Cytochrome P450 17α-hydroxylase gene expression in differentiating rat trophoblast cells

Abstract

Trophoblast giant cells of the rat placenta express cytochrome P450 17 alpha-hydroxylase (P450c17) and synthesize androgens. The purpose of this study was to investigate androgen production and expression of P450c17 in the Rcho-1 trophoblast cell line. These cells are capable of differentiating along the trophoblast giant cell lineage. Androstenedione production increased approximately 70-fold as Rcho-1 trophoblast cells progressed from the proliferation to the differentiation state. P450c17 enzyme activity and mRNA also showed significant increases associated with trophoblast giant cell differentiation. To study the transcriptional regulation of the P450c17 gene, the activities of a series of P450c17 promoter-luciferase reporter constructs were evaluated following transient transfection into Rcho-1 trophoblast cells. A DNA region located-98 bp upstream of the P450c17 gene transcriptional start site was the shortest promoter DNA construct consistently possessing activity in Rcho-1 trophoblast cells. Activities of longer constructs (-156 to -1560 bp) in this population of cells were significantly greater than the -98 bp promoter-reporter construct. The -476 bp P450c17 construct showed maximal promoter activity in transiently transfected Rcho-1 trophoblast cells and was developmentally activated in stably transfected Rcho-1 trophoblast cells. Activation of the cyclic AMP/protein kinase A pathway did not significantly affect P450c17 promoter activity in Rcho-1 trophoblast cells, in contrast to its effects in mouse MA-10 Leydig cells. In summary, Rcho-1 trophoblast cells are capable of endocrine differentiation and are a useful in vitro system for studying the regulation of trophoblast androgen production and P450c17 gene expression.