オキシトシン受容体から見た子宮収縮制御機構の解明(1子宮収縮機構とその制御)

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Oxytocin receptor (OTR) expressionshould play a crucial role for initiation and regulation of labor in the pregnant uterus. To elucidate the molecular mechanism of the OTR regulation in the uterus, we attempted to clone the human OTR cDNA, gene and transcription factor for the gene. We also analyse the pharmacological character and desensitization mechanism of OTR. Term uterine myometrium was obtained from uterine rupture under informed consent, mRNA was extracted and cDNA library was constructed. OTR was successfully reconstructed on Xenopus oocytes by microinjection of the mRNA, followed by electrophysiological detection of the oxytocin-elicited inward current. Using this detection system, OTR cDNA was cloned from the cDNA library and cRNA from cDNA injected Xenopus oocytes. Human OTR cDNA was 4.1kb, encoding 389 aminoacids polypeptide belongs to the 7 transmembrane receptor family. Northern blotting indicated OTR mRNA was highly upregulated (〜300-fold) in term myometrium compared with non-pregnant myometrium. We established anti OTR monoclonal antibodies and performed Western blotting. The protein expression pattern was similar to that of OTR mRNA, suggesting that the OTR expression is mainly regulated at the transcription level. Immunoreactive OTR and OTR mRNA positive myometrial cells were diffusely scattered in the term myometrium, suggesting cell-to-cell communication system is required for coordinative uterine contraction to the oxytocin stimuli. Uterine myometrial tissue, which had been used for pharmacological analysis of OTR, contains significant amount of vasopressin Vla receptor, that can cross-react to oxytocin related ligands. Pharmacological analysis of cloned OTR, using OTR transfected COS cells and Xenopus oocyted, revealed that Arg^8-vasopressin was not physiological agonist to OTR. Parmanent cell line, OTR-Hela appears to be a good tool for pharmacological characterization because it express as much amount of OTR protein as observed in term myometrium. OTR desensitization occurred in cultured myometrial cells after 12 hour treatment of oxytocin. FACS analysis with anti OTR antibody and ^3H-oxytocin binding study revealed that the desensitization of OTR does not require the internalization of the receptor protein. The expression of OTR mRNA was also downregulated during the desensitization period, suggesting the lack of newly translated OTR protein also caused the receptor desensitization. OTR gene was cloned from human genomic library. Chromosomal localization was determined by FISH. It spans 17kb, locates on the chromosome 3(3p26.2) and contains 3 introns and 4 exons. Computer analysis indicated several consensus transcription factor-binding DNA sequences, such as for SP-1, ERE-half sites, NF-IL6 and Stat 3. Reporter-gene co-transfection experiments indicated estrogen receptor and NF-IL6 do not have direct effect on the OTR transcription. Nuclear proteins to bind the OTR gene promoter were characterized by gel shift assay and methylation interference footprinting. Footprinting experiments revealed term myometrium contains at least 2 apparent nuclear protein binding elements (US-1 and US-2) to which the binding proteins upregulated in term uterine myometrium. To clone the binding proteins, yeast one hybrid system was applied. For US-1 element, the system gave only false positive clones. For US-2 site, we have cloned a human homologue of chicken Maf F (hMafF). hMafF has leucine zipper structure, and has no transactivation activity to the OTR gene promoter. Northern blot indicated hMafF mRNA was highly upregulated in term myometrium as well as in the kidney. This molecule might play an important role for the regulation of OTR transcription through heterodimer formation with another leucine-zipper transcription factor. We have clarified the molecular structure and mechanism of OTR expression and desensitization in human myometrium. We also established the tool for pharmacological analysis of OTR. These result
社団法人日本産科婦人科学会の論文
2000-08-01

オキシトシン受容体から見た子宮収縮制御機構の解明(1子宮収縮機構とその制御)

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