Synergistic Activation of the Human Type II 3β-Hydroxysteroid Dehydrogenase/Δ5-Δ4 Isomerase Promoter by the Transcription Factor Steroidogenic Factor-1/Adrenal 4-binding Protein and Phorbol Ester

Susan Leers-Sucheta,Ken-Ichirou Morohashi,J Ian Mason,Michael H Melner

doi:10.1074/jbc.272.12.7960

Susan Leers-Sucheta, Ken-Ichirou Morohashi + Show 2 more

Open Access

https://doi.org/10.1074/jbc.272.12.7960

Copy DOI

Abstract

Steroidogenic factor-1/adrenal 4-binding protein (SF-1/Ad4BP) is an orphan nuclear receptor/transcription factor known to regulate the P450 steroid hydroxylases; however, mechanisms that regulate the activity of SF-1/Ad4BP are not well defined. In addition, little is known about the mechanisms that regulate the human steroidogenic enzyme, type II 3beta-hydroxysteroid dehydrogenase (3beta-HSD II), the major gonadal and adrenal isoform. Regulation of the 3beta-HSD II promoter was examined using human adrenal cortical (H295R; steroidogenic) and cervical (HeLa; non-steroidogenic) carcinoma cells. H295R cells were transfected with a series of 5' deletions of 1251 base pairs (bp) of the 3beta-HSD II 5'-flanking region fused to a chloramphenicol acetyltransferase (CAT) reporter gene followed by treatment with or without phorbol ester (phorbol 12-myristate 13-acetate; PMA). CAT assay data indicated that the region from -101 to -52 bp of the promoter was required for PMA-induced expression. A putative SF-1/Ad4BP regulatory element, TCAAGGTAA, was identified by sequence homology at -64 to -56 bp of the promoter. Cotransfection of HeLa cells with the -101 3beta-HSD-CAT construct and an expression vector for SF-1/Ad4BP increased CAT activity 49-fold. Subsequent treatment with PMA induced an unexpected synergistic increase in transcriptional activity 540-fold over basal. Mutation of the putative response element (TCAAGGTAA to TCAATTTAA) abolished SF-1-induced CAT activity and the synergistic response to PMA. Gel mobility shift assays confirmed that SF-1/Ad4BP interacts with the putative element and transcripts for SF-1/Ad4BP were detected in H295R cells by Northern analysis. These data are the first to demonstrate 1) regulation of a non-cytochrome P450 steroidogenic enzyme promoter by SF-1/Ad4BP, 2) a powerful synergistic effect of PMA on SF-1/Ad4BP-induced transcription, and 3) the importance of the SF-1/Ad4BP regulatory element in the regulation of the 3beta-HSD II promoter.

Highlights

Steroidogenic factor-1/adrenal 4-binding protein (SF1/Ad4BP) is an orphan nuclear receptor/transcription factor known to regulate the P450 steroid hydroxylases; mechanisms that regulate the activity of steroidogenic factor-1 (SF-1)/ Ad4BP are not well defined
To determine the region(s) of the promoter that confer phorbol ester-mediated transcriptional regulation of the type II 3␤-hydroxysteroid dehydrogenase/ ⌬5-⌬4-ene-isomerase (3␤-HSD) gene, a series of 5Ј deletions of the promoter fused to a chloramphenicol acetyltransferase (CAT) reporter gene were used to transiently transfect human adrenocortical carcinoma (H295R) cells followed by treatment for 24 h in the absence or presence of PMA
Using deletion mutagenesis we determined that the region from Ϫ101 to Ϫ52 bp of the promoter was essential for PMA-mediated transcription of the reporter gene and contained a putative SF-1/Ad4BP regulatory element TCAAGGTAA from Ϫ64 to Ϫ52 bp

Summary

Introduction

Steroidogenic factor-1/adrenal 4-binding protein (SF1/Ad4BP) is an orphan nuclear receptor/transcription factor known to regulate the P450 steroid hydroxylases; mechanisms that regulate the activity of SF-1/ Ad4BP are not well defined. Control of 3␤-HSD Promoter by SF-1/Ad4BP and Phorbol Ester scription factor steroidogenic factor-1 (SF-1; called adrenal 4-binding protein; Ad4BP) in the cAMP-mediated transactivation of cytochrome P450 steroid hydroxylase genes (10 –15) in adrenal and gonadal tissues This transcription factor is a member of the steroid hormone receptor superfamily (11, 16) and is classified as an orphan nuclear receptor because an endogenous ligand has not been identified. In addition to its regulatory actions on steroid hydroxylase gene expression, targeted disruption of the Ftz-F1 gene in mice proved this transcription factor to be essential for sexual differentiation and development of the adrenal gland and gonads (20) and critical for normal development of the ventromedial hypothalamus and pituitary gonadotrophs (21) This nuclear receptor mediates transcriptional control over a number of genes that are involved in various aspects of reproductive function including the ␣-subunit of pituitary glycoprotein hormones (22), Mullerian inhibiting substance (23), and oxytocin (24) genes. Pituitaries of Ftz-F1-disrupted mice lack transcripts for gonadotropin-releasing hormone receptor, as well as ␤-subunits of luteinizing hormone and follicle-stimulating hormone (25), all of which are requisite for reproductive competence

Objectives

Methods

Results

Conclusion