The Transcriptional Regulating Protein of 132 kDa (TReP-132) Enhances P450scc Gene Transcription through Interaction with Steroidogenic Factor-1 in Human Adrenal Cells

Florence Gizard,Bernard Lavallée,Fredérique Dewitte,Elisabeth Teissier,Bart Staels,Dean W Hum

doi:10.1074/jbc.m205786200

Florence Gizard, Bernard Lavallée + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m205786200

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Abstract

The human P450scc gene is regulated by the tissue-specific orphan nuclear receptor, steroidogenic factor-1 (SF-1), which plays a key role in several physiologic processes including steroid synthesis, adrenal and gonadal development, and sexual differentiation. Several studies have demonstrated the interaction of SF-1 with different proteins. However, it is clear that additional factors not yet identified are involved with SF-1 to regulate different target genes. Recently, it was demonstrated that a novel transcriptional regulating protein of 132 kDa (TReP-132) regulates expression of the human P450scc gene. The overexpression of TReP-132 in adrenal cells increases the production of pregnenolone, which is associated with the activation of P450scc gene expression. Considering the colocalization of TReP-132 and SF-1 in steroidogenic tissues such as the adrenal and testis, and the presence of two putative LXXLL motifs in TReP-132 that can potentially interact with SF-1, the relationship between these two factors on the P450scc gene promoter was determined. The coexpression of SF-1 and TReP-132 in adrenal NCI-H295 cells cooperates to increase promoter activity. Pull-down experiments demonstrated the interaction between TReP-132 and SF-1, and this was further confirmed in intact cells by coimmunoprecipitation/Western blot and two-hybrid analyses. Deletions and mutations of the TReP-132 cDNA sequence demonstrate that SF-1 interaction requires the LXXLL motif found at the amino-terminal region of the protein. Also, the "proximal activation domain" and the "AF-2 hexamer" motif of SF-1 are involved in interaction with TReP-132. Consistent with previous studies showing interaction between CBP/p300 and SF-1 or TReP-132, the coexpression of these three proteins results in a synergistic effect on P450scc gene promoter activity. Taken together the results in this study identify a novel function of TReP-132 as a partner in a complex with SF-1 and CBP/p300 to regulate gene transcription involved in steroidogenesis.

Highlights

The human P450 side chain cleavage (P450scc) gene is regulated by the tissuespecific orphan nuclear receptor, steroidogenic factor-1 (SF-1), which plays a key role in several physiologic processes including steroid synthesis, adrenal and gonadal development, and sexual differentiation
TReP-132 Increases Pregnenolone and P450scc Transcript Levels in Human Adrenal Cells—In a previous study it was demonstrated that TReP-132 increases P450scc gene promoter activity [42], which would indicate that this transcriptional regulating protein is potentially able to increase steroid synthesis
To confirm that an increased level of TReP-132 can stimulate the conversion of cholesterol to pregnenolone catalyzed by P450scc, NCI-H295 cells were transfected with a pcDNA3 expression plasmid encoding TReP-132 or with the empty pcDNA3 vector alone

Summary

The abbreviations used are

P450scc, P-450 side chain cleavage; ACTH, adrenocorticotropic hormone; MIS, Mullerian inhibitory substance; LH, luteinizing hormone; FSH, follicle-stimulating hormone; SF-1, steroidogenic factor-1; CBP, CREB-binding protein; CREB, cAMP-response element-binding protein; TReP-132, transcriptional regulating protein of 132 kDa; HA, hemagglutinin; NR, nuclear receptor; CMV, cytomegalovirus; GST, glutathione S-transferase; HPLC, high performance liquid chromatography; AF-2, activation function 2; PBS, phosphate-buffered saline. In agreement with the role of SF-1 in tissues involved in steroidogenesis and sexual differentiation, targeted SF-1 gene disruption produced knockout mice which completely lacked the ventromedial hypothalamic nucleus, adrenal glands, and gonads (reviewed in Ref. 23) These developmental defects were associated with phenotypic male-to-female sex reversal and adrenocortical insufficiency, resulting in neonatal death [36, 37]. SF-1 and TReP-132 transcripts colocalize in many tissues, including the testis and adrenal cortex, which is consistent with their functional interaction in steroidogenesis [5, 42] This present study addresses the role of TReP-132 in the regulation of the human P450scc gene mediated by SF-1 in human adrenal carcinoma NCI-H295 cells.

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION