kidney, not in the sham-operated kdney, CHO cells expressing BLT1 migrated into the peritubal portion of the kidney. Control cells transfected with empty vector did not migrate at all. Prefeeding of the rats with BLT antagonist (Ono 4057) not only inhibited the cells migration, but also reduced the severity of the renal failure, suggesting that LTB4-BLT1 interaction plays important roles in the initiation and the progression of ischemic renal failure.(Noiri, E., et al Proc.Natl.Acad.Sci.U.S.A., 2000)To clarify the molecular mechanisms of the cell-specific expression of BLT1, we examined the transcriptional regulation of BLT1. BLT1 gene is a small gene within 4 kbps, and its promoter contains no TATA-box, but an Initiator sequence. Serial deletion analyses revealed that Sp-1 at-50 from the initiation site plays a pivotal role in BLT1 transcription. Moreover, the promoter region was totally methylated in BLT1-nonexpressing cells, but not in the expressing cells. In vitro methylation of BLT1 promoter almost completely inhibited its promoter activity.(Kato, K., et al.J.Exp.Med.2000)On the course of analyzing BLT1 promoter, we found a novel gene with structural similarities with BLT1. The encoded protein was a novel G-protein-coupled receptor, and exhibited a low-affinity, but significant binding to LTB4. This receptor efficiently transduces intracellular signaling toward calcium increase, inhibition of ademylyl cyclase, and chemotaxis by application of LTB4, thus we named it a second LTB4 receptor, BLT2.(Yokomizo, et al.J.Exp.Med.2000, Yokomizo, T.et al.J.Biol.Chem.2001) Less

为了研究白三烯B4受体在体内的生物学功能，我们建立了高亲和力白三烯B4受体（BLT 1）缺陷小鼠系。用BLT 1的靶向载体转染胚胎干（ES）细胞，分离几个克隆，并通过Southern印迹法确认它们的同源重组。将这些ES细胞显微注射到囊胚中，转移到假孕雌性小鼠中，随后获得嵌合小鼠。这些嵌合小鼠在它们之间杂交，最终产生了BLT 1缺陷小鼠。稳定表达BLT 1的CHO细胞在体外对LTB 4表现出强烈的趋化性，因此我们试图在体内检测这些细胞的趋化活性。大鼠急性缺血-再灌注肾损伤由肾动脉阻断45分钟开始。24小时后，将细胞注射到损伤的肾脏中，并使其化学适应4小时。仅在 ...更多信息表达BLT 1的CHO细胞迁移到缺血肾的输卵管周围部分，而在假手术肾中没有。用空载体转染的对照细胞根本不迁移。预先给予BLT拮抗剂Ono 4057不仅能抑制细胞迁移，而且能减轻肾功能衰竭的严重程度，提示LTB 4-BLT 1相互作用在缺血性肾功能衰竭的发生和发展中起重要作用。（Noiri，E.，等Proc.Natl.Acad.Sci.U.S.A.，为了阐明BLT 1细胞特异性表达的分子机制，我们研究了BLT 1的转录调控。BLT 1基因是一个长度在4kbps以内的小基因，其启动子不含TATA盒，但含有一个Initiator序列。序列缺失分析表明，Sp-1在起始位点的-50处起着关键作用的BLT 1转录。此外，启动子区完全甲基化的BLT 1-nonexpressing细胞，但不是在表达细胞。BLT 1启动子的体外甲基化几乎完全抑制其启动子活性。(Kato，K.，等J.Exp.Med.2000）在分析BLT 1启动子的过程中，我们发现了一个与BLT 1具有结构相似性的新基因。编码的蛋白质是一种新的G蛋白偶联受体，并表现出低亲和力，但显着结合LTB 4。该受体通过应用LTB 4有效地将细胞内信号传导至钙增加、腺苷酸环化酶的抑制和趋化性，因此我们将其命名为第二LTB 4受体，BLT 2。（Yokomizo等人，J.Exp.Med.2000，Yokomizo，T.et al.J.Biol.Chem.2001）减