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¹ Departments of Biochemistry and
² Obstetrics and Gynaecology, Hokkaido University Graduate School of Medicine, Kita-ku, Kita 15, Nishi 7, Hokkaido, Sapporo 060-8638, Japan

(Requests for offprints should be addressed to T Sugawara; Email: terusuga{at}med.hokudai.ac.jp)

	Abstract

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Steroidogenic acute regulatory (StAR) protein plays a critical role in steroid hormone synthesis. Tropic hormones induce human StAR gene expression by a cAMP-dependent pathway. Steroidogenic factor-1/adrenal-4-binding protein (SF-1/Ad4BP) plays an important role in the expression of human StAR gene. We investigated the mechanism of cAMP responsiveness in human StAR gene expression in NCI-H295R cells. The StAR promoter activity and protein levels in cells subjected to various treatments were examined. Anti-SF-1/Ad4BP IgG transfection treatment resulted in decreases in the basal StAR promoter activity and StAR protein levels, but did not affect cAMP-stimulated promoter activity and protein levels. The basal and cAMP-stimulated StAR promoter activity levels were reduced in SF-1/Ad4BP mutant (G35E)-transfected cells, but the cAMP induction of StAR promoter activity in response to 1 mM 8-Br-cAMP was not inhibited when G35E SF-1/Ad4BP mutant expression vectors were co-transfected with cAMP-response element-binding (CREB) expression vectors. Although the basal StAR mRNA expression and protein levels were decreased by SF-1/Ad4BP-siRNA treatment, the cAMP-stimulated StAR mRNA expression and protein levels did not change. The basal StAR promoter activity level was not decreased by cAMP-response element modulator (CREM)-siRNA treatment, but the cAMP-stimulated StAR promoter activity level, the magnitude of cAMP induction of StAR promoter, and the cAMP-stimulated StAR protein level were decreased. The cAMP induction of StAR promoter activity in cells was inhibited when S117ACREM mutant expressionvectors were transfected. We conclude that inhibition of the function of SF-1/Ad4BP does not reduce the cAMP induction of StAR promoter activity and protein level. CREM is needed to confer cAMP responsiveness in human StAR protein expression.

	Introduction

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The first step in the biosynthesis of steroid hormones is the conversion of cholesterol into pregnenolone. Mitochondria are membrane-enclosed organelles in a cell, separated from the outside by lipid double lumens. The membranes contain phospholipids, and the inter-membrane space is an aqueous space. Cholesterol is hydrophobic and requires some factors to pass through the aqueous inter-membrane space. Steroidogenic acute regulatory (StAR) protein is a 30 kDa phosphorylated protein that binds to cholesterol and plays a key role in the movement of cholesterol from the outer mitochondrial membrane to the inner membrane, where P450scc resides (Stocco & Clark 1996, Strauss et al. 1999, Christenson et al. 2000). StAR mutations cause congenital lipoid adrenal hyperplasia, which impairs production of all steroid hormones (Lin et al. 1995, Bose et al. 1996). StAR-knockout mice have the same phenotype as humans with congenital lipoid adrenal hyperplasia, and StAR protein has been shown to mediate the true rate-limiting step in the synthesis of steroid hormones (Caron et al. 1997b).

StAR gene expression is present mainly in steroidogenic organs, such as adrenal gland, ovary, and testis (Clark et al. 1994, Sugawara et al. 1995). Production of steroid hormones in steroidogenic organs is rapidly increased by the stimulation of tropic hormones (adrenocorticotropic hormone (ACTH), luteinizing hormone (LH), and follicle-stimulating hormone (FSH). When the tropic hormones bind to their cognate receptors, intra-cellular cAMP levels increase via a G-protein-coupled mechanism and protein kinase A (PKA) is activated. The promoter activity of human StAR gene is increased by cAMP stimulation (Caron et al. 1997a, Sugawara et al. 1997b, Sandhoff et al. 1998, Clark & Combs 1999). The phosphorylation of StAR protein is also associated with PKA and increases steroid hormone production (Arakane et al. 1997). The human StAR promoter contains several response elements of transcription factors: steroidogenic factor-1/adrenal-4-binding protein (SF-1/Ad4BP), Sp1, CAAT/enhancer-binding protein ß (C/EBPß ), sterol regulatory element-binding protein-1a, and GATA-4 factors (Christenson et al. 1999, Sugawara et al. 2000, Tremblay et al. 2002). The transcription factors bind to the response elements of human StAR promoter and recruit co-activators or co-repressors and cooperate with other factors to control the human StAR promoter activity (Christenson et al. 1999, Sugawara et al. 2000, 2001). Although there is species difference in cAMP responsiveness, SF-1/Ad4BP is associated with basal and cAMP-stimulated human StAR promoter activities (Kiriakidou et al. 1996, Sugawara et al. 1996, LaVoie et al. 1999).

