The transcription factor SF-1 (steroidogenic factor-1) is a master regulator of steroidogenesis. Previously, we have found that SF-1 induces the differentiation of mesenchymal stem cells into steroidogenic cells. To elucidate the molecular mechanisms of SF-1-mediated functions, we attempted to identify protein components of the SF-1 nuclear protein complex in differentiated cells. SF-1 immunoaffinity chromatography followed by MS/MS analysis was performed, and 24 proteins were identified. Among these proteins, we focused on C/EBPβ (CCAAT/enhancer-binding protein β), which is an essential transcription factor for ovulation and luteinization, as the transcriptional mechanisms of C/EBPβ working together with SF-1 are poorly understood. C/EBPβ knockdown attenuated cAMP-induced progesterone production in granulosa tumour-derived KGN cells by altering STAR (steroidogenic acute regulatory protein), CYP11A1 (cytochrome P450, family 11, subfamily A, polypeptide 1) and HSD3B2 (hydroxy-δ-5-steroid dehydrogenase, 3β- and steroid δ-isomerase 2) expression. EMSA and ChIP assays revealed novel C/EBPβ-binding sites in the upstream regions of the HSD3B2 and CYP11A1 genes. These interactions were enhanced by cAMP stimulation. Luciferase assays showed that C/EBPβ-responsive regions were found in each promoter and C/EBPβ is involved in the cAMP-induced transcriptional activity of these genes together with SF-1. These results indicate that C/EBPβ is an important mediator of progesterone production by working together with SF-1, especially under tropic hormone-stimulated conditions.

INTRODUCTION

SF-1 [steroidogenic factor-1; also known as Ad4BP (adrenal 4-binding protein); encoded by NR5A1 (nuclear receptor subfamily 5, group A, member 1)] is one of the essential transcription factors for gonadal and adrenal development and differentiation [1,2]. SF-1 regulates the expression of steroidogenesis-related genes, hence it is considered a master regulator of steroid hormone production. Previously, we have reported that introduction of SF-1 into MSCs (mesenchymal stem cells) resulted in the differentiation of the cells to the steroidogenic lineage [3]. In these cells, SF-1 must form a protein complex to facilitate recruitment of RNA polymerase II at the promoter sites of its target genes, which, in turn, induces expression of the steroidogenesis-related genes.

Transcription factors recognize and bind to specific DNA sequences and recruit transcriptional machinery to regulate gene transcription. This machinery forms a large protein complex including histone modifiers, chromatin remodellers and general transcription factors. In the present study, we identified components of the SF-1 nuclear protein complex using SF-1 immunoaffinity chromatography followed by MALDI–TOF-MS/MS analysis. Among the nuclear protein components identified, we focused on C/EBPβ (CCAAT/enhancer-binding protein β), a basic leucine zipper family transcription factor.

C/EBPβ is one of the six C/EBP family members that play pivotal roles in various cellular processes, such as adipocyte differentiation, liver gluconeogenesis and lipolysis, mammary gland development, and inflammation [48]. In the ovary, C/EBPβ is known as a critical factor for ovulation and luteinization. Targeted disruption of the Cebpb gene caused infertility in female mice, because of failure of ovulation and luteinization [9]. GC (granulosa cell)-specific conditional knockout studies revealed that expression of C/EBPβ is induced by LH (luteinizing hormone) and activated in an ERK1/2 (extracellular-signal-regulated kinase 1/2)-dependent manner in preovulatory follicles [10]. Moreover, analysis of Cebpa/bgc−/− double-mutant mice revealed that C/EBPα is also involved in this process [11]. However, little is known about the C/EBP-dependent mechanisms of target gene transcription in this process.

Progesterone production is one of the most important functions of luteal cells. Cholesterol is transported into mitochondria by StAR (steroidogenic acute regulatory protein), and subsequently converted into progesterone by the cytochrome P450 side-chain cleavage enzyme [encoded by CYP11A1 (cytochrome P450, family 11, subfamily A, polypeptide 1)] and 3β-hydroxysteroid dehydrogenase/isomerase [encoded by HSD3B2 (hydroxy-δ-5-steroid dehydrogenase, 3β- and steroid δ-isomerase 2) in the human gonads and adrenal glands]. These genes are mainly regulated by pituitary trophic hormones [12] and nuclear receptor 5A family, SF-1 and LRH-1 (liver receptor homologue-1) [13]. In the present study, we showed that C/EBPβ is also involved in the regulation of these genes. C/EBPβ knockdown attenuated cAMP-induced progesterone production and the expression of genes related to progesterone production. EMSA and ChIP assays revealed novel C/EBPβ-binding sites upstream of the TSS (transcription start site) of CYP11A1 and HSD3B2. Interestingly, each C/EBPβ-binding site is close to the SF-1-binding sites of these genes. Luciferase assays showed that C/EBPβ, together with SF-1, is involved in the cAMP-induced gene expression of STAR, CYP11A1 and HSD3B2. We showed that C/EBPβ is one of the factors in the cAMP-mediated progesterone production.

MATERIAL AND METHODS

Antibodies

Dynabeads coated with Protein G or M-280 sheep anti-(mouse IgG) were purchased from Life Technologies. An anti-FLAG M2 (F1804) antibody was purchased from Sigma–Aldrich. Anti-C/EBPβ (sc-150) and anti-c-Jun (sc-44) antibodies were purchased from Santa Cruz Biotechnology. Anti-SF-1 antibody (07-618) was purchased from Millipore. Anti-Ku (p70) (MS-329-P1) and anti-DNA-PKcs (protein kinase catalytic subunits) (MS-423-P1) antibodies were purchased from Thermo Scientific. Anti-HSP70 (heat-shock protein 70; SPA-812) and anti-HSC70 (heat-shock cognate protein 70) (SPA-816) antibodies were purchased from Enzo Life Sciences. Horseradish peroxidase-conjugated anti-[rabbit IgG, goat, F(ab′)2] (013-17941) and anti-[mouse IgG, goat, F(ab′)2] antibodies (019-17921) were purchased from Wako Pure Chemical.

Preparation and infection of retroviruses

Retrovirus preparation and infection was performed as described previously [14]. The human MSC-hTERT (human telomerase reverse transcriptase)-E6/E7 line was infected with retroviruses expressing FLAG-tagged SF-1 (FLAG–SF-1 hMSCs) for 48 h, followed by selection with puromycin to generate stable cell lines. The cell line expressing FLAG–SF-1 was finally chosen from approximately 20 cell lines for further experiments.

Cell culture

FLAG–SF-1 hMSCs were maintained in DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% FBS, gentamycin and puromycin [14]. KGN cells [15], a human granulosa tumour cell line, and Y1 cells, a mouse adrenocortical tumour cell line, were maintained in DMEM/Ham's F-12 medium supplemented with 10% FBS and gentamycin.

Immunoaffinity chromatography

The soluble chromatin fraction from the FLAG–SF-1 hMSCs was isolated as follows. All operations were carried out at 4 °C. Approximately 109 FLAG–SF-1 hMSCs were homogenized in a glass Dounce homogenizer in five volumes of buffer A [15 mM Hepes/KOH (pH 7.8), 60 mM KCl, 15 mM NaCl, 14 mM 2-mercaptoethanol, 0.15 mM spermine, 0.5 mM spermidine, 1 mM PMSF and 0.2% Nonidet P40] containing 0.3 M sucrose. The homogenate was layered on top of a cushion of 0.9 M sucrose in buffer A and centrifuged at 2500 g for 10 min. The precipitated crude nuclei were resuspended in buffer A containing 0.9 M sucrose and re-centrifuged. The resulting pellet was resuspended and digested with 3000 IU of MNase (micrococcal nuclease) in MNase digestion buffer [20 mM Hepes/KOH (pH 7.8), 100 mM NaCl, 1.5 mM MgCl2, 5 mM CaCl2, 0.1 mM EDTA, 1 mM Na3VO4, 0.02% Nonidet P40 and 20% glycerol] containing protease inhibitors for 3 h at 4 °C. The digested nuclei were centrifuged at 27000 g for 5 min, and then the supernatant was collected as the soluble chromatin fraction. An 8 mg aliquot of the soluble chromatin fraction was pre-cleared with Sepharose CL-4B (GE Healthcare) for 1 h at 4°C, and then incubated with anti-FLAG M2 agarose (A2220; Sigma–Aldrich) for 4 h at 4°C with gentle rotation. The resin was washed with 200 column volumes of MNase digestion buffer. Bound proteins were eluted with a buffer containing 0.5 mg/ml 3× FLAG peptide (F4799; Sigma–Aldrich) for 20 min at 4°C. The eluate was divided into two aliquots and each were subjected to SDS/PAGE using 5–12.5% gradient gels (DRC). The gel was silver-stained by SilverQuest™ (Life Technologies). For identification of the protein components, the gel was cut equally lengthwise and divided into 34 gel slices. Each gel slice was placed in an Eppendorf tube for in-gel digestion.

