PPARγ co-activator-1α co-activates steroidogenic factor 1 to stimulate the synthesis of luteinizing hormone and aldosterone

Biochem. J. (2010) 432, 473–483 (Printed in Great Britain) doi:10.1042/BJ20100460 473

PPARγ co-activator-1α co-activates steroidogenic factor 1 to stimulate thesynthesis of luteinizing hormone and aldosteroneLiuluan ZHU*†1, Yaojun KE‡1, Di SHAO*, Ying CUI*, Aijun QIAO*, Xiaojun LIU*, Fude FANG*2 and Yongsheng CHANG*2

*National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005,China, †Institute of Infectious Diseases, Beijing Ditan Hospital, Capital Medical University, Beijing 100015, China, and ‡Department of Interventional Radiology, Tianyou Hospital,Wuhan University of Science and Technology, Wuhan 430064, China

The orphan nuclear receptor SF-1 (steroidogenic factor 1) ishighly expressed in the pituitary, gonad and adrenal glandsand plays key roles at all levels of the hypothalamic–pituitary–steroidogenic tissue axis. In the present study, we show thatPGC-1α [PPARγ (peroxisome-proliferator-activated receptorγ ) co-activator 1α] interacts with and co-activates SF-1 toinduce LHβ (luteinizing hormone β) and αGSU (α-glycoproteinsubunit) gene expression, subsequently leading to the increasedsecretion of LH in pituitary gonadotrope-derived αT3-1 cells.PGC-1α co-activation of LHβ expression requires an SF-1-binding element [GSE (gonadotrope-specific element)] mappedto the promoter region of LHβ. Mammalian two-hybrid andco-immunoprecipitation assays, as well as GST (glutathionetransferase) pull-down experiments demonstrated that PGC-1αinteracts with SF-1 in vivo and in vitro. Additionally, PGC-1α sti-mulates the expression of Cyp11b2 (aldosterone synthase

gene), Cyp11b1 (steroid 11β-hydroxylase gene) and P450scc(cholesterol side-chain cleavage enzyme), and the synthesisof aldosterone in adrenal-cortex-derived Y-1 cells. Chromatinimmunoprecipitation assays confirmed that endogenous PGC-1αco-localizes with SF-1 in the LHβ and Cyp11b2 promoter region.Knockdown of endogenous SF-1 by siRNA (small interferingRNA) abolished the PGC-1α induction of LHβ and Cyp11b2gene expression in αT3-1 and Y-1 cells respectively. Finally,we demonstrated that PGC-1α induces SF-1 gene expression inboth αT3-1 and Y-1 cells. Taken together, our findings revealthe potential role of PGC-1α and suggest that it may playimportant roles in steroidogenesis, gonad development and sexdifferentiation through SF-1.

Key words: gene expression, luteinizing hormone, pituitary,steroidogenesis, transcription.

INTRODUCTION

PGC-1α [PPARγ (peroxisome-proliferator-activated receptor γ )co-activator 1α] is a member of a family of transcription co-activators which play central roles in regulating cellular energymetabolism. PGC-1α is highly induced in the liver and heartduring fasting, in brown adipose tissue after exposure to coldtemperature and in skeletal muscle while exercising [1,2]. Currentevidence suggests that PGC-1α can interact directly with and co-activate several nuclear receptors including, but not limited to,all ERR (oestrogen-related receptor) [3,4] and PPAR subtypes[5,6], FOXO1 (forkhead box O1) [7] and NRF-1 (nuclearrespiratory factor 1) [8]. Generally, the interactions between PGC-1α and nuclear receptors involve an LXXLL motif located at theN-terminal amino acids 142–146 of PGC-1α and the C-terminalAF-2 domain of nuclear receptors. PGC-1α (Ppargc1a)-knockoutmice exhibit multiple metabolic defects, such as obesity,neurodegeneration and cardiomyopathy [9,10]. PGC-1α playsimportant roles in adaptive thermogenesis in brown adiposetissue, gluconeogenesis in the liver, mitochondrial biogenesis andrespiration in muscle cells, and heart development [1,11].

SF-1 (steroidogenic factor 1; encoded by Nr5a1), a memberof the nuclear hormone receptor superfamily, is highly expressedin steroidogenic tissues, including adrenal cortex and gonads, as

well as non-steroidogenic tissues, such as pituitary gonadotropesand hypothalamus [12]. SF-1 plays key roles in severalphysiological processes, including steroid synthesis, adrenal andgonadal development, and sex differentiation [13]. In pituitarygonadotropes, SF-1 stimulates the expression of the genesencoding αGSU (α-glycoprotein subunit), LHβ (luteinizinghormone β) and FSHβ (follicle-stimulating hormone β), bywhich SF-1 influences the reproductive axis [12]. LH is apituitary-derived glycoprotein hormone that belongs to a family ofhormones that includes FSH, TSH (thyroid-stimulating hormone)and CG (chorionic gonadotropin) [14]. LH is encoded by twogenes located on different chromosomes, so it is a heterodimericglycoprotein, consisting of an αGSU common to all familymembers and a unique β-subunit that associates non-covalently[14]. Sequence analysis of LHβ gene promoters from a broadrange of species reveals the presence of a consensus GSE(gonadotrope-specific element; TGACCTTGT). SF-1 binds thiselement, and overexpression of Nr5a1 (mouse SF-1 gene) inthe murine gonadotrope-derived αT3-1 cell line activates bovineLHβ promoter activity [15,16]. The expression of LHβ mRNAis markedly decreased in SF-1 (mouse gene symbol Nr5a1)− /−