SF-1/Ad4BP is a nuclear receptor and combines with a specific DNA consensus site (Parker & Schimmer 1997). SF-1/Ad4BP expression is present in steroidogenic tissues and is essential for the development of steroidogenic tissues, where it plays a major role in the regulation of the expression of steroidogenic P450 enzymes, Müllerian-inhibiting substance and DAX-1 (Morohashi et al. 1993, Shen et al. 1994, Michael et al. 1995, Yu et al. 1998). Although the StAR gene expression level is increased by cAMP stimulation, SF-1/Ad4BP expression and protein levels are not increased by PKA induction (Nomura et al. 1998). SF-1/Ad4BP is phosphorylated by a PKA or mitogen-activated protein kinase (MAPK) and recruits nuclear receptor cofactor (Zhang & Mellon 1996, Hammer et al. 1999), but mutations of PKA and MAPK phosphorylation sites did not affect transactivity (Aesoy et al. 2002, Zheng & Jefcoate 2005). In patients, several mutations in SF-1/Ad4BP have been reported (Achermann et al. 1999, 2002, Biason-Lauber & Schoenle 2000, Correa et al. 2004, Mallet et al. 2004). SF-1/Ad4BP mutants of DNA binding are located in the P-box G35E and A-box R92Q. Patients had an XY sex-reversed female and adrenal insufficiency owing to a heterozygous mutation (G35E) and to a homozygous mutation (R92Q) in loss of function to SF-1/Ad4BP-binding sites.

The 1.3 kb human StAR promoter has three SF-1/Ad4BP-binding sites: a distal (– 926 to– 918), a middle (– 105 to – 96), and a proximal site (– 43 to – 36) (Sugawara et al. 1996, 1997a). The middle SF-1/Ad4BP-binding site contains a 5'-TGAC-3' sequence. Although this sequence is not a canonical cAMP-response element (CRE; 5'-TGACGTCA-3' ), it has recently been reported to be a CRE half-site and to have responsiveness to cAMP in the mouse StAR promoter (Manna et al. 2002, 2004). Although SF-1/Ad4BP is important in human StAR promoter activity, the mechanism of the cAMP response of SF-1/Ad4BP is not clear. In this study, we examined whether SF-1/Ad4BP and cAMP response element modulator (CREM) confer cAMP responsiveness in human StAR gene expression in NCI-H295R cells.

	Materials and Methods

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Plasmid constructs

A luciferase reporter gene for 1.3 kb human StAR promoter is the pGL₂-1.3 kb StAR vector as previously described (Sugawara et al. 1996). A 150 bp StAR promoter (pGL₂-150StAR) was constructed by inserting a HindIII fragment prepared by PCR from the human 1.3 kb StAR promoter as a template into the pGL₂ vector (Promega Corp., Madison, WI, USA). Mutations were produced using a Transformer Site-Directed Mutagenesis Kit (Clontech Laboratories, Inc., Palo Alto, CA, USA). pGL₂-150StAR/Mut1 has a mutation (5'-TATCCTTGAC-3' to 5'-TATCCTCGAC-3' ) in the sequence that encodes the SF-1/Ad4BP-binding site containing a 5'-TGAC-3' sequence, which is a CRE-binding half-site. pGL₂-150StAR/Mut2 has a mutation (5'-CAG- CCTTC-3' to 5'-CAGAATTC-3' ) in the sequence of the SF-1/Ad4BP-binding site. pGL₂-150StAR/Mut1/Mut2 has both the mutations in the sequence. Mutant StAR reporter constructs are depicted in Fig. 1A. Human SF-1/Ad4BP cDNA (pcDNAd4BP/SF-1) was kindly provided by Dr Yanase, Graduate School of Medical Sciences, Kyushu University. SF-1/Ad4BP mutants (G35E in the P-box and R92Q in the A-box) were produced using a Transformer Site-Directed Mutagenesis Kit (Clontech). The ß-galactosidase expression vector pCH110 was used for normalization of luciferase data, as previously described (Sugawara et al. 1996). RNA was isolated from human granulosa-like tumor KGN cells (Nishi et al. 2001). cDNA synthesis was carried out using 1 µg total RNA and 200 units SuperScript II reverse transcriptase (Life Technologies, Inc./BRL, Washington, DC, USA). Human cAMP-response element binding (CREB) cDNA was generated by PCR reactions carried out with 1 µl of reverse transcription reaction product using the following primers (sense, 5'-GCAGT-GACGGAGGAGCTTGTAC-3' ; antisense, 5'-TCTGAT-TTGTGGCAGTAAAG-3' ). The PCR reaction was subjected to 30 cycles of denaturing at 94 ° C for 45 s, annealing at 65 ° C for 45 s, and extension at 72 ° C for 1 min. Human CREM cDNA was prepared by PCRs with Marathon-Ready cDNA, 5'-stretched human testis cDNA (Clontech), using the following primers (sense, 5'-AC-TGGGCAAATTTCAATCCCTGC-3' ; antisense, 5'-CAA-ACTTCCGGGCGATGCAGCCATC-3' ). PCR was performed according to the supplier’s protocol. The PCR was subjected to 30 cycles of denaturation at 94 ° C for 30 s and extension at 68 ° C for 4 min. The PCR products were electrophoresed, and DNA fragments were cut from the gels. The fragments were ligated to PCR 2.1 vectors using a manual protocol (Invitrogen, Carlsbad, CA, USA). The PCR products were sequenced, and the sequences were compared with the previously published findings (Yoshimura et al. 1990, Ruppert et al. 1992, Masquilier et al. 1993, Gellersen et al. 1997). Human CREB expression vector (pCREB) was prepared by inserting an EcoRI fragment from human CREB cDNA into pSV.SPORT 1. Human CREM expression vector (pCREB) was prepared by inserting an EcoRI fragment from human CREM cDNA into pCMV5. Human CREM mutants (S117A in the P-box) were produced using a Transformer Site-Directed Mutagenesis Kit (Clontech). The plasmids were prepared for transfection studies using a Qiagen Maxiprep system (QIAGEN, Hilden, Germany).

View larger version (13K): [in this window] [in a new window]   Figure 1 cAMP response of the middle SF-1/Ad4BP site. (A) Schema of the human 150 bp StAR gene promoter. The human 150 bp StAR promoter has two SF-1/Ad4BP-binding sites (– 105 to – 96 and – 43 to – 36). One of the SF-1/Ad4BP-binding sites (– 105 to – 96) contain 5'-TGAC-3' sequences. (B) The indicated plasmids and pCH110 were transfected into human adrenocortical carcinoma NCI-H295R cells. Cells were harvested after a 48-h culture period, and the cell lysate was subjected to a luciferase assay. Cells were treated with 8-Br-cAMP (1 mM) during the final 24-h culture (black bars) or not treated with 8-Br-cAMP (open bars). Promoter activity is expressed as a percentage of that of the 1.3 kb human StAR promoter. The results are presented as mean ± S.E.M. from three separate experiments with each treatment group consisting of three replicate cultures.

Human adrenocortical carcinoma NCI-H295R cells were a gift from Dr Mitsuhiro Okamoto, Osaka University Medical School, Osaka, Japan. NCI-H295R cells were grown in DMEM/F12 containing 2% ULTROSER G (BioSepra, Cergy-Pontoise, France) and 1% ITS Premix (Becton Dickinson and Co., Franklin Lakes, NJ, USA). Cultures of sub-confluent NCI-H295R cells were plated so that 35 mm tissue culture dishes received equal numbers of cells.

