Analysis of cytochrome P-450 side-chain cleavage gene promoter activation during trophoblast cell differentiation

• Published in: Molecular and cellular endocrinology 1995-09, Vol.113 (2), p.183-194
• Main Authors: Yamamoto, Toshiya, Chapman, Belinda M., Clemens, Jeffrey W., Richards, JoAnne S., Soares, Michael J.
• Format: Article
• Language: English
• Subjects: 8-Bromo Cyclic Adenosine Monophosphate - pharmacology; Animals; Base Sequence; Cell Differentiation; Cell Line; Cholesterol Side-Chain Cleavage Enzyme - genetics; Colforsin - pharmacology; Cyclic AMP - pharmacology; Cyclic AMP-Dependent Protein Kinases - metabolism; Cytochrome P-450; DNA-Binding Proteins - analysis; DNA-Binding Proteins - genetics; Enzyme Activation - drug effects; Fushi Tarazu Transcription Factors; Gene Expression; Gene promoter activation; Homeodomain Proteins; Molecular Sequence Data; Promoter Regions, Genetic; Rats; Receptors, Cytoplasmic and Nuclear; RNA, Messenger - analysis; Side-chain cleavage; Steroidogenic Factor 1; Transcription Factors - analysis; Transcription Factors - genetics; Transfection; Trophoblast cell differentiation; Trophoblasts - cytology; Trophoblasts - enzymology
• ISSN: 0303-7207; 1872-8057
• DOI: 10.1016/0303-7207(95)03628-K

Trophoblast giant cell differentiation is accompanied by transcriptional activation of the cytochrome P-450 side-chain cleavage (P450scc) gene. The Rcho-1 trophoblast cell line has the capacity to differentiate along the trophoblast giant cell lineage and has been used to study trophoblast-specific P450scc gene expression. In this report, P450scc gene promoter activities in trophoblast cells have been mapped and the involvement of known modulators of steroid hydroxylase gene expression, the cyclic AMP/protein kinase A pathway and steroidogenic factor-1 (SF-1), evaluated. Comparisons were made with Y-1 adrenal and R2C Leydig cells. The cumulative results from transient and stable transfection experiments implicate the region between −428 and −511 bp of 5′-flanking DNA in the developmental activation of the P450scc promoter during trophoblast giant cell differentiation. Differences in basal activities of the P450scc promoter constructs were also observed in Y-1 adrenal and R2C Leydig cells; however, the magnitude of the differences was modest. Activators of the protein kinase A pathway stimulated P450scc promoter activity in Y-1 cells, whereas similar treatment of Rcho-1 trophoblast cells did not stimulate but actually inhibited P450scc promoter activity. The inhibitory activity was localized between −639 and −894 bp of the P450scc promoter. SF-1 mRNA and protein were detected in adrenal and gonadal cells but not in rat placenta or Rcho-1 trophoblast cells by Northern and Western blotting, respectively. Thus, P450scc gene activation during trophoblast cell differentiation involves an 83-bp region of its 5′-flanking DNA between −428 and −511 but does not appear to involve cyclic AMP-activated pathways or SF-1. In conclusion, the mechanism of P450scc gene activation during trophoblast cell differentiation appears different from the regulation of P450scc gene activation in other steroidogenic tissues.