MS and data analyses

Proteins in each gel slice were digested with trypsin (sequencing grade modified; Promega) overnight at 37°C. Tryptic peptides were extracted from each slice by previously described methods [16], and analysed using a 4800 MALDI–TOF/TOF mass spectrometer (AB Sciex). Samples were analysed in the MS positive-ion reflector mode in the mass range 900–4000 Da, and peak lists were generated as described previously [17]. Peak lists were searched against NCBInr Human database using Mascot (version 2.2), with trypsin specification. Carbamidomethylation of cysteine residues was set as a fixed modification. Peptides with a score above the identity threshold (corresponding to an expectation value below 0.05) were considered as identified.

Preparation of nuclear extracts

Nuclear extracts were prepared from Y1 or KGN cells by the method described by Hagenbüchle et al. [18] with minor modifications [19]. Briefly, the harvested cells were washed and homogenized in a glass Dounce homogenizer in five volumes of buffer A. After centrifugation at 2500 g for 5 min, the nuclear pellet was extracted with high-salt buffer [20 mM Hepes/NaOH (pH 7.9), 420 mM NaCl, 1.5 mM MgCl2, 0.1 mM EDTA, 1 mM DTT, 1 mM PMSF and 10% glycerol], and then gently stirred with a magnetic stirrer on ice for 45 min. After centrifugation at 27000 g for 5 min, nuclear extracts were collected. For IP (immunoprecipitation) assays, the nuclear extract was further dialysed twice for 2 h against 50 volumes of buffer C [25 mM Tris/HCl (pH 8.0), 1 mM EDTA, 5 mM MgCl2, 0.1% Nonidet P40, 1 mM DTT and 10% glycerol] containing 0.1 M KCl. The dialysate was centrifuged at 27000 g for 10 min at 4 °C to remove insoluble materials. The protein concentration of the supernatants was determined using a protein assay kit (Bio-Rad Laboratories).

IP assays and Western blotting

IPs with anti-FLAG M2–agarose using the chromatin fractions from FLAG–SF-1 hMSCs with or without 1 mM 8-Br-cAMP treatment were performed as described above. Because the expression level of SF-1 was different between the 8-Br-cAMP-stimulated and unstimulated FLAG–SF-1 hMSCs (Supplementary Figure S1 at http://www.biochemj.org/bj/460/bj4600459add.htm), different amounts of chromatin fractions (40 μg for the 8-Br-cAMP-stimulated cells and 100 μg for the unstimulated cells) were used for IP. IP assays with anti-C/EBPβ antibody using the nuclear extracts or the chromatin fractions from Y1 cells were performed as follows. Protein G-coated Dynabeads were incubated with anti-C/EBPβ antibody in 0.1% BSA/PBS at 4°C with rotation for 6 h, and then washed. The soluble chromatin fraction or nuclear extract (0.75 mg of protein) was then incubated with the antibody-bound Dynabeads at 4°C with rotation overnight. Beads were washed five times with MNase digestion buffer or buffer C. After removing the wash buffer, beads were mixed with sample buffer [62.5 mM Tris/HCl (pH 6.8), 2% SDS, 10% glycerol and 0.01% Bromophenol Blue] and eluted. DTT and 2-mercaptoethanol were added to the eluent and boiled.

Briefly, Western blotting was performed as follows. Aliquots of the nuclear extracts or the chromatin fractions containing the same amount (10 μg) of protein or immunoprecipitant were resolved by SDS/PAGE (7.5, 10 or 12.5% gels) and transferred on to PVDF membranes. Western blot analysis was performed with the antibodies described above. All immune complexes were visualized and quantified using Clarity Western ECL Substrate (Bio-Rad Laboratories) and an LAS4000 mini imager (Fujifilm).

Steroid hormone measurements

Medium progesterone concentrations were measured using an ELISA kit (Oxford Biomedical Research) according to the manufacturer's instructions. C/EBPβ (D-006423-25 and D-006423-27; Thermo Scientific) or non-targeting (D-001210-02) siRNAs were transfected into KGN cells (7.5×104 cells) with Lipofectamine™ RNAiMAX (Life Technologies), and then cells were seeded into a 24-well plate. The final siRNA concentration in the medium was 10 nM. At 48 h after transfection, media, with or without 8-Br-cAMP, were replaced and the cells were further incubated for 24 h. Culture media and cells were collected and used for progesterone and protein concentration measurements.

qPCR (quantitative real-time PCR)

qPCR was performed using StepOnePlus™ Real-Time PCR System (Applied Biosystems) as described previously [20]. C/EBPβ or non-targeting siRNAs were transfected into KGN cells (3×105 cells) with Lipofectamine™ RNAiMAX, and then cells were seeded into a six-well plate. The final siRNA concentration in the medium was 10 nM. At 48 h after transfection, media, with or without 8-Br-cAMP, were replaced and the cells were further incubated for 24 h. Knockdown efficiencies were confirmed by Western blotting as described above. The primers used for qPCR have been described previously [14].

Plasmids

Human HSD3B2 upstream regions consisting of various 5′-ends cloned into the pGL4.11 luciferase basic vector (Promega) were generated as follows. The 5′-flanking region (−4092/+23) plasmid was amplified by PCR using KOD FX (Toyobo), with genomic DNA from KGN cells as the template, and the PCR product was ligated into pTA2 (TaKaRa Bio). The resulting pTA2/hHSD3B2(−4092/+23) plasmid was digested with Acc65I/SpeI and ligated into a Acc65I/NheI-digested pGL4.11 plasmid, which was designated pGL4/hHSD3B2(−4092/+23). pGL4/hHSD3B2(−4092/+23) was digested with Acc65I/SphI, blunt-ended and was self-ligated, and the resultant plasmid was designated pGL4/hHSD3B2(−3733/+23). The BglII fragment from pGL4/hHSD3B2(−4092/+23) was ligated into a BglII-digested pGL4.11 plasmid, which was designated as pGL4/hHSD3B2(−351/+23). Mutations of three C/EBP-binding sites (as shown in Figure 2D) in the HSD3B2 upstream region (−4092/+23) were created using the QuikChange® Site-Directed Mutagenesis Kit (Agilent Technologies), and designated pGL4/hHSD3B2(−4092/+23)mutC/EBP. The region (−4092/−3757), in which Acc65I at the 5′-end was flanked, was amplified by PCR using KOD-Plus-Neo (Toyobo), with pGL4/hHSD3B2(−4092/+23) as the template. The PCR product was digested with Acc65I and then ligated into an Acc65I/EcoRV-digested pGL4.24 plasmid (a reporter vector with minimal promoter activity; Promega), which was designated pGL4.24/ hHSD3B2(−4092/−3,757).

The human CYP11A1 upstream region (−2298/+72) was inserted into pGL3 as described previously [21]. The KpnI/HindIII fragment (−2298/+72) was ligated into a KpnI/HindIII-digested pGL4.11, which was designated pGL4/hCYP11A1(−2298/+72). The SpeI/HindIII fragment from pGL4/hCYP11A1(−2298/+72) was ligated into a NheI/HindIII-digested pGL4.11, which was designated pGL4/hCYP11A1(−1711/+72). Deletion constructs (−2094, −1937, −1657, −1545, −1327, −828, −654, −160 and −73/+72) were generated by PCR using KOD-Plus-Neo, with pGL4/hCYP11A1(−2298/+72) as a template, and then ligated into pGL4.11. Fragments (−2094/−1932 and −1644/−1542) were amplified by PCR using KOD-Plus-Neo with pGL4/hCYP11A1(−2298/+72) as a template and then ligated into pGL4.24. Mutations in AP1 or SF-1 sites in the CYP11A1 upstream region (−1644/−1542) were created using the QuikChange® Site-Directed Mutagenesis Kit. The sequences of these mutations were from Guo et al. [22].