Transfection and luciferase assays

NCI-H295R cells were transfected with pGL₂ plasmids and pCH110 using 3 µl FuGENE 6 (Roche Molecular Biochemicals, Mannheim, Germany)/1 µg DNA. Cultured cells at 40–60% confluence were transfected with 0.5 µg pGL₂ plasmid and 0.1 µg PCH110. The cells were cultured for 48 h after transfection and then harvested. Some cells were co-transfected with a pGL₂ plasmid, pCH110, and various kinds of plasmids. Some dishes were treated with 8-Br-cAMP (1 mM) during the final 24-h culture. Luciferase assays were performed using a Luciferase Assay System (Promega). The assay results were normalized to ß-galactosidase activity to compensate for variation in transfection efficiency as described previously (Sugawara et al. 2000). Each treatment group contained triplicate cultures, and each experiment was repeated three or four times.

Transfection of SF-1/Ad4BP antibody

NCI-H295R cells were plated in 35 mm tissue culture dishes and grown to 40–60% confluence. Cultured cells at 40–60% confluence were transfected with 0.5 µg pGL₂ plasmid and 0.1 µg pCH110. The cells were transfected on the next day with anti-mouse SF-1/Ad4BP rabbit IgG (Upstate Bio-technology, Lake Placid, NY, USA) using Chariot reagent (Active Motif, Carlsbad, CA, USA). Anti-SF-1/Ad4BP, 1 µl was diluted with 100 µl PBS, and 6 µl Chariot reagent was diluted with water (1:1000). The diluted IgG and the diluted reagent were mixed and incubated at room temperature for 30 min. Growth medium was aspirated from the cells, and cells were washed with PBS. Transfection complex was added to the cells. The volume was adjusted to 600 µl with serum-free medium, and the cells were incubated for 1 h. Cells were supplemented with 1 ml culture medium, incubated for 4 h, and then harvested. A luciferase assay and western blotting were performed using cell extracts.

Western blot analysis

NCI-H295R cell extracts were harvested with RIPA buffer (50 mM Tris–HCl, 1% Nonidet P-40, 0.1% deoxycholate, 0.1% SDS, 150 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 0.1 mM PMSF, and 1 x proteinase inhibitor) after transfection. Western blot analysis was performed as previously described (Sugawara et al. 2004). Ten micrograms of cell extract were subjected to 12% SDS-PAGE. After electrophoresis, the gels were transferred to nitrocellulose membranes for immunodetection with anti-StAR rabbit serum, a rabbit polyclonal anti-CREM IgG (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and a mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (American Research Products, Inc., Belmont, MA, USA). Anti-human StAR protein serum was generously provided by Dr Jerome F Strauss III of the University of Pennsylvania, Philadelphia, PA, USA.

Small interfering RNA (siRNA)

