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Papers In Press, published online ahead of print July 5, 2002
J. Biol. Chem, 10.1074/jbc.M205786200
Submitted on June 11, 2002
Revised on July 5, 2002
Accepted on July 5, 2002
Genfit, Loss 59120
Corresponding Author: Dean.Hum{at}genfit.com
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 demonstrate 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. As well, 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 partner in a complex with SF-1 and CBP/p300 to regulate gene transcription involved in steroidogenesis.
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