Insulin stimulates placental leucine aminopeptidase/oxytocinase/insulin-regulated membrane aminopeptidase expression in BeWo choriocarcinoma cells.
Placental leucine aminopeptidase (P-LAP), a cystine aminopeptidase that is identical to insulin-regulated membrane aminopeptidase, hydrolyzes oxytocin, which results in the loss of oxytocin activity. We previously isolated genomic clones containing the human P-LAP promoter region, which included two sites homologous to the 10-bp-insulin responsive element (IRE) that was identified on the phosphoenolpyruvate carboxinase gene. We therefore postulated that insulin regulates P-LAP expression via these IREs and investigated this notion using BeWo choriocarcinoma trophoblastic cells cultured in the presence of insulin. Insulin increased P-LAP activity in a time- and dose-dependent manner. Physiological concentrations of insulin at 10(-7) M exhibited the most potent effect on P-LAP activity. Western blotting demonstrated that 10(-7) M insulin increased P-LAP protein levels. Semi-quantitative RT-PCR and Southern blotting showed that insulin also increased P-LAP mRNA, which was abrogated by prior exposure to cycloheximide. Luciferase assay did not reveal any regulatory regions within 1.1 kb upstream of the P-LAP gene that could explain the insulin-induced P-LAP mRNA accumulation. These findings indicate that insulin induces P-LAP expression in trophoblasts, and that it acts via de novo synthesis of other proteins, which partially contradicts our initial hypothesis.
Nakata
Masayuki
M
Department of Obstetrics and Gynecology, Graduate School of Medicine, Nagoya University, 65 Tsurumai-cho, Showa, Nagoya, 466-8550, Japan.
Nomura
Seiji
S
Ikoma
Yoko
Y
Sumigama
Seiji
S
Shido
Fumi
F
Ito
Tomomi
T
Okada
Mayumi
M
Kikkawa
Fumitaka
F
Tsujimoto
Masafumi
M
Mizutani
Shigehiko
S
D001483:Base Sequence; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D002822:Choriocarcinoma; D003513:Cycloheximide / Q000494:pharmacology; D010122:Cystinyl Aminopeptidase / Q000235:genetics / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D005260:Female; D006801:Humans; D007328:Insulin / Q000494:pharmacology* / Q000502:physiology; D008156:Luciferases / Q000378:metabolism; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D013997:Time Factors; D014327:Trophoblasts / Q000201:enzymology*; D014594:Uterine Neoplasms
D007328:Insulin; D012333:RNA, Messenger; D003513:Cycloheximide; D008156:Luciferases; D010122:Cystinyl Aminopeptidase; C105964:leucyl-cystinyl aminopeptidase