Regulation of 3 beta-hydroxysteroid dehydrogenase and 17 beta-hydroxysteroid dehydrogenase messenger ribonucleic acid levels by cyclic adenosine 3',5'-monophosphate and phorbol myristate acetate in human choriocarcinoma cells.

Abstract

3 beta-Hydroxysteroid dehydrogenase (3 beta HSD) in human placenta converts 3 beta-hydroxy-5-ene steroids producing progesterone, whereas 17 beta-hydroxysteroid dehydrogenase (17 beta HSD) mediates the interconversion of estrone and estradiol. We first showed that the expression of type I 17 beta HSD (17 beta HSD-I) gene was undetectable in human JEG-3 cells. We then studied the effects of cAMP- and protein kinase-C-dependent pathways on the expression of 3 beta HSD-I and 17 beta HSD-II genes using an analog of cAMP [8-(4-chlorophenylthio)cAMP (8CPTcAMP)] and a protein kinase-C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), in JEG-3 cells. Novel inhibitors of protein kinase-A (PKA) and PKC were also used. The 3 beta HSD cDNA probe hybridized to a single 1.7-kilobase (kb) 3 beta HSD mRNA species corresponding to the transcript of the 3 beta HSD-I gene. The 17 beta HSD cDNA probe hybridized to two 17 beta HSD transcripts of 1.3 and 2.2 kb. The 1.3-kb 17 beta HSD mRNA species was regulated, whereas the 2.2-kb species was constitutively expressed in JEG-3 cells. When JEG-3 cells were exposed to 8CPTcAMP or PMA, 3 beta HSD-I and 17 beta HSD-II gene transcriptions were increased in a dose- and time-dependent manner. Moreover, the combined effects of PMA and 8CPTcAMP on 3 beta HSD-I mRNA levels was additive and synergistic on 17 beta HSD-II mRNA levels. The mechanism by which cAMP activated accumulation of 3 beta HSD-I and 17 beta HSD-II mRNAs involved an activation of the cyclase. The effects of a cAMP-dependent kinase inhibitor and a diacylglycerol-dependent kinase inhibitor in JEG-3 cells indicated that cAMP acts on 3 beta HSD-I mRNA via a PKA-dependent mechanism, but on 17 beta HSD-II mRNA via another nonclassical cAMP-dependent mechanism. Finally, the effect of activation of both signaling pathways on expression of the 17 beta HSD-II gene as well as the effect of PMA on the 3 beta HSD-I gene did not require protein synthesis. These data provide strong evidence for the regulation of the 3 beta HSD-I and 17 beta HSD-II genes by cAMP and PKC and, thus, indicate an important endocrine and/or paracrine regulation of steroid hormone production in human placenta.

Potential endocrine disrupting effect of ochratoxin A on human placental 3β-hydroxysteroid dehydrogenase/isomerase in JEG-3 cells at levels relevant to human exposure

“…It is more likely that the observed effects are due to the interaction with receptor or cell signalling rather than cytotoxic responses. Moreover, gene transcription of 3␤-HSD1 has been reported to be affected by cellular adenosine 3 ,5 -cyclic monophosphate (cAMP) via a protein kinase (PK) A-dependent mechanism [20] and by cellular calcium content via a PKC-dependent pathway [29]. Perturbation of calcium homeostasis [30] and induction of cAMP level [31] by OTA may contribute to the modulation of 3␤-HSD1 mRNA expression.…”

“…Although the two isoforms of human 3␤-HSD are regulated by different transcription factors [for a review, see 28], previous studies have demonstrated that similar signal transduction pathways such as cAMP-related protein kinase A and phorbol ester/phorbol myristate acetate-related PKC pathways are involved in the regulation of human 3␤-HSD gene expression [20,32,33]. OTA may possibly induce similar effects in other steroidogenic organs/tissues.…”

“…Carrier solvents never exceed 0.1% in the experimental media. 8CPTcAMP, analogue of cAMP, has been shown to induce 3␤-HSD1 expression in a dose-and time-dependent manner in JEG-3 cells [20].…”

Molecular Cloning and Expression of Guinea Pig Cytochrome P450C17 cDNA (Steroid 17α-Hydroxylase/17,20 Lyase): Tissue Distribution, Regulation, and Substrate Specificity of the Expressed Enzyme

“…Total RNA from guinea pig tissues and adrenal cells were isolated by homogenization in 4 M guanidinium thiocyanate, centrifuged through a 5.7 M CsCl cushion (Chirgwin et al, 1979), and fractionated by electrophoresis as described (Tremblay et al, 1989;Tremblay and Beaudoin, 1993).…”

“…A rat 18S ribosomal RNA oligodeoxyribonucleotide probe (Clements et al, 1988), synthesized (Bioresearch DNA synthesizer), and end-labeled with [-/-"PJATP, was used to assess the amount and integrity of RNA loaded (Tremblay and Beaudoin, 1993).…”