The expression vectors for the C/EBPβ isoforms, LAP (liver-enriched activator protein) and LIP (liver-enriched inhibitory protein), which acts as a dominant-negative C/EBP, were generated as follows. Human LAP and LIP fused to a C-terminal in-frame Myc tag, in which KpnI at the 5′-end and NotI at the 3′-end were flanked, were amplified by PCR using KOD FX, with cDNA from KGN cells as the template, and then subcloned into the KpnI/NotI-digested pShuttle vectors, which were designated pShuttle/LAP and pShuttle/LIP respectively.

Human STAR upstream regions (−3402/+39 and −235/+39) in pGL4.11 and the pCMV-FLAG-SF-1 expression vector were as described previously [20].

Nucleotide numbering is relative to the TSS of each gene. The nucleotide sequences of all PCR products were confirmed by DNA sequencing.

Luciferase assay

Luciferase assays were performed as described previously [23] using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's instructions. Briefly, 1 day before transfection KGN cells were dispensed into 24-well plates at 7×104 cells/well. A 200 ng aliquot of each reporter plasmid and 1 ng of pRL-CMV Renilla luciferase control vector (for normalization of transfection efficiency; Promega) were transfected with 1 ng of LIP expression vector (pShuttle/LIP) or control vector (pShuttle; TaKaRa Bio using Lipofectamine™ and Lipofectamine™ Plus Reagent (Life Technologies) according to the manufacturer's instructions. For the siRNA experiments, 200 ng of each reporter plasmid and 1 ng of pRL-CMV Renilla luciferase control vector were transfected with 10 nM SF-1 (sc-37901; Santa Cruz Biotechnology), C/EBPβ or non-targeting siRNA using Lipofectamine™ and Lipofectamine™ Plus Reagent. At 36 h after transfection, cells were incubated with 8-Br-cAMP (1 mM) or medium (Control) for 12 h, and then luciferase activities were measured using a Lumat LB9501 luminometer (Berthold). Firefly luciferase activities (relative light units) were normalized to Renilla luciferase activities. Each data point represents the mean for at least four independent experiments.

ChIP assay

ChIP assays were performed as described previously [20]. Briefly, cells [(1–2)×106] were cross-linked with 1% formaldehyde in PBS at room temperature for 10 min, and then glycine was added, and cells were washed and harvested in buffer [50 mM Tris/HCl (pH 8.0), 2 mM EDTA, 0.1% Nonidet P40, 10 mM NaCl, 1.5 mM MgCl2, 10% glycerol and 1 mM PMSF]. Pellets were collected by centrifugation (1500 g for 5 min) and resuspended in SDS lysis buffer [50 mM Tris/HCl (pH 8.0), 10 mM EDTA and 1% SDS]. Samples were sonicated using a Bioruptor (Cosmo Bio). After centrifugation (20000 g for 5 min) to remove debris, supernatants were diluted 10-fold in dilution buffer [16.7 mM Tris/HCl (pH 8.0), 1.2 mM EDTA, 167 mM NaCl, 1.1% Triton X-100, 0.01% SDS and protease inhibitors mixture]. Aliquots of diluted supernatants were used as input and the remaining volumes were subjected to ChIP. Protein G or M-280 sheep anti-(mouse IgG)-coated Dynabeads were incubated with the antibodies of interest in 0.1% BSA/PBS at 4°C with rotation for 6 h and then washed. The ChIP samples (1–2 mg of protein) were then incubated with the antibody-bound Dynabeads at 4°C with rotation overnight. After incubation, beads were washed, eluted and incubated at 65°C overnight to reverse cross-linking. Samples were then treated with proteinase K. DNA was recovered after phenol/chloroform treatment and precipitated with ethanol, using glycogen as a carrier.

Each sample was analysed by qPCR (Power SYBR Green PCR Master Mix; Life Technologies) using a StepOnePlus Real-Time PCR System (Life Technologies) and results are presented as the percentage of input DNA. Each data point represents the mean for at least three independent experiments. Primer sequences are shown in Supplementary Table S1 (http://www.biochemj.org/bj/460/bj4600459add.htm) and in a previous paper [20].

EMSA

EMSAs using in vitro-synthesized proteins were performed as described previously [20]. Briefly, 1 μg of the pCMV-FLAG-SF-1 or pShuttle/LAP plasmids was incubated at 30°C for 60 min with the TnT® Coupled Reticulocyte Lysate System using RNA polymerase (Promega). The in vitro-translated product (1 μl) was added to the binding mixture with a γ-32P-labelled probe (10 fmol). For competition analysis, a 200-fold molar excess of competitor DNA was added to the binding mixture. After completion of binding, the mixture was subjected to 4% PAGE and the gel was dried and autoradiographed.

Statistical analysis

Results are given as means±S.E.M. Data were subjected to one-way ANOVA for means of comparison, and significant differences were calculated according to Duncan's multiple range test. Differences at P<0.05 were considered statistically significant.

RESULTS

Identification of the components of SF-1 nuclear complex

Previously, we have reported that introduction of SF-1 into MSCs resulted in the differentiation of the cells to the steroidogenic lineage [3]. FLAG-tagged SF-1 was introduced into hMSCs (human MSCs) and nuclear proteins were extracted to identify components that form a complex with SF-1. To obtain the native SF-1 nuclear complex, the chromatin fraction was isolated by MNase digestion under low-salt conditions (Supplementary Figure S2 at http://www.biochemj.org/bj/460/bj4600459add.htm), followed by IP with an anti-FLAG antibody. The eluent was separated by SDS/PAGE (Figure 1) and then identified by MALDI–TOF-MS/MS analysis. A total of 24 proteins were identified as components of the SF-1 complex, including transcription factors, chromatin remodelling factors and DNA repair proteins (Table 1). Among these proteins, C/EBPβ and HSP70 showed a marked increase in association with SF-1 upon 8-Br-cAMP treatment (Figure 1B). Because steroid hormone production is immediately stimulated by tropic hormones, such as LH, FSH (follicle-stimulating hormone) and ACTH (adrenocorticotropic hormone), through the cAMP/PKA (protein kinase A) pathway, C/EBPβ and HSP70 may be involved in the cAMP-mediated stimulation of steroidogenesis. We focused on the analysis of C/EBPβ, because C/EBPβ is known as a critical factor for ovulation, luteinization and steroidogenesis in the ovary, but little is known about the molecular mechanisms by which it regulates gene transcription in co-operation with SF-1.

Table 1
SF-1 nuclear complex proteins identified by MS/MS of their tryptic peptides

ND, no protein was identified.

