2002 Fiscal Year Final Research Report Summary
Physiological roles of placental leucine aminopeptidase/oxytocinase and its clinical application using molecular biological technique.
| Project/Area Number |
12470341
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Obstetrics and gynecology
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| Research Institution | Nagoya University |
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Principal Investigator |
MIZUTANI Shigehiko Nagoya University, Graduate School of Medicine, Professor, 大学院・医学系研究科, 教授 (00159162)
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| Co-Investigator(Kenkyū-buntansha) |
NOMURA Seiji Nagoya University, Graduate School of Medicine, Associate Professor, 大学院・医学系研究科, 助教授 (20242860)
TSUJIMOTO Masafumi RIKEN, Chief Scientist, 理化学研究所・細胞生化学研究室, 主任研究員 (00281668)
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| Project Period (FY) |
2000 – 2002
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| Keywords | oxytocinase / molecular biology / recombinant protein / placenta / preterm delivery / aminopeptidase / gene targeting / transgenic animal |
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| Research Abstract |
Placental leucine aminopeptidase (P-LAP), which is identical to cystine aminopeptidase, is the predominant and functional oxytocinase in the placenta and maternal serum. The aim of this study is to evaluate the clinical significance and application of P-LAP using molecular biological technique. We have succeeded in establishing the CHO cells producing recombinant soluble form of P-LAP. Recombinant P-LAP hydrolyses not only oxytocin and vasopressin, but also dynorphin A and enkephalin in the brain. Although further in vivo studies are required, recombinant P-LAP could be used to inhibit the labor. P-LAP exists in both membrane-bound and soluble forms, which means that the soluble form is released by a specific proteolytic cleavage. P-LAP secretase has a metalloprotease activity and recognizes the amino acid sequence of cleavage site to attack. We have isolated genomic clones containing the 5'-upstream region of the P-LAP gene. We demonstrated that transcription factors activating protein 2 (AP-2) and Ikaros cooperatively enhance P-LAP transcription in trophoblastic cells. Interleukin-lbeta (IL-1β) increased P-LAP activity and proteins. Semi-quantitative RT-PCR and Southern blotting showed that IL-1β also increased P-LAP mRNA, which was abrogated by prior exposure to cycloheximide. Luciferase assays did not reveal any regulatory regions that could explain IL-1β-induced P-LAP mRNA accumulation. These findings indicated that prolonged exposure to IL-1β induces P-LAP in the placenta, possibly via other de novo protein synthesis. Transmission immunoelectron microscopy revealed that P-LAP was expressed on the surface of apical microvilli of syncytiotrophoblast cells. During this study, we could obtain both P-LAP-deficient mice and P-LAP overexpressing rats. Now the studies employing these animals to evaluate the changes of the spontaneous and oxytocin-induced labor are on the way. The results would elucidate the roles of oxytocinase during pregnancy.
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