Regulation of aromatase cytochrome P-450 and 17 beta-hydroxysteroid dehydrogenase messenger ribonucleic acid levels in choriocarcinoma cells.
In human placenta the enzyme complex aromatase catalyzes the conversion of androgens to estrogens and 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) mediates the reversible interconversion of, e.g. estrone to estradiol. We studied the effects of cholera toxin (CT), an activator of adenylate cyclase, and 12-O-tetradecanoyl phorbol 13-acetate (TPA), a phorbol ester protein kinase C activator, on the levels of messenger (m) RNAs encoding aromatase cytochome P-450 (P-450AROM) and 17 beta-HSD in cultured JEG-3 choriocarcinoma cells. With the use of oligonucleotide probes designed according to known complementary DNA sequences, hybridizable mRNA transcripts of 3.0, 2.4, and 1.6 kilobases for P-450AROM were found in Northern blot analysis of JEG-3 cell RNA. A single 1.4-kilobase transcript was detected for 17 beta-HSD. Time-dependent increases in P-450AROM mRNA levels in JEG-3 cells were observed for both CT and TPA with maximal effects at 24-48 h. CT and TPA increased P-450AROM mRNA levels in a concentration-dependent manner. The maximal effects, about 4.8-fold and 3.3-fold stimulations above basal levels, were obtained with 10 ng/ml of CT and 100 ng/ml of TPA, respectively. The effects of CT and TPA were additive. CT induced 17 beta-HSD mRNA levels in a time- and concentration-dependent manner and its maximal effect of 10.1-fold above basal levels was obtained within a similar time and concentration-dependence as for P-450AROM mRNA. TPA itself had no clear effect but it approximately doubled the effect of CT on 17 beta-HSD mRNA expression. Inhibition of protein synthesis by cycloheximide decreased basal, CT and TPA stimulated P-450AROM mRNA levels but increased the expression of 17 beta-HSD mRNA. This result is consistent with the hypothesis that induction of P-450AROM gene expression is mediated by a labile protein regulator resembling to most other steroidogenic P-450 enzymes, whereas 17 beta-HSD as a non-P450 enzyme appears to be controlled in a different manner. The present results suggest that: 1) induction of P-450AROM mRNA may at least partly be responsible for our previously reported increases in the rate of conversion of androgens to estrogens by CT and TPA in JEG-3 cells; 2) 17 beta-HSD mRNA expression is mainly controlled through a cAMP-dependent mechanism in contrast to the multifactorial control of P-450AROM mRNA; and 3) protein synthesis inhibition by cycloheximide has opposite effects on the mRNA levels of these two key enzymes in placental estrogen metabolism.
Ritvos
O
O
Department of Pathology, University of Helsinki, Finland.
Voutilainen
R
R
D015067:17-Hydroxysteroid Dehydrogenases / Q000235:genetics*; D001141:Aromatase / Q000235:genetics*; D001483:Base Sequence; D002772:Cholera Toxin / Q000494:pharmacology; D002822:Choriocarcinoma / Q000737:chemistry*; D000242:Cyclic AMP / Q000032:analysis; D003513:Cycloheximide / Q000494:pharmacology; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D005260:Female; D006801:Humans; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000032:analysis*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014594:Uterine Neoplasms / Q000737:chemistry*
D012333:RNA, Messenger; D002772:Cholera Toxin; D003577:Cytochrome P-450 Enzyme System; D003513:Cycloheximide; D000242:Cyclic AMP; D015067:17-Hydroxysteroid Dehydrogenases; D001141:Aromatase; D013755:Tetradecanoylphorbol Acetate