Slice number Protein name UniProtKB accession number Number of identified peptides Theoretical molecular mass (kDa) 
ND    
DNA-dependent protein kinase catalytic subunit (DNA-PKcs) P78527 469 
ND    
ND    
ND    
ND    
ND    
BRG1 (SWI/SNF-related matrix-associated actin-dependent regulator of chromatin a4 variant) Q59FZ6 130 
BRG1 (SWI/SNF-related matrix-associated actin-dependent regulator of chromatin a4 variant) Q59FZ6 130 
10 ND    
11 BAF155 (SWI/SNF complex subunit SMARCC1) Q92922 123 
12 ND    
13 ND    
14 ND    
15 SNF2h (SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 5) O60264 122 
 hnRNP U (heterogeneous nuclear ribonucleoprotein U) Q00839 91 
16 hnRNP U (heterogeneous nuclear ribonucleoprotein U) Q00839 91 
17 KAP-1 (transcription intermediary factor 1-β) Q13263 89 
18 KAP-1 (transcription intermediary factor 1-β) Q13263 89 
19 KAP-1 (transcription intermediary factor 1-β) Q13263 89 
20 ND    
21 KAP-1 (transcription intermediary factor 1-β) Q13263 89 
 Ku80 (X-ray repair cross-complementing protein 6) P12956 70 
22 Ku80 (X-ray repair cross-complementing protein 6) P12956 70 
23 HSP70 Q53GZ6 71 
 hnRNP M (heterogeneous nuclear ribonucleoprotein M) P52272 78 
24 ND    
25 SF-1 Q13285 52 
26 SF-1 Q13285 52 
 HDAC1 (histone deacetylase 1) Q13547 55 
27 SF-1 Q13285 52 
28 Hornerin Q86YZ3 282 
29 Protein DEK P35659 43 
 hnRNP F (heterogeneous nuclear ribonucleoprotein F) P52597 46 
30 Actin, cytoplasmic 1 P60709 42 
 C/EBPβ P17676 36 
 hnRNP C1/C2 (heterogeneous nuclear ribonucleoproteins C1/C2) P07910 34 
31 hnRNP C1/C2 (heterogeneous nuclear ribonucleoproteins C1/C2) P07910 34 
 hnRNP A3 (heterogeneous nuclear ribonucleoprotein A3) P51991 40 
 Transcription factor AP-1 (proto-oncogene c-Jun) P05412 36 
32 hnRNP A2/B1 (heterogeneous nuclear ribonucleoprotein A2/B1) P22626 37 
 Replication factor C subunit 4 P35249 40 
 RNA-binding protein Raly Q9UKM9 32 
33 hnRNP A2/B1 (heterogeneous nuclear ribonucleoprotein A2/B1) P22626 37 
34 hnRNP A1 (heterogeneous nuclear ribonucleoprotein A1) P09651 39 
 ELAV-like protein 1 Q15717 36 
Slice number Protein name UniProtKB accession number Number of identified peptides Theoretical molecular mass (kDa) 
ND    
DNA-dependent protein kinase catalytic subunit (DNA-PKcs) P78527 469 
ND    
ND    
ND    
ND    
ND    
BRG1 (SWI/SNF-related matrix-associated actin-dependent regulator of chromatin a4 variant) Q59FZ6 130 
BRG1 (SWI/SNF-related matrix-associated actin-dependent regulator of chromatin a4 variant) Q59FZ6 130 
10 ND    
11 BAF155 (SWI/SNF complex subunit SMARCC1) Q92922 123 
12 ND    
13 ND    
14 ND    
15 SNF2h (SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 5) O60264 122 
 hnRNP U (heterogeneous nuclear ribonucleoprotein U) Q00839 91 
16 hnRNP U (heterogeneous nuclear ribonucleoprotein U) Q00839 91 
17 KAP-1 (transcription intermediary factor 1-β) Q13263 89 
18 KAP-1 (transcription intermediary factor 1-β) Q13263 89 
19 KAP-1 (transcription intermediary factor 1-β) Q13263 89 
20 ND    
21 KAP-1 (transcription intermediary factor 1-β) Q13263 89 
 Ku80 (X-ray repair cross-complementing protein 6) P12956 70 
22 Ku80 (X-ray repair cross-complementing protein 6) P12956 70 
23 HSP70 Q53GZ6 71 
 hnRNP M (heterogeneous nuclear ribonucleoprotein M) P52272 78 
24 ND    
25 SF-1 Q13285 52 
26 SF-1 Q13285 52 
 HDAC1 (histone deacetylase 1) Q13547 55 
27 SF-1 Q13285 52 
28 Hornerin Q86YZ3 282 
29 Protein DEK P35659 43 
 hnRNP F (heterogeneous nuclear ribonucleoprotein F) P52597 46 
30 Actin, cytoplasmic 1 P60709 42 
 C/EBPβ P17676 36 
 hnRNP C1/C2 (heterogeneous nuclear ribonucleoproteins C1/C2) P07910 34 
31 hnRNP C1/C2 (heterogeneous nuclear ribonucleoproteins C1/C2) P07910 34 
 hnRNP A3 (heterogeneous nuclear ribonucleoprotein A3) P51991 40 
 Transcription factor AP-1 (proto-oncogene c-Jun) P05412 36 
32 hnRNP A2/B1 (heterogeneous nuclear ribonucleoprotein A2/B1) P22626 37 
 Replication factor C subunit 4 P35249 40 
 RNA-binding protein Raly Q9UKM9 32 
33 hnRNP A2/B1 (heterogeneous nuclear ribonucleoprotein A2/B1) P22626 37 
34 hnRNP A1 (heterogeneous nuclear ribonucleoprotein A1) P09651 39 
 ELAV-like protein 1 Q15717 36 

Identification of the components of SF-1 nuclear complex in FLAG–SF-1 hMSCs

Figure 1
Identification of the components of SF-1 nuclear complex in FLAG–SF-1 hMSCs

(A) Affinity-purified SF-1 complexes from the chromatin fraction of FLAG–SF-1 hMSCs were subjected to SDS/PAGE (5–12.5% gels) followed by silver staining. The chromatin fractions were immunoprecipitated with anti-FLAG M2–agarose (+) or with Protein G–agarose (−). The numbers indicate the position of gel slices. M, protein marker. (B) The chromatin fractions of FLAG–SF-1 hMSCs (Input) stimulated with or without 8-Br-cAMP (8Br) for 24 h and immunoprecipitated fractions (IP) using anti-(FLAG M2) agarose were subjected to Western blot analysis. Because the level of SF-1 was different between the 8-Br-cAMP-stimulated and unstimulated FLAG–SF-1 hMSCs (Supplementary Figure S1 at http://www.biochemj.org/bj/460/bj4600459add.htm), different amounts of chromatin fractions were used for IP. The indicated proteins were detected by specific antibodies. (C) Association of C/EBPβ with SF-1 in Y1 cells. Nuclear extracts (High salt) or chromatin fractions (MNase) prepared from Y1 cells were subjected to IP with an anti-C/EBPβ antibody or a normal rabbit IgG. SF-1 and C/EBPβ were detected by Western blot analysis. DNA-PK, DNA-dependent protein kinase; HSC70, heat-shock cognate protein 70.

Figure 1
Identification of the components of SF-1 nuclear complex in FLAG–SF-1 hMSCs

(A) Affinity-purified SF-1 complexes from the chromatin fraction of FLAG–SF-1 hMSCs were subjected to SDS/PAGE (5–12.5% gels) followed by silver staining. The chromatin fractions were immunoprecipitated with anti-FLAG M2–agarose (+) or with Protein G–agarose (−). The numbers indicate the position of gel slices. M, protein marker. (B) The chromatin fractions of FLAG–SF-1 hMSCs (Input) stimulated with or without 8-Br-cAMP (8Br) for 24 h and immunoprecipitated fractions (IP) using anti-(FLAG M2) agarose were subjected to Western blot analysis. Because the level of SF-1 was different between the 8-Br-cAMP-stimulated and unstimulated FLAG–SF-1 hMSCs (Supplementary Figure S1 at http://www.biochemj.org/bj/460/bj4600459add.htm), different amounts of chromatin fractions were used for IP. The indicated proteins were detected by specific antibodies. (C) Association of C/EBPβ with SF-1 in Y1 cells. Nuclear extracts (High salt) or chromatin fractions (MNase) prepared from Y1 cells were subjected to IP with an anti-C/EBPβ antibody or a normal rabbit IgG. SF-1 and C/EBPβ were detected by Western blot analysis. DNA-PK, DNA-dependent protein kinase; HSC70, heat-shock cognate protein 70.

To assess whether SF-1 directly interacts with C/EBPβ, we performed IP assays. Consistent with a previous paper describing an in vitro pull-down assay of C/EBPβ and SF-1 [24], direct interactions between SF-1 and C/EBPβ were confirmed by IP using nuclear extracts prepared from Y1 cells either by a conventional extraction procedure or by MNase digestion under low-ionic strength conditions (Figure 1C).

C/EBPβ is involved in progesterone production

Because at least six members of C/EBP family have been identified, we examined the involvement in transcriptional activity of the members in KGN cells. Western blotting experiments showed that C/EBPβ, δ and ζ, but not α, γ and ε, were detected in KGN cells (Figure 2A, and Supplementary Figure S3A at http://www.biochemj.org/bj/460/bj4600459add.htm). To identify which members were bound to a binding site of C/EBP in the human StAR promoter region [25], EMSA supershift and ChIP assays were performed. A supershifted band was observed only in an anti-C/EBPβ antibody lane when nuclear extracts prepared from 8-Br-cAMP-stimulated KGN cells were used (Supplementary Figure S3B). ChIP assay revealed that C/EBPβ bound strongly to the promoter in KGN cells after both basal and 8-Br-cAMP treatment, whereas weak or no binding of C/EBPδ or C/EBPζ was observed (Supplementary Figure S3C). These results strongly suggest that C/EBPβ is a major C/EBP family working in KGN cells.

C/EBPβ is involved in 8-Br-cAMP-induced progesterone production through regulation of gene expression in KGN cells

Figure 2
C/EBPβ is involved in 8-Br-cAMP-induced progesterone production through regulation of gene expression in KGN cells

KGN cells were transfected with one of two independent C/EBPβ siRNAs (siC/EBPβ) or non-targeting siRNA (siCon). At 48 h after transfection, medium with or without 1 mM 8-Br-cAMP was replaced and cells were further incubated for 24 h. (A) Knockdown efficiencies were assessed by Western blot analysis. (B) Effect of C/EBPβ knockdown on progesterone production by KGN cells. 8-Br-cAMP-induced progesterone production markedly decreased by siC/EBPβ. Progesterone levels were measured by ELISA. (CE) Effect of C/EBPβ knockdown on progesterone production-related gene expression [HSD3B2 (C), STAR (D) and CYP11A1 (E)] in KGN cells. mRNA levels were measured by qPCR and normalized against GAPDH (glyceraldehyde-3-phosphate dehydrogenase). The ratio of non-targeting-siRNA (siCon)-transfected KGN cells was arbitrarily defined as 1. ND, not detected. Results are means±S.E.M. for at least four independent experiments. a, b and c indicate significant differences among groups at P<0.05 by Duncan's multiple range test.