Endogenous SF-1/Ad4BP mRNA was targeted in cells with transfection by the addition of a 23-nucleotide duplex (siRNA-SF-1/Ad4BP) (Dharmacon, Inc., Lafayette, CO, USA). These duplex RNAs target 71 nucleotides downstream of the start codon of SF-1/Ad4BP. SiRNAs were constructed using the ribooligonucleotide pairs SF-1/AD4BP with the following sequences: 5'-CUACGGA-CUGCUCACGUGUGAdTdT-3' and 5'-UCACACGUG-AGCAGUCCGUAGdTdT-3' . As a control for the specificity of these duplexes, we used a scrambled ribooligonucleotide pair (siRNA-Scramble) with the following sequences: 5'-GCGCGCUUUGUAGGAUUCGdTdT-3' and 5'-CGAA-TCCTACAAAGCGCGCdTdT-3' . CREB mRNA was targeted in cells with transfection by the addition of a 23-nucleotide duplex. These duplex RNAs target 66 nucleotides downstream of the start codon of CREB. SiRNAs were constructed using the ribooligonucleotide pairs CREB with the following sequences: 5'-CAAAUGACAGUU-CAAGCCCAGdTdT-3' and 5'-CUGGGCUUGAACU-GUCAUUUGdTdT-3' . CREM mRNA was targeted in cells with transfection by the addition of a 21-nucleotide duplex. These duplex RNAs target 42 nucleotides downstream of the start codon of CREM. SiRNAs were constructed using the ribooligonucleotide pairs CREM with the following sequences: 5'-CAGCUUCUUUGACAGAGAGCAdTdT- 3 ' and 5'-UGCUCUCUGUCAAAGAAGCUGdTdT-3' . The oligonucleotides were annealed according to the Dharmacon protocol. Three hundred picomoles of each duplex was introduced into cells using 15 µl metafectene (Biontex Laboratories GmbH, Munich, Germany) as recommended by the manufacturer. At 48 h after transfection, total RNA was extracted and reverse transcriptase (RT)-PCR was performed, as previously described (Sugawara et al. 2004), for SF-1/AD4BP (27 cycles) and for human StAR (27 cycles) with GAPDH (24 cycles) as a control using the primers 5'-GCGGACGCGGCGGGCATGGACTATT-3' (sense) and 5'-AACAGAGGCTCTCCCTCCTCCTGGTCTC-3' (antisense) for SF-1/Ad4BP and the primers 5'-GCAGC-AGCAGCGGCGGCAGCAG-3' (sense) and 5'-ATGAG-CGTGTGTACCAGTG-3' (antisense) for StAR. RT-PCR for GAPDH as a control was performed using the primers 5'-TGCCGTCTAGAAAAACCTGC-3' (sense) and 5'-ACCCTGTTGCTGTAGCCAAA-3' (antisense). RT-PCR was also performed for human CREM (25 cycles) with GAPDH (25 cycles) as a control using the primers 5 '-GACCATGGAAACAGTTGAATCCCA-3' (sense) and 5'-CGTCGACATTCTTTGGCAGC-3' (antisense) for human CREM (Groussin et al. 2000). In some experiments, cell extracts were harvested after transfection and then western blot analysis or luciferase assay was performed.

Data analysis

Values are presented as mean ± S.E.M. Significance between experimental values was determined by Student’s unpaired t-test, and one-way ANOVA was used to test differences in repeated measures across experiments. Significant results from ANOVA were further analyzed by Tukey’s post hoc test. P < 0.05 was considered significant.

	Results

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The human StAR promoter has three SF-1/Ad4BP-binding sites. Mutations were introduced into SF-1/Ad4BP-binding sites, two of the mutations being within 150 bp upstream of the transcriptions start site (– 105 to – 96 and – 43 to – 36). The indicated plasmids were transfected into human adrenocortical carcinoma NCI-H295R cells, and luciferase assays were performed. The basal and cAMP-stimulated promoter activity levels of pGL₂-150StAR were increased compared with the pGL₂-1.3 kb StAR promoter activity level. The mutated human StAR promoter activity level was decreased compared with that of pGL₂-150StAR. Basal and cAMP-stimulated promoter activity levels in cells transfected with pGL₂-150StAR/Mut1 were reduced to 8.6 and 4.2% respectively. Basal and cAMP-stimulated promoter activity levels in cells transfected with pGL₂-150StAR/Mut2 were reduced to 30 and 21.3% respectively. Mutations in both SF- 1/Ad4BP-binding sites (pGL₂-150StAR/Mut1/Mut2) reduced promoter activity to nearly background levels (Fig. 1B). The cAMP induction with pGL₂-150StAR did not significantly differ from that of pGL₂-1.3 kb StAR. Although the cAMP induction with pGL₂-150StAR/Mut2 did not significantly differ from that with pGL₂-150StAR, the cAMP induction with pGL₂-150StAR/Mut1 and pGL₂-150/StARMut1/Mut2 were significantly (P < 0.05) different from that of pGL₂-150StAR (Table 1).

View larger version (20K): [in this window] [in a new window]   Figure 2 Transfection of an SF-1/Ad4BP antibody into NCI-H295R cells. (A) The indicated plasmids were transfected into NCI-H295R cells. The next day, cells were transfected with an SF-1/Ad4BP antibody using a reagent. Cells were treated with 8-Br-cAMP during the final 24-h culture or not treated with 8-Br-cAMP. Promoter activity is expressed as RLU (luciferase/ß-galactosidase). *Significant difference for basal promoter activity compared with that of control; *P < 0.05. (B) The cells were collected from each dish by scraping. The cell extracts were subjected to 12% SDS-PAGE, and then western blot analysis was performed using anti-StAR.

