Stat5-mediated regulation of the human type II 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase gene:: Activation by prolactin

Altered PRL levels are associated with infertility in women. Molecular targets at which PRL elicits these effects have yet to be determined. These studies demonstrate transcriptional regulation by PRL of the gene encoding the final enzymatic step in progesterone biosynthesis: 3 beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4) isomerase (3 beta-HSD). A 9/9 match with the consensus State response element was identified at -110 to -118 in the human Type II 3 beta-HSD promoter. 3 beta-HSD chloramphenicol acetyltransferase (CAT) reporter constructs containing either an intact or mutated State element were tested for PRL activation. Expression vectors for State and the PRL receptor were cotransfected with a -300 --> +45 3 beta-HSD CAT reporter construct into HeLa cells, which resulted in a 21-fold increase in reporter activity in the presence of PRL. Promoter activity showed an increased response with a stepwise elevation of transfected State expression or by treatment with increasing concentrations of PRL (max, 250 ng/ml). This effect was dramatically reduced when the putative State response element was removed by 5'-deletion of the promoter or by the introduction of a 3-bp mutation into critical nucleotides in the element. Furthermore, P-32-labeled promoter fragments containing the State element were shifted in electrophoretic mobility shift assay experiments using nuclear extracts from cells treated with PRL, and this complex was supershifted with antibodies to State. These results demonstrate that PRL has the ability to regulate expression of a key human enzyme gene (type II 3 beta-HSD) in the progesterone biosynthetic pathway, which is essential for maintaining pregnancy.