Figure 2
C/EBPβ is involved in 8-Br-cAMP-induced progesterone production through regulation of gene expression in KGN cells

KGN cells were transfected with one of two independent C/EBPβ siRNAs (siC/EBPβ) or non-targeting siRNA (siCon). At 48 h after transfection, medium with or without 1 mM 8-Br-cAMP was replaced and cells were further incubated for 24 h. (A) Knockdown efficiencies were assessed by Western blot analysis. (B) Effect of C/EBPβ knockdown on progesterone production by KGN cells. 8-Br-cAMP-induced progesterone production markedly decreased by siC/EBPβ. Progesterone levels were measured by ELISA. (CE) Effect of C/EBPβ knockdown on progesterone production-related gene expression [HSD3B2 (C), STAR (D) and CYP11A1 (E)] in KGN cells. mRNA levels were measured by qPCR and normalized against GAPDH (glyceraldehyde-3-phosphate dehydrogenase). The ratio of non-targeting-siRNA (siCon)-transfected KGN cells was arbitrarily defined as 1. ND, not detected. Results are means±S.E.M. for at least four independent experiments. a, b and c indicate significant differences among groups at P<0.05 by Duncan's multiple range test.

To investigate whether C/EBPβ is involved in 8-Br-cAMP-induced steroidogenesis, progesterone production assays and gene expression analysis were performed. Treatment of KGN cells with 8-Br-cAMP resulted in induced C/EBPβ expression in the nucleus (Figure 2A). The expression of C/EBPβ was abolished by its knockdown using two independent siRNAs against C/EBPβ in KGN cells after both basal and 8-Br-cAMP treatment, whereas the expression of SF-1 was not affected (Figure 2A). As shown in Figure 2(B), the knockdown of C/EBPβ dramatically attenuated the 8-Br-cAMP-induced progesterone production in KGN cells. This result allowed us to study the effect of C/EBPβ knockdown on the expression of STAR, CYP11A1 and HSD3B2, which are critical factors for progesterone production (Figures 2C–2E). Consistent with the results of the knockout mice experiments [10], the 8-Br-cAMP-induced STAR and CYP11A1 expression decreased to half of the previous level in C/EBPβ-knockdown cells. Additionally, the 8-Br-cAMP-induced HSD3B2 expression was attenuated. These results indicate that C/EBPβ is involved in progesterone production through the induction of STAR, CYP11A1 and HSD3B2 expression under 8-Br-cAMP-stimulated conditions in KGN cells.

C/EBPβ is involved in the transcriptional activity of progesterone production-related genes together with SF-1

The gene expression profiles observed in the present study were clearly affected by C/EBPβ; however, it was still unknown whether C/EBPβ directly or indirectly activates the expression of these genes. To address this issue, the C/EBPβ-binding sites, which are near the TSSs of these genes, were explored by ChIP assays. Because transcriptional regulation has been characterized only within the 1-kb region upstream of the HSD3B2 TSS [26], we examined the recruitment of SF-1 and C/EBPβ over a wide region encompassing the HSD3B2 gene by ChIP-on-chip assays. These assays revealed that both SF-1 and C/EBPβ strongly bound to the region of −4 kb upstream from the TSS of HSD3B2 in FLAG–SF-1 hMSCs (Supplementary Figure S4 at http://www.biochemj.org/bj/460/bj4600459add.htm). These interactions were confirmed by conventional ChIP assays (Figures 3A and 3B). Similar results were obtained in KGN cells. Moreover, the binding of C/EBPβ was enhanced by treatment with 8-Br-cAMP (Figure 3C). These results indicate that C/EBPβ directly binds upstream of the HSD3B2 gene to regulate its expression in a cAMP-dependent manner. Because there are several putative SF-1- and C/EBPβ-binding sites between −4092 and −3757 bp upstream of HSD3B2, we performed EMSA assays to identify these binding sites. Interestingly, an identical 43-bp direct repeat sequence exists between −4067 and −3975 bp upstream of HSD3B2, which contains both a putative SF-1- and a putative C/EBPβ-binding site (Figure 3D). The in vitro-translated SF-1 or C/EBPβ bound to the radiolabelled probes containing the −4068/−4038 bp or the −4018/−3976 bp region. SF-1 binding was prevented by the addition of an excess of unlabelled probe or a probe mutated in the C/EBPβ-binding site, but not by the addition of a probe mutated in the SF-1-binding site. Similar results were obtained in the case of C/EBPβ binding. These results indicate that the binding of SF-1 and C/EBPβ to this region is independent and not prevented by each other. To assess whether the binding of C/EBPβ to this region is involved in the 8-Br-cAMP-induced transcriptional activation of HSD3B2, we performed luciferase assays in KGN cells using the C/EBPβ isoform LIP, which acts as a dominant-negative C/EBP (Figure 3E). Luciferase activities of all constructs were induced by treatment with 8-Br-cAMP. Overexpression of LIP dramatically decreased the 8-Br-cAMP-induced reporter activity of the construct containing the −4092/+23 bp region. Similar results were observed with the construct containing the upstream region −4092/−3757 with a minimal promoter. No effect was observed by overexpression of LIP with the mutated (−4092/+23) or deleted (−3773/+23 and −351/+23) constructs. Among the C/EBP family members, C/EBPβ predominantly had an effect on HSD3B2 transcription (Supplementary Figure S5 at http://www.biochemj.org/bj/460/bj4600459add.htm). These results strongly suggest that 8-Br-cAMP-induced HSD3B2 transcription is mediated, at least in part, by the binding of C/EBPβ to its promoter region.

Identification of the novel SF-1- and C/EBPβ-binding sites in the upstream region of the human HSD3B2 gene and its transcriptional activity

Figure 3
Identification of the novel SF-1- and C/EBPβ-binding sites in the upstream region of the human HSD3B2 gene and its transcriptional activity

(AC) ChIP analysis of SF-1 or C/EBPβ binding to the region upstream of the human HSD3B2 gene. (A) An anti-FLAG antibody or a normal rabbit IgG (negative control) was used to detect SF-1 binding to this region in FLAG–SF-1 hMSCs. (B) An anti-C/EBPβ antibody was used to detect C/EBPβ binding to this region in FLAG–SF-1 hMSCs. (C) An anti-C/EBPβ antibody was used to detect C/EBPβ binding to this region in KGN cells treated with or without 1 mM 8-Br-cAMP for 24 h. (D) EMSA analysis of SF-1 or C/EBPβ binding to the HSD3B2 upstream region. The upper panel shows the sequence of the HSD3B2 upstream region (from −4068 to −3973 bp; nucleotide numbering is relative to the TSS). Underlined nucleotides are the identical 43 bp direct repeat sequence that contain both SF-1- and C/EBPβ-binding sites. This panel also shows the probe sequences and the sites where mutations were introduced. The lower panel shows the EMSA results. Probes containing binding sites for both SF-1 and C/EBPβ (−4068/−4038) or only C/EBPβ (−3808/−3781) were used. Recombinant SF-1 and C/EBPβ proteins were prepared by in vitro translation. Unlabelled wild-type probes (W) and mutated probes (M1, M2, M3 and M4) were used as competitors. (E) Luciferase reporter assays of the upstream region of the HSD3B2 gene. Reporter constructs are depicted schematically on the left-hand side of the Figure. Mutations were introduced in three putative C/EBPβ-binding sites of the longest construct [pGL4/hHSD3B2(−4092/+23)mutC/EBP]. Transcriptional activity of the upstream region was also examined using a reporter construct containing a DNA fragment (−4092/−3757) upstream of the HSD3B2 gene fused to a minimal promoter. The indicated reporter and Renilla luciferase control vector were transfected with LIP expression vector (pShuttle/LIP) or control vector (pShuttle) into KGN cells. At 36 h after transfection, cells were incubated with 8-Br-cAMP (1 mM) or medium (control) for 12 h. Results are means±S.E.M. for at least four independent experiments. a, b and c indicate significant differences among groups at P<0.05 by Duncan's multiple range test.

