IN VIVO AND INTRACELLULAR ROLES OF LEUKOTRIENE B4 RECEPTOR.

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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