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

Oncology and Molecular Endocrinology Research Center, Laval University, Québec GIK 7P4, Canada.
Journal of Biological Chemistry (Impact Factor: 4.6). 11/2002; 277(42):39144-55. DOI: 10.1074/jbc.M205786200
Source: PubMed

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.

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    • "s shown in Fig. 3B, TdIF1 unexpectedly bound to truncated TReP-132 residues ranging from 387 to 444 without the LXXLL motif and did not bind to del7, suggesting that TdIF1 binds to a confined region from 387 to 407 of TReP-132. Recently, TReP-132 has been reported to bind to SF-1 only through its N-terminal LXXLL, not through its C-terminal LXXLL (Gizard et al . 2002b). Thus, we then examined the possibility of binding through the N-terminal LXXLL. As shown in Fig. 3B (del8), no binding through the N-terminal LXXLL was observed. We therefore conclude that TdIF1 mainly binds to the novel confined region between residues 387 and 407 in TReP-132."
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    ABSTRACT: N regions at the junction of V, D and J DNA segments are synthesized with large protein complexes including terminal deoxynucleotidyltransferase (TdT) during V(D)J recombination in B- or T-cells. TdT directly binds to TdIF1, TdIF2, PCNA and the Ku70/86 heterodimer. Using a yeast two-hybrid system, we isolated a cDNA clone encoding the gene for TReP-132, which is involved in P450scc gene expression in steroid-hormone-producing cells or lymphoid cells. Interaction between TReP-132 and TdIF1 was confirmed by pull-down assay and immunoprecipitation assay using specific antibodies against TReP-132 both in vitro and in vivo. TdT also directly bound to TReP-132 through its confined N-terminal region. Furthermore, the co-expression of TdIF1 and TReP-132 or TdT and TReP-132 in COS7 cells showed that these proteins are co-localized within the nucleus. TReP-132 reduces TdT activity to 2.5% of its maximum value in the in vitro assay system using double-stranded DNA with a 3' protrusion as a primer. These findings suggest that TdT synthesizes N region under a negative control of TReP-132 during V(D)J recombination.
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    • "Plasmid Gal4-tk80-luc was a luciferase reporter gene under the control of the Gal4-responsive element and plasmid pGal4-Smad1 was a human Smad1 sequence fused with the DNA-binding domain of Gal4 (Pearson et al. 1999). P450 scc luciferase reporter constructs containing fragments of the human P450 scc gene spanning from nucleotides –110 to +49, with or without the mutated SF-1-binding site, subcloned in pGL3 vector, were kindly provided by Dr B Staels (Gizard et al. 2002). StAR luciferase reporter construct containing fragments of human StAR gene spanning nucleotides –235 to +39 was a gift from Dr J Strauss (Sugawara et al. 1996). "
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    • "Depression has been reported to be characterized by elevated corticosteroidal activity and HPA-axis activation [41] [42]. Both HELO1 and TreP-132 are highly expressed in adrenals and testis [32] [40]. Cloning of the mouse orthologue of human TReP-132, indicated that expression of the gene was highest in thymus, testis and brain structures such as the hypothalamus, basal ganglia, hippocampus, and piriform and cerebral cortex. "
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