IN VIVO AND INTRACELLULAR ROLES OF LEUKOTRIENE B4 RECEPTOR.

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(BLT1)缺陷小鼠系。将BLT1靶向载体导入胚胎干细胞，经Southern blotting鉴定，获得多个克隆，证实其同源重组。将这些ES细胞显微注射到囊胚中，转移到假孕母鼠体内，获得嵌合体小鼠。这些嵌合小鼠在它们之间杂交，最终出生了BLT1缺陷小鼠。目前这些小鼠正在进行回交以检测其表型。稳定表达BLT1的CHO细胞在体外对LTB4具有很强的趋化活性，因此我们试图在体内检测这些细胞的趋化活性。夹闭大鼠肾动脉45分钟，造成大鼠急性肾缺血再灌注损伤。24小时后，将细胞注入损伤的肾脏，并允许化疗4小时。仅限于ca…假手术组肾组织中表达BLT1的CHO细胞迁移至输卵管周部，而缺血肾组织中表达BLT1的CHO细胞迁移至输卵管周围。转染空载体的对照细胞完全不迁移。预先给予BLT拮抗剂(ONO 4057)不仅抑制了细胞的迁移，而且减轻了肾功能衰竭的严重程度，提示LTB4-BLT1的相互作用在缺血性肾功能衰竭的发生和发展中起着重要的作用(Noiri，E.，et al Proc.Natl.Academy.Science U.S.，2000)为了阐明BLT1细胞特异性表达的分子机制，我们检测了BLT1的转录调控。BLT1基因是一个4kbps的小基因，它的启动子没有TATA盒，而是一个启动子序列。序列缺失分析表明，起始部位的Sp-1 at-50在BLT1转录中起着关键作用。此外，在不表达BLT1的细胞中，启动子区域完全甲基化，而在表达BLT1的细胞中则没有。在体外，BLT1启动子的甲基化几乎完全抑制了它的启动子活性(Kato，K.，et al.J.Exp.Med.2000)在分析BLT1启动子的过程中，我们发现了一个与BLT1结构相似的新基因。该编码蛋白是一种新的G蛋白偶联受体，与LTB4亲和力低，但有明显的结合作用。这种受体通过应用LTB4有效地传递细胞内钙增加、抑制ADM环酶和趋化的信号，因此我们将其命名为第二个LTB4受体BLT2(Yokomizo等人，J.Exp.Med.2000，Yokomizo，T.et al.J.Biol.Chem.2001)Less

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