ANALYSIS OF CYTOCHROME-P-450 SIDE-CHAIN CLEAVAGE GENE PROMOTER ACTIVATION DURING TROPHOBLAST CELL-DIFFERENTIATION

Trophoblast giant cell differentiation is accompanied by transcription al activation of the cytochrome P-450 side-chain cleavage (P450scc) ge ne. The Rcho-1 trophoblast cell line has the capacity to differentiate along the trophoblast giant cell lineage and has been used to study t rophoblast-specific P450scc gene expression. In this report, P450scc g ene 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 Leydi g cells. The cumulative results from transient and stable transfection experiments implicate the region between -428 and -511 bp of 5'-flank ing DNA in the developmental activation of the P450scc promoter during trophoblast giant cell differentiation. Differences in basal activiti es of the P450scc promoter constructs were also observed in Y-1 adrena l 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 tr ophoblast cells did not stimulate but actually inhibited P450scc promo ter 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 trophobla st cells by Northern and Western blotting, respectively. Thus, P450scc gene activation during trophoblast cell differentiation involves an 8 3-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 dif ferentiation appears different from the regulation of P450scc gene act ivation in other steroidogenic tissues.