Figure 3
Identification of the novel SF-1- and C/EBPβ-binding sites in the upstream region of the human HSD3B2 gene and its transcriptional activity

(AC) ChIP analysis of SF-1 or C/EBPβ binding to the region upstream of the human HSD3B2 gene. (A) An anti-FLAG antibody or a normal rabbit IgG (negative control) was used to detect SF-1 binding to this region in FLAG–SF-1 hMSCs. (B) An anti-C/EBPβ antibody was used to detect C/EBPβ binding to this region in FLAG–SF-1 hMSCs. (C) An anti-C/EBPβ antibody was used to detect C/EBPβ binding to this region in KGN cells treated with or without 1 mM 8-Br-cAMP for 24 h. (D) EMSA analysis of SF-1 or C/EBPβ binding to the HSD3B2 upstream region. The upper panel shows the sequence of the HSD3B2 upstream region (from −4068 to −3973 bp; nucleotide numbering is relative to the TSS). Underlined nucleotides are the identical 43 bp direct repeat sequence that contain both SF-1- and C/EBPβ-binding sites. This panel also shows the probe sequences and the sites where mutations were introduced. The lower panel shows the EMSA results. Probes containing binding sites for both SF-1 and C/EBPβ (−4068/−4038) or only C/EBPβ (−3808/−3781) were used. Recombinant SF-1 and C/EBPβ proteins were prepared by in vitro translation. Unlabelled wild-type probes (W) and mutated probes (M1, M2, M3 and M4) were used as competitors. (E) Luciferase reporter assays of the upstream region of the HSD3B2 gene. Reporter constructs are depicted schematically on the left-hand side of the Figure. Mutations were introduced in three putative C/EBPβ-binding sites of the longest construct [pGL4/hHSD3B2(−4092/+23)mutC/EBP]. Transcriptional activity of the upstream region was also examined using a reporter construct containing a DNA fragment (−4092/−3757) upstream of the HSD3B2 gene fused to a minimal promoter. The indicated reporter and Renilla luciferase control vector were transfected with LIP expression vector (pShuttle/LIP) or control vector (pShuttle) into KGN cells. At 36 h after transfection, cells were incubated with 8-Br-cAMP (1 mM) or medium (control) for 12 h. Results are means±S.E.M. for at least four independent experiments. a, b and c indicate significant differences among groups at P<0.05 by Duncan's multiple range test.

It has been reported that C/EBPs are involved in STAR expression in several species [24,25,2729]; however, it has not been investigated whether endogenously expressed C/EBPs bind to and activate the STAR promoter in human cells. According to our previous study, both the distal control region and the promoter of the human STAR gene are important for its expression [20]. Therefore SF-1 and C/EBPβ binding was analysed from the distal control region to the promoter by ChIP assays. As expected, SF-1 binding was detected at these regions in hMSCs (Figure 4A). In the case of C/EBPβ, binding was detected only at the promoter in SF-1-transduced hMSCs (Figure 4B) and KGN cells (Figure 4C). Furthermore, treatment with 8-Br-cAMP enhanced C/EBPβ binding to the region. Consistent with these results, when using constructs −3402/+39 and −235/+39, which contain C/EBPβ-binding sites, the reporter activities clearly decreased by overexpression of LIP in both unstimulated and 8-Br-cAMP-stimulated KGN cells (Figure 4D). These results indicate that endogenously expressed C/EBPβ participates in the stimulation of STAR gene expression, especially after 8-Br-cAMP stimulation.

Transcriptional activity of the SF-1- and C/EBPβ-binding sites in the promoter region of the human STAR gene

Figure 4
Transcriptional activity of the SF-1- and C/EBPβ-binding sites in the promoter region of the human STAR gene

(AC) ChIP analysis of SF-1 or C/EBPβ binding to the region upstream of the human STAR gene. (A) An anti-FLAG antibody or a normal rabbit IgG (negative control) was used to detect SF-1 binding to this region in FLAG–SF-1 hMSCs. (B) An anti-C/EBPβ antibody was used to detect C/EBPβ binding to this region in FLAG–SF-1 hMSCs. (C) An anti-C/EBPβ antibody was used to detect C/EBPβ binding to this region in KGN cells treated with or without 1 mM 8-Br-cAMP for 24 h. (D) Luciferase reporter assays of the upstream region of the STAR gene. Reporter constructs are depicted schematically on the left-hand side. The indicated reporter and Renilla luciferase control vector were transfected with the LIP expression vector (pShuttle/LIP) or the control vector (pShuttle) into KGN cells. At 36 h after transfection, cells were incubated with 8-Br-cAMP (1 mM) or medium (control) for 12 h. Results are means±S.E.M. for at least four independent experiments. a, b and c indicate significant differences among groups at P<0.05 by Duncan's multiple range test.

Figure 4
Transcriptional activity of the SF-1- and C/EBPβ-binding sites in the promoter region of the human STAR gene

(AC) ChIP analysis of SF-1 or C/EBPβ binding to the region upstream of the human STAR gene. (A) An anti-FLAG antibody or a normal rabbit IgG (negative control) was used to detect SF-1 binding to this region in FLAG–SF-1 hMSCs. (B) An anti-C/EBPβ antibody was used to detect C/EBPβ binding to this region in FLAG–SF-1 hMSCs. (C) An anti-C/EBPβ antibody was used to detect C/EBPβ binding to this region in KGN cells treated with or without 1 mM 8-Br-cAMP for 24 h. (D) Luciferase reporter assays of the upstream region of the STAR gene. Reporter constructs are depicted schematically on the left-hand side. The indicated reporter and Renilla luciferase control vector were transfected with the LIP expression vector (pShuttle/LIP) or the control vector (pShuttle) into KGN cells. At 36 h after transfection, cells were incubated with 8-Br-cAMP (1 mM) or medium (control) for 12 h. Results are means±S.E.M. for at least four independent experiments. a, b and c indicate significant differences among groups at P<0.05 by Duncan's multiple range test.

Because it has been demonstrated that the −2.3-kb region upstream of the CYP11A1 gene is sufficient for its tissue-specific and tropic hormone-dependent expression in steroidogenic cells [30], we examined the effect of C/EBPβ on CYP11A1 expression in this region. Although it has been demonstrated that LH surge-induced CYP11A1 expression was eliminated in the C/EBPβ-deficient mouse ovary, direct action of C/EBPβ on the transcription of CYP11A1 has not been shown [10]. In the present study, three SF-1-binding sites (two are consistent with the EMSA study of Hu et al. [30] and the other is novel) were detected in FLAG–SF-1 hMSCs (Figure 5A). Furthermore, a novel C/EBPβ-binding site was detected at approximately −2 kb upstream of the CYP11A1 TSS in FLAG–SF-1 hMSCs (Figure 5B). In the case of KGN cells, a C/EBPβ-binding site was also detected in that region (Figure 5C), and stimulation with 8-Br-cAMP enhanced the C/EBPβ binding. EMSA demonstrated that C/EBPβ bound to the region between −1961 and −1939 bp from the TSS of the CYP11A1 gene (Figure 5D). To examine whether this binding site is involved in the transcriptional activation of CYP11A1, luciferase assays were performed in KGN cells. Luciferase activity of the construct containing −2298/+72 bp was induced by treatment with 8-Br-cAMP, which was inhibited by overexpression of LIP (Figure 5E). LIP also showed effects on the reporter activity of the construct containing the fragment of the binding site of C/EBP (−2094/−1932 bp) (Figure 5F). Unexpectedly, 8-Br-cAMP-induced luciferase activity was also inhibited by overexpression of LIP with the construct containing the −1937/+72 bp region, which lacks the binding site of C/EBP. No effect was observed by overexpression of LIP on the −1545/+72 bp construct, which lacks the upstream CRS (cAMP-responsive sequence) (Figure 5E). This region is known as a site important for cAMP-mediated stimulation of CYP11A1 transcription [31]. Within the CRS, an SF-1-binding site is flanked at both sides with AP1-binding sites, but there is no C/EBP-binding site. Nevertheless, as shown in Figure 5(F), overexpression of LIP dramatically decreased the 8-Br-cAMP-induced reporter activity of the construct containing the CRS fragment (−1644/−1542 bp). Furthermore, both the AP1 and SF-1 sites in the CRS were important for the transcriptional activation by C/EBPβ (Figure 5F). However, C/EBPβ did not directly bind to the sites (Supplementary Figure S6 at http://www.biochemj.org/bj/460/bj4600459add.htm). These results suggest that C/EBPs could act not only via binding to its preferable sites, but also as a regulator without direct DNA binding at the CRS of the CYP11A1 promoter.

