Nakata Masayuki, Nomura Seiji, Ikoma Yoko, Sumigama Seiji, Shido Fumi, Ito Tomomi, Okada Mayumi, Kikkawa Fumitaka, Tsujimoto Masafumi, Mizutani Shigehiko
Department of Obstetrics and Gynecology, Graduate School of Medicine, Nagoya University, 65 Tsurumai-cho, Showa, Nagoya, 466-8550, Japan.
Regul Pept. 2004 Mar 15;117(3):187-93. doi: 10.1016/j.regpep.2003.10.015.
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.