mice [17].To date, SF-1 is known to directly enhance the transcription

of more than 20 genes. Numerous target genes are required for

Abbreviations used: ALD, aldosterone; ChIP, chromatin immunoprecipitation; CRE, cAMP-response element; DMEM, Dulbecco’s modified Eagle’smedium; ERR, oestrogen-related receptor; FBS, fetal bovine serum; FSH, follicle-stimulating hormone; GFP, green fluorescent protein; GSE, gonadotrope-specific element; GST, glutathione transferase; αGSU, α-glycoprotein subunit; HA, haemagglutinin; HEK, human embryonic kidney; LH, luteinizing hormone;NRF-1, nuclear respiratory factor 1; PGC-1α, PPARγ (peroxisome-proliferator-activated receptor γ) co-activator 1α; PPARγ, peroxisome-proliferator-activated receptor γ; pRL-TK, thymidine kinase promoter-Renilla luciferase reporter plasmid; SF-1, steroidogenic factor 1; siRNA, small interfering RNA;SOX9, SRY (sex-determining region Y)-box 9.

1 These authors contributed equally to this work.2 Correspondence may be addressed to either of these authors (email [email protected] or [email protected]).

c© The Authors Journal compilation c© 2010 Biochemical Society

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474 L. Zhu and others

cholesterol mobilization and steroid hormone biosynthesis insteroidogenic cells, including cytochrome P450 steroidhydroxylases, StAR (steroidogenic acute regulatory protein) andHMG-CoA (3-hydroxy-3-methylglutaryl-CoA) synthase [12,18].ALD (aldosterone), the principal human mineralocorticoid, isproduced in the zona glomerulosa of the adrenal gland. Thefinal key steps in ALD synthesis are sequential 11-hydroxylation,18-hydroxylation and 18-oxidation of the precursor steroiddeoxycorticosterone in zona glomerulosa cells. A single enzyme,ALD synthase, carries out all of these steps and is encodedby the gene Cyp11b2 [19]. Human CYP11B2 transcription isstimulated by angiotensin II, potassium ions and cAMP signallingpathways using the SF-1 and CRE (cAMP-response element)-like cis-elements, which are located at positions − 71/ − 64(TGACGTGA) and − 129/ − 114 (CTCCAGCCTTGACCTT)respectively [20].

SF-1-knockout mice exhibit adrenal and gonadal agenesis,male-to-female sex reversal of internal and external genitalia,impaired expression of pituitary gonadotropins and structuralabnormalities of ventromedial hypothalamic nucleus [17,21]. Inaddition, the structure of SF-1 has also been characterized tounderstand the molecular mechanism of transcriptional activity.The AF-2 transactivation domain is absolutely conserved in allSF-1 proteins from several species, including human, bovine,rat and mouse [13]. The proximal activation domain locatedat C-terminal residues 187–245 in SF-1 is required for itstranscriptional activity and can physically interact with itsco-activators, such as SRC-1 (steroid receptor co-activator-1)and TReP-132 (transcriptional regulating protein of 132 kDa)[22–24].

A recent study revealed that PGC-1α can induce the expressionof CYP11A1 and CYP17A1, which encode the key rate-limitingenzymes involved in the initial steps of steroidogenesis in hepaticcells [25], suggesting that PGC-1α may function in a muchbroader aspect in endocrine, more than energy, metabolism. Inthe present study, we show that PGC-1α induced αGSU and LHβgene expression in αT3-1 cells and the expression of severalkey enzymes involved in steroidogenesis, including Cyp11b2 andP450scc, in Y-1 cells. These results imply that PGC-1α mayinfluence steroid synthesis, adrenal and gonadal development andsex differentiation by co-activating SF-1.

MATERIALS AND METHODS

Plasmids and adenoviruses

Full-length mouse SF-1 (GenBank® accession numberNM_139051) was obtained by PCR amplification of cDNAfrom mouse ovary and cloned into pCMV-HA (Clontech)with EcoRI/BglII restriction sites and pACT (Promega) withBamHI/XbaI sites respectively. The different truncated versionsof SF-1 encoding amino acids 187–451, amino acids 187–462, amino acids 246–451 and amino acids 246–462 were allcloned into pCMV-HA (Clontech). Full-length mouse PGC-1α (GenBank® accession number NM_008904) was subclonedinto pBIND (Promega) from a construct expressing PGC-1α–Myc, which was a gift from Dr Teresa C. Leone (Center forCardiovascular Research, Washington University School of Medi-cine, St. Louis, MO, U.S.A.). Full-length ERRα (GenBank® ac-cession number NM_004451) was cloned into pcDNA3.1as described previously [26]. Full-length ERRγ (GenBank®