View larger version (13K): [in this window] [in a new window]   Figure 3 Effect of SF-1/Ad4BP mutant on human StAR promoter. The pGL2-1.3 kb StAR vector (0.5 µg) and SF-1/Ad4BP (0.2 µg) or an SF-1/Ad4BP mutant (R92Q or G35E) expression vector (0.2 µg) were transfected into NCI-H295R cells. Cells were treated with 8-Br-cAMP during the final 24-h culture or not treated with 8-Br-cAMP. Values presented are the mean ± S.E.M. of the promoter activities, expressed as a percentage of that of the 1.3 kb human StAR promoter with an empty vector. *Significant difference for basal promoter activity compared with that of the empty vector. Significant difference for cAMP-stimulated promoter activity compared with that of the empty vector. *,P < 0.05.

View larger version (50K): [in this window] [in a new window]   Figure 4 Reduction in basal level of StAR protein by siRNA-SF-1/Ad4BP treatment. Endogenous SF-1/Ad4BP gene expression in NCI-H295R cells was eliminated by transfection of a 23-nucleotide RNA duplex (siRNA-SF-1/Ad4BP). As a control, scrambled sequences were introduced into the cells. The next day, 8-Br-cAMP (1 mM) was added to some of the culture media. After 48-h culture, RNA was extracted from the cells. (A) The effects of siRNA treatment were assayed by RT-PCR for SF-1/Ad4BP and GAPDH gene. (B) Some cells were collected from each dish by scraping. The cell extracts were subjected to 12% SDS-PAGE, and then western blot analysis was performed using anti-StAR.

View larger version (13K): [in this window] [in a new window]   Figure 5 Effect of CREB protein on StAR promoter. (A) StAR promoter plasmids (0.5 µg) and CREB expression vectors (0.5 µg) were over-expressed in NCI-H295R cells. The cells were treated with 8-Br-cAMP (1 mM) during the final 24-h culture or not treated with 8-Br-cAMP. Promoter activity is expressed as RLU (luciferase/ß-galactosidase). *Significant difference for basal promoter activity compared with that of the empty vector. Significant difference for cAMP-stimulated promoter activity compared with that of the empty vector. *,P < 0.05. (B) The pGL2-1.3 kb StAR vector (0.5 µg), SF-1/Ad4BP (0.2 µg) or an SF-1/Ad4BP mutant (R92Q or G35E) expression vector (0.2 µg) and CREB expression vectors (0.5 µg) were transfected into NCI-H295R cells. Cells were treated with 8-Br-cAMP (1 mM) during the final 24-h culture or not treated with 8-Br-cAMP. Values presented are the mean ± S.E.M. of the promoter activities, expressed as a percentage of that of pGL2- 1.3 kb StAR with an empty vector. *Significant difference for basal promoter activity compared with that of the empty vector. Significant difference for cAMP-stimulated promoter activity compared with that of the empty vector. *,P < 0.05.

View larger version (25K): [in this window] [in a new window]   Figure 6 Effects of siRNA–CREM treatment on StAR promoter and protein levels. Endogenous CREB gene expression in NCI-H295R cells was eliminated by treatment with siRNA. As a control, scrambled sequences were introduced into the cells. The next day, 8-Br-cAMP (1 mM) was added to some of the culture media. (A) The effects of siRNAs were assayed by promoter activity. Promoter activity is expressed as RLU (luciferase/ß-galactosidase). *Significant difference for cAMP-stimulated promoter activity compared with that of siRNA scramble. *P < 0.05. (B) The effects of siRNAs were assayed by RT-PCR for CREM and GAPDH gene. Direct sequences of PCR products amplified with CREM primers correspond to CREM 2 (1131 bp) and CREM (942 bp) (Groussin et al. 2000). (C) Some cells were collected from each dish by scraping. The cell extracts were subjected to 12% SDS-PAGE, and then western blot analysis was performed using anti-StAR, anti-CREM, and anti-GAPDH.

