Yamamoto T, Chapman B M, Johnson D C, Givens C R, Mellon S H, Soares M J
Department of Physiology, University of Kansas Medical Center, Kansas City 66160, USA.
J Endocrinol. 1996 Jul;150(1):161-8. doi: 10.1677/joe.0.1500161.
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.
大鼠胎盘的滋养层巨细胞表达细胞色素P450 17α-羟化酶（P450c17）并合成雄激素。本研究的目的是调查Rcho-1滋养层细胞系中雄激素的产生及P450c17的表达。这些细胞能够沿着滋养层巨细胞谱系分化。随着Rcho-1滋养层细胞从增殖状态发展到分化状态，雄烯二酮的产生增加了约70倍。P450c17酶活性和mRNA也显示出与滋养层巨细胞分化相关的显著增加。为了研究P450c17基因的转录调控，在将一系列P450c17启动子-荧光素酶报告基因构建体瞬时转染到Rcho-1滋养层细胞后，评估了它们的活性。位于P450c17基因转录起始位点上游-98 bp的DNA区域是在Rcho-1滋养层细胞中始终具有活性的最短启动子DNA构建体。在这群细胞中，更长的构建体（-156至-1560 bp）的活性显著高于-98 bp启动子-报告基因构建体。-476 bp的P450c17构建体在瞬时转染的Rcho-1滋养层细胞中显示出最大的启动子活性，并在稳定转染的Rcho-1滋养层细胞中被发育激活。与环磷酸腺苷/蛋白激酶A途径对小鼠MA-10 Leydig细胞的作用相反，该途径的激活对Rcho-1滋养层细胞中P450c17启动子活性没有显著影响。总之，Rcho-1滋养层细胞能够进行内分泌分化，是研究滋养层雄激素产生调控和P450c17基因表达的有用体外系统。