C/EBPβ is involved in transcriptional activity of the human CYP11A1 gene

Figure 5
C/EBPβ is involved in transcriptional activity of the human CYP11A1 gene

(AC) ChIP analysis of SF-1 or C/EBPβ binding to the region upstream of the human CYP11A1 gene. (A) An anti-FLAG antibody or a normal rabbit IgG (negative control) was used to detect SF-1 binding to this region in FLAG–SF-1 hMSCs. (B) An anti-C/EBPβ antibody was used to detect C/EBPβ binding to this region in FLAG–SF-1 hMSCs. (C) An anti-C/EBPβ antibody was used to detect C/EBPβ binding to this region in KGN cells treated with or without 1 mM 8-Br-cAMP for 24 h. (D) EMSA analysis of C/EBPβ binding to the CYP11A1 upstream region. The upper panel shows the probe sequences and the sites where mutations were introduced. The lower panel shows the EMSA results. Probes for a C/EBPβ-binding site (−1961/−1939) detected by ChIP assays were used. Recombinant C/EBPβ protein was prepared by in vitro translation. Unlabelled wild-type probes (W) and mutated probes (M) were used as competitors. (E) Luciferase activities of deletion constructs of the CYP11A1 promoter. Reporter constructs are schematically drawn on the left-hand side. (F) Luciferase activities of DNA fragments (−2094/v1932 and −1644/−1542) upstream of the CYP11A1 gene. The effects of mutations in AP1 or SF-1-binding site of the reporter (−1644/−1542) were also examined. Reporter constructs are schematically drawn on the left-hand side. The indicated reporter and Renilla luciferase control vectors were transfected with the LIP expression vector (pShuttle/LIP) or the control vector (pShuttle) into KGN cells. At 36 h after transfection, cells were incubated with 8-Br-cAMP (1 mM) or medium (control) for 12 h. Results are means±S.E.M. for at least four independent experiments. a, b and c indicate significant differences among groups at P<0.05 by Duncan's multiple range test.

Figure 5
C/EBPβ is involved in transcriptional activity of the human CYP11A1 gene

(AC) ChIP analysis of SF-1 or C/EBPβ binding to the region upstream of the human CYP11A1 gene. (A) An anti-FLAG antibody or a normal rabbit IgG (negative control) was used to detect SF-1 binding to this region in FLAG–SF-1 hMSCs. (B) An anti-C/EBPβ antibody was used to detect C/EBPβ binding to this region in FLAG–SF-1 hMSCs. (C) An anti-C/EBPβ antibody was used to detect C/EBPβ binding to this region in KGN cells treated with or without 1 mM 8-Br-cAMP for 24 h. (D) EMSA analysis of C/EBPβ binding to the CYP11A1 upstream region. The upper panel shows the probe sequences and the sites where mutations were introduced. The lower panel shows the EMSA results. Probes for a C/EBPβ-binding site (−1961/−1939) detected by ChIP assays were used. Recombinant C/EBPβ protein was prepared by in vitro translation. Unlabelled wild-type probes (W) and mutated probes (M) were used as competitors. (E) Luciferase activities of deletion constructs of the CYP11A1 promoter. Reporter constructs are schematically drawn on the left-hand side. (F) Luciferase activities of DNA fragments (−2094/v1932 and −1644/−1542) upstream of the CYP11A1 gene. The effects of mutations in AP1 or SF-1-binding site of the reporter (−1644/−1542) were also examined. Reporter constructs are schematically drawn on the left-hand side. The indicated reporter and Renilla luciferase control vectors were transfected with the LIP expression vector (pShuttle/LIP) or the control vector (pShuttle) into KGN cells. At 36 h after transfection, cells were incubated with 8-Br-cAMP (1 mM) or medium (control) for 12 h. Results are means±S.E.M. for at least four independent experiments. a, b and c indicate significant differences among groups at P<0.05 by Duncan's multiple range test.

To further clarify how SF-1 and C/EBPβ transactivate progesterone production-related genes, we examined the effect of siRNA against SF-1 or C/EBPβ on the transcription of HSD3B2, STAR and CYP11A1 in KGN cells (Figures 6A–6C). Consistent with the luciferase assay experiments with LIP, the 8-Br-cAMP-induced luciferase activities of all constructs decreased to 50–70% of the original level by C/EBPβ knockdown. These results further support the concept that the transcription of these genes is mediated, at least in part, by C/EBPβ, especially under 8-Br-cAMP-stimulated conditions. Knockdown of SF-1 resulted in increased C/EBPβ expression in KGN cells after both basal and 8-Br-cAMP treatment (Figure 6D). However, SF-1 knockdown completely abolished the luciferase activities of STAR and CYP11A1, and decreased that of HSD3B2 to 30% of the original level. These results suggest that SF-1 is a critical factor for the expression of these genes, whereas C/EBPβ is a mediator of their expression in co-operation with SF-1, especially under tropic hormone-stimulated conditions.

Effect of SF-1 or C/EBPβ knockdown on progesterone production-related gene expression in KGN cells

Figure 6
Effect of SF-1 or C/EBPβ knockdown on progesterone production-related gene expression in KGN cells

Luciferase reporter assays of the upstream region of the HSD3B2 (A), STAR (B) and CYP11A1 (C) genes. The indicated reporter and Renilla luciferase control vectors were transfected with siSF-1, siC/EBPβ or siControl into KGN cells. At 36 h after transfection, cells were incubated with 8-Br-cAMP (1 mM) or medium (control) for 12 h. Results are means±S.E.M. for at least four independent experiments. a, b and c indicate significant differences among groups at P<0.05 by Duncan's multiple range test. (D) Knockdown efficiencies were assessed by Western blot analysis. KGN cells were transfected with SF-1-siRNA (siSF-1) or non-targeting-siRNA (siControl). At 48 h after transfection, medium with or without 1 mM 8-Br-cAMP was replaced and cells were further incubated for 24 h.

Figure 6
Effect of SF-1 or C/EBPβ knockdown on progesterone production-related gene expression in KGN cells

Luciferase reporter assays of the upstream region of the HSD3B2 (A), STAR (B) and CYP11A1 (C) genes. The indicated reporter and Renilla luciferase control vectors were transfected with siSF-1, siC/EBPβ or siControl into KGN cells. At 36 h after transfection, cells were incubated with 8-Br-cAMP (1 mM) or medium (control) for 12 h. Results are means±S.E.M. for at least four independent experiments. a, b and c indicate significant differences among groups at P<0.05 by Duncan's multiple range test. (D) Knockdown efficiencies were assessed by Western blot analysis. KGN cells were transfected with SF-1-siRNA (siSF-1) or non-targeting-siRNA (siControl). At 48 h after transfection, medium with or without 1 mM 8-Br-cAMP was replaced and cells were further incubated for 24 h.

DISCUSSION

To regulate the expression of their target genes, transcription factors bind to their target sequences and form a complex with numerous nuclear proteins. Therefore it is critical to identify the components of a nuclear complex of a certain transcription factor to elucidate its transcriptional regulatory mechanism. SF-1 is known as a master regulator of adrenal and gonadal development and differentiation, including regulation of the expression of steroidogenic genes [1,2]. The transcriptional regulation of individual SF-1 target genes by SF-1 has been well characterized [32]; however, identification of the components of the native SF-1 nuclear complex has not been performed as yet. Previously, it has been found that SF-1 transduction into MSCs resulted in cell differentiation into steroidogenic cell lineages [3]. In the present paper, we tried to identify the components of the SF-1 nuclear complex by biochemical approaches.

Because the reconstituted SF-1 complex was isolated by a traditional extraction method, the chromatin fraction was isolated from FLAG–SF-1 hMSCs under low-salt conditions to isolate the native SF-1 complex (Supplementary Figure S2). We identified 24 proteins as components of the SF-1 nuclear complex using affinity purification followed by MALDI–TOF-MS/MS analysis (Table 1). Unexpectedly, in addition to transcription-related proteins, DNA repair factors and splicing factors were identified. We speculated that SF-1 may have many functions, not only transcriptional regulation, but also some important adrenal and/or gonadal functions. Consistent with this speculation, it has been reported that SF-1 works together with the DNA repair proteins Ku70/80 as a regulator of centrosome homoeostasis in the centrosome, but not in the nucleus [33,34]. Recently, we have also shown that SF-1 is important for altering the chromatin structure in the GSTA (glutathione transferase Alpha) family gene locus via its hinge region [23]. Further studies will elucidate the various potential functions of SF-1.