View larger version (12K): [in this window] [in a new window]   Figure 7 Effects of CREM on cAMP responsive StAR transcription. pGL2-1.3 kb StAR promoter plasmids (1 µg), pCH110 (0.5 µg) and CREM expression vectors (0.5 µg), or S117A CREM mutant expression vector (0.5 µg) were transfected into NCI-H295R cells. The cells were treated with 8-Br-cAMP (1 mM) during the final 24-h culture or not treated with 8-Br-cAMP. Values presented are the mean ± S.E.M. of the promoter activities, expressed as a percentage of that of pGL2-1.3 kb StAR with an empty vector. *, Significant difference from each other at P < 0.05.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Analysis of mutations of the SF-1/Ad4BP sites showed reductions of basal and cAMP-stimulated promoter activity levels. Of all the SF-1/Ad4BP-binding sites, the most important was the – 105 to – 95 site. The results obtained for NCI-H295R are consistent with those of human granulosa–lutein cells, which undergo terminal differentiation (luteinization) in response to human chorionic gonadotropin (Kiriakidou et al. 1996, Sugawara et al. 1997a). It has been reported that forskolin, which stimulates adenyl cyclase, could reorganize SF-1/Ad4BP from a diffuse distribution pattern to formation in the nucleus of granulosa tumor KGN cells (Fan et al. 2004). PKA stimulation modifies the interaction of SF-1/Ad4BP with its activator, transformation/transcription domain-associated protein (TRRAP), and suppressor, dosage-sensitive sex reversal (DAX-1) protein (Fan et al. 2004).

SF-1/Ad4BP is translated in the cytoplasm and enters the nuclei of cells, where it binds to a DNA consensus sequence. When an antibody is introduced into the cell nucleus by a protein transfection reagent, transcription activation is reduced (Cheriyath et al. 2002). Although the transfection reagent decreased the basal promoter activity and protein levels, the cAMP-stimulated promoter activity and protein levels did not change. The affinity of the proximal SF-1/Ad4BP-binding sites (– 105 to – 95 and – 42 to – 35) is lower than that of the distal SF-1/Ad4BP-binding site (– 926 to – 918) (Sugawara et al. 1997a). One possibility is that IgG is not sufficient for formation of a complex with SF-1/Ad4BP, and a small amount of free SF-1/Ad4BP can respond to cAMP stimulation. Alternatively, SF-1/Ad4BP antibody might not completely obstruct the interaction between other transcription factors, co-activators and co-repressors, or other factors might compensate the dysfunction of SF-1/Ad4BP. Other factors, GATA-4, C/EBPß , or CREB, may compensate the loss of the effect of SF-1/Ad4BP with IgG transfection on cAMP-stimulated StAR promoter activity or LRH-1/NR5A2 transcription factor may induce cAMP-stimulated promoter activity. It has been reported that LRH-1 also binds SF-1/Ad4BP-response elements and controls promoter activity in steroidogenic cells, including NCI-H295R cells and granulosa cells (Wang et al. 2001, Kim et al. 2004).

Several mutations of SF-1/Ad4BP in patients, who have adrenal insufficiency and sex-reversal with retained Müllerian structures have been reported (Achermann et al. 1999, 2002, Biason-Lauber & Schoenle 2000). The R92Q A-box mutation is a homozygous mutation with an autosomal recessive mode of inheritance. The G35E P-box mutation is a heterozygous mutation. The P-box of SF-1/Ad4BP interacts directly with the half-site sequence of SF-1/Ad4BP-binding (PyCA AGGTCA) motif, which is located in the major groove of the DNA helix. On the other side, the A-box region interacts with the minor groove of the DNA helix (PyCA AGGTCA) and stabilizes the SF-1-DNA interaction. Mutation in the A-box region (R92Q) has little effect on DNA binding (Ito et al. 2000). Although P-box mutation (G35E) results in complete loss of SF-1/Ad4BP-binding activity, A-box mutation (R92Q) results only in partial impairment of binding (Achermann et al. 2002). P-box mutation resulted in reduction of cAMP-stimulated promoter activity and impairment of cAMP induction. The SF-1/AD4BP-binding motif in the major groove of the DNA helix (P-box) seems to be associated with the induction of cAMP simulation. Although a P-box mutant cannot bind DNA, the protein interaction domains, which bind other transcription factors, are intact. Other transcription factors may not compensate the promoter activity because the transcription factors may form a complex with mutant SF-1/Ad4BP and because there is supposed to be a shortage of co-activators.