accession number NM_001438) was cloned into pCMV-HAwith EcoRI/XhoI sites. The 5′-end of the mouse SF-1 geneextending from position − 1892 bp (relative to the transcriptionstart site) to +147 was cloned into the pGL3-Basic (Promega)

luciferase reporter plasmid with the SacI/SmaI sites. Themouse LHβ gene promoter (− 948 to +264 bp) was amplifiedusing mouse genomic DNA and inserted into pGL3-Basic(− 948Luc). A series of 5′-deletion and corresponding mutationconstructs of LHβ (− 948mut, − 160, − 160mut, − 100 to +264)were prepared by PCR using − 948Luc as a template. Theplasmid pGEX-PGC-1α-180 expressing the GST (glutathionetransferase)-fused N-terminal 180 amino acids of PGC-1α wasa gift from Dr Bruce Spiegelman (Dana Farber Cancer Institute,Harvard Medical School, Boston, MA, U.S.A.). The constructcontaining siRNA (small interfering RNA) against mouse SF-1was generated by subcloning a double-stranded oligomer targetedto nucleotides 321–341 of mouse SF-1 mRNA (GenBank®

accession number NM_139051) [27] into the BbsI site ofpBabe-Dual (from Dr Ye Zhang, Institute of Basic MedicalSciences, Chinese Academy of Medical Sciences and PekingUnion Medical College, Beijing, China) [28]. All constructswere confirmed by direct nucleotide sequencing, and the primersused for plasmid construction are shown in Supplementary TableS1 at http://www.BiochemJ.org/bj/432/bj4320473add.htm. Ad-PGC-1α (adenovirus expressing PGC-1α) was constructed bycloning full-length PGC-1α into the pAd-Track vector at SalI andEcoRV sites. Recombination with pAdEasy-1 and propagationof adenovirus expressing PGC-1α in HEK (human embryonickidney)-293A cells was performed as described previously [26].

Cell culture, transfection and adenovirus infection

Murine adrenocortical Y-1 cells were obtained from A.T.C.C.and cultured in F-12 medium containing 15 % horse serumand 2.5% FBS (fetal bovine serum), supplemented with 2 mML-glutamine, 100 units/ml penicillin and 0.1 mg/ml streptomycinincubated at 37 ◦C with 5% CO2. Murine gonadotrope-derivedαT3-1 cells were maintained at 37 ◦C with 5% CO2 in DMEM(Dulbecco’s modified Eagle’s medium)/F-12 supplemented with10% FBS, 100 units/ml penicillin and 0.1 mg/ml streptomycin.HEK-293ET, HEK-293A and CV-1 cells were cultured in high-glucose DMEM with 10 % FBS and antibiotics.

Generally, cells were plated 24 h or 48 h prior to transienttransfection using LipofectamineTM 2000 reagent (Invitrogen)according to the manufacturer’s protocol. For adenoviralinfection, Y-1 and αT3-1 cells were infected for 48 h at amultiplicity of infection sufficient to infect >95% of cells.

Luciferase assays

All experiments were performed using the Dual-LuciferaseReporter Assay System (Promega) with an internal Renillaluciferase control plasmid to normalize transfection efficiencies.In general, αT3-1, 293ET and CV-1 cells were plated in 24-wellplates. After 12–24 h, cells were transfected with 0.25 μg of fireflyluciferase reporter constructs of serial 5′-deletions or single-bp mutation promoters, 10 ng of pRL-TK (thymidine kinasepromoter-Renilla luciferase reporter plasmid) and expressionplasmids (0.5 μg of PGC-1α; 0.25 μg of SF-1, ERRα and ERRγ ;or as specifically described). Cells were harvested 24–48 h aftertransfection. Reporter gene activity was calculated as activity overbasal levels generated from transfection of the firefly luciferasereporter gene, pRL-TK and pcDNA3.1. The results represent themeans +− S.D. of at least three independent experiments.

Mammalian two-hybrid assays

The CheckMateTM Mammalian Two-Hybrid System (Promega)was used according to the manufacturer’s protocol. Briefly,


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PGC-1α interacts with and co-activates SF-1 475

1 × 105 cells were plated in a 24-well plate. On the following day,0.25 μg of the pACT (VP16) and pBIND (GAL4) fusionconstructs and 0.25 μg of pG5luc were transfected. Cells wereincubated for 24 h before harvesting, and luciferase activitywas screened using the Dual-Luciferase Reporter Assay System.These experiments were performed in triplicate and repeated inthree independent experiments.

ChIP (chromatin immunoprecipitation)

ChIP assays in mouse αT3-1 cells and Y-1 cells were performed asdescribed previously [29]. Cells were cultured on 100-mm dishesand cross-linked with 1% formaldehyde, lysed and subjected tochromatin shearing by sonication. Input samples were obtainedat 1% (v/v), followed by immunoprecipitation