CEBPα and β are transiently induced by LH synergistically or independently via the cAMP/PKA and ERK pathways in the preovulatory follicles of rodent ovaries [10]. In humans, it has also been demonstrated that C/EBPα and β exist in granulosa-luteal cells [25]. In GC-specific C/EBPα and β double-deficient mice, numerous genes, which are related to steroidogenesis, luteal maintenance and vascular development, were directly and indirectly affected [11]. C/EBPα and β have been shown as essential factors for ovulation and luteinization; nevertheless, little is known about the transcriptional regulation of these target genes in the ovary. In steroidogenic-related genes, even though the transcriptional regulation of STAR by C/EBPs has been well characterized in several species [24,25,2729], the regulation of HSD3B2 (rodent counterpart Hsd3b1) and CYP11A1 is poorly understood. In the present study, we have demonstrated that C/EBPβ is involved in cAMP-induced progesterone production by directly regulating these genes together with SF-1 in KGN cells. Because KGN cells dominantly express C/EBPβ rather than C/EBPα, it appeared that progesterone production was markedly decreased by C/EBPβ knockdown alone. Consistent with this result, the phenotype of the Cebpbgc−/− mice was more severe than that of the Cebpagc−/− mice [11].

C/EBP family proteins interact not only with SF-1, but also with other transcription factors, such as the GR (glucocorticoid receptor) [35] and PPARγ (peroxisome-proliferator-activated receptor γ) [36]. More than half of the GR-binding sites are preoccupied by C/EBPβ, and disruption of C/EBPβ binding resulted in decreased chromatin accessibility and reduced GR recruitment in 3134 cells that originated from murine mammary epithelial cells [35]. C/EBPα binds in close proximity to over 60% of the locations bound by PPARγ in mouse 3T3-L1 adipocytes [36]. Thus C/EBP family proteins may be essential cofactors for gene expression of GR or PPARγ target genes and/or regulators for maintaining preferable chromatin structures. On the other hand, it is not probable that C/EBP family proteins have a similar role in regulating SF-1 target gene expression in the ovary. Because, in the ovary, C/EBP family proteins are not abundantly expressed until the preovulatory LH surge, it is probable that the SF-1-binding elements are not constantly occupied by C/EBP family proteins to maintain their preferable chromatin structures for SF-1 binding. Furthermore, the overlap between SF-1 and C/EBPβ binding was only 12.5% in the SF-1-transduced hMSCs (Supplementary Figure S7 at http://www.biochemj.org/bj/460/bj4600459add.htm). As mentioned above and in some previous papers [37,38], co-occupancy of more than half of the regulatory elements seems essential for C/EBP family proteins to function as co-factors or regulators of SF-1. Collectively, these data suggest that both SF-1 (LRH-1) and C/EBP family proteins function as transcription factors to co-operatively and synergistically regulate some of their common target genes, including progesterone production-related genes.

In agreement with previous studies [39,40], SF-1 knockdown markedly decreased the expression of progesterone production-related genes in KGN cells (Figure 6). Therefore SF-1 is thought to be a master regulator of steroidogenic-related genes including these genes. In the present study, C/EBPβ has been shown to be a common transcriptional regulator of these genes in co-operation with SF-1. C/EBPβ binds to its target elements and interacts with SF-1 via a large complex, subsequently enhancing SF-1-regulated transcription, especially under cAMP-stimulated conditions. LH is essential for ovulation and luteinization by activating the cAMP/PKA and MAPK (mitogen-activated protein kinase) pathways. C/EBPβ has been shown to be induced by LH and activated in an ERK1/2-dependent manner in preovulatory follicles [10]. These findings suggest that LH induces and activates C/EBPβ, which in turn stimulates transcription of progesterone production-related genes in co-operation with SF-1.

We have shown that C/EBPβ regulates CYP11A1 transcription through both DNA-binding-dependent and -independent pathways (Figure 5 and Supplementary Figure S6). The DNA-binding-independent pathway of a certain transcription factor has been described as a tethering mechanism through direct and indirect interactions with another transcription factor [4143]. On the other hand, C/EBPβ-activated HSD3B2 and STAR expression was DNA-binding-dependent [25] (Figure 3). Although C/EBPβ is a common regulator of progesterone production-related genes, two different mechanisms of transcriptional regulation may work.

In summary, we have found that C/EBPβ is a component of the SF-1 nuclear complex and a common regulator of progesterone production-related genes. Novel C/EBP-binding sites have been identified in the human HSD3B2 and CYP11A1 upstream regions. The SF-1 and C/EBPβ synergistic augmentation of the expression of these genes led to an effective increase in progesterone production, especially under tropic hormone-stimulated conditions. Identification of the novel regulatory regions of these genes may help future studies to identify gene mutations in corpus luteum insufficiency and infertility.

Abbreviations

     
  • 8-Br-cAMP

    8-bromo-cAMP

  •  
  • C/EBP

    CCAAT/enhancer-binding protein

  •  
  • CRS

    cAMP-responsive sequence

  •  
  • CYP11A1

    cytochrome P450, family 11, subfamily A, polypeptide 1

  •  
  • DMEM

    Dulbecco’s modified Eagle’s medium

  •  
  • ERK

    extracellular-signal-regulated kinase

  •  
  • GC

    granulosa cell

  •  
  • GR

    glucocorticoid receptor

  •  
  • HSD3B2

    hydroxy-δ-5-steroid dehydrogenase, 3β- and steroid δ-isomerase 2

  •  
  • hMSC

    human MSC

  •  
  • HSP70

    heat-shock protein 70

  •  
  • IP

    immunoprecipitation

  •  
  • LAP

    liver-enriched activator protein

  •  
  • LH

    luteinizing hormone

  •  
  • LIP

    liver-enriched inhibitory protein

  •  
  • LRH-1

    liver receptor homologue-1

  •  
  • MNase

    micrococcal nuclease

  •  
  • MSC

    mesenchymal stem cell

  •  
  • PKA

    protein kinase A

  •  
  • PPARγ

    peroxisome-proliferator-activated receptor γ

  •  
  • qPCR

    quantitative real-time PCR

  •  
  • SF-1

    steroidogenic factor-1

  •  
  • StAR

    steroidogenic acute regulatory protein

  •  
  • TSS

    transcription start site

AUTHOR CONTRIBUTION

Kaoru Miyamoto and Tetsuya Mizutani conceived the project and designed the experiments. Tetsuya Mizutani performed experiments, analysed data and wrote the paper. Yunfeng Ju and Yoshitaka Imamichi carried out the luciferase assay and created the plasmids. Tsukasa Osaki and Naoto Minamino performed the MS/MS analysis and analysed data. Takashi Yazawa, Shinya Kawabe, Shin Ishikane, Takehiro Matsumura and Masafumi Kanno performed the ChIP assay. Yasue Kamiki and Kohei Kimura performed the qPCR experiments. Kaoru Miyamoto wrote the paper. All authors contributed to the review of the paper before submission.

We thank Dr T. Yanase (Fukuoka University, Fukuoka, Japan) for providing KGN cells, Dr J. Toguchida (Kyoto University, Kyoto, Japan) for providing hMSC-hTERT-E6/E7 line, Dr M. Oki (University of Fukui, Fukui, Japan) and Dr J. Nakayama (Nagoya City University, Nagoya, Japan) for technical advice on immunoaffinity chromatography and ChIP assay, Ms K. Matsuura, H. Fujii, Y. Yamazaki and Y. Usami for technical assistance, and Y. Inoue for administrative assistance.

FUNDING

This work was supported by the Ministry of Education, Culture, Sports, Science and Culture [grant numbers 24590347 (to T.M.), 25861482 (to Y.I.), 25861481 (to S.K.) and 25670440 (to K.M.)], a Health and Labour Sciences Research grant from the Ministry of Health, Labour and Welfare, Research on Development of New Drugs and the Smoking Research Foundation.

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Author notes

2

Present address: Department of Molecular Patho-Biochemistry and Patho-Biology of Hematology and Circulation, Yamagata University School of Medicine, Iida-Nishi 2-2-2, Yamagata 990-9585, Japan.

3

Present address: Division of Cellular Signal Transduction, Department of Biochemistry, Asahikawa Medical University, Asahikawa 078-8510, Japan.

Supplementary data