Transfection with siRNA is a widely used method for reducing endogenous gene expression (Elbashir et al. 2001). When endogenous SF-1/Ad4BP expression level was decreased, basal StAR protein level was reduced but cAMP-stimulated StAR protein level did not change. This is because siRNA treatment cannot completely inhibit the transcription of SF-1/Ad4BPand a small amount of SF-1/Ad4BP protein might be sufficient to increase cAMP-stimulated StAR protein level. This result is consistent with the results for the R92Q A-box mutant. The mRNA and protein levels of SF-1/Ad4BP remain constant after an increase or decrease in the cAMP level (Nomura et al. 1998). Since an association of SF-1/Ad4BP with the StAR promoter has been reported without an increase in SF-1/Ad4BP within 15 min after cAMP stimulation (Hiroi et al. 2004), cAMP-stimulated promoter activity is independent of SF-1/Ad4BP protein level. Other factors, such as GATA-4, have been reported to be phosphorylated in response to cAMP stimulation and cooperate to increase cAMP-stimulated StAR promoter activity level (Tremblay et al. 2002). Phosphorylated transcription factors might cooperate with SF-1/Ad4BP and recruit co-activators.

Transcriptional activation by cAMP is mediated through the interaction of the CREB protein in many target genes. Although the human StAR gene promoter regions lack the consensus CRE, CRE half-sites are found in SF-1/Ad4BP-binding sites. CREB over-expression affects the basal and cAMP-stimulated StAR promoter activity levels in NCI-H295R cells, which lack endogenous CREB expression. Mutant G35E with CREB over-expression had no transcription activity of human StAR. The CBP acts as a bridging protein with other transcription factors, including CREB, CREM, SF-1/Ad4BP, c-Fos, and c-Jun (Bannister & Kouzarides 1995, Bannister et al. 1995). In the mouse StAR promoter, CREM was found by EMSA using nuclear extracts from MA-10 mouse Leydig tumor cells to be the predominant protein binding to the cAMP responsive region (Manna et al. 2002). Using DNA-affinity chromatography, CREB has been found to interact with the mouse StAR promoter with dependence on AP-1-binding sites, which are present near SF-1/Ad4BP sites (Clem et al. 2005). Although the antibodies used in these two studies are different, the results show that both CREB and CREM bind to the mouse StAR promoter.

Phosphorylation of CREB by PKA permits binding of CBP, and then the CREB/CBP complex enhances DNA accessibility by HAT activity and binds to the CRE and the TATA box regions of the promoter (Parker et al. 1996, Wolfl et al. 1999). (Bu)₂cAMP treatment has been reported to increase phosphorylated CREM and CBP interaction with the mouse StAR promoter (Clem et al. 2005). SiRNA-CREM treatment reduced the cAMP-stimulated promoter activity levels of human StAR. The human cAMP-stimulated promoter activity decreased with S117A CREM mutant transfection. In the human StAR promoter, as in the mouse StAR promoter, PKA may phosphorylate CREB/CREM and bind CBP to control transcription activity. Although CREB is not expressed in NCI-H295R cells, CREM, which is a CREB family member, is needed to confer cAMP responsiveness in human StAR protein expression.

Recently, phospholipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, and phosphatidyl-inositol-phosphates) have been reported to be ligands of SF-1/Ad4BP and lipid binding has been shown to be required for maximum activity (Krylova et al. 2005, Li et al. 2005, Ortlund et al. 2005, Wang et al. 2005). To clarify the species difference in cAMP response to the StAR promoter, detailed analysis of the human StAR promoter taking into account the presence of SF-1/Ad4BP ligands is needed.

	Acknowledgements

 
The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

	References

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Received 26 October 2005
Received in final form 12 July 2006
Accepted 19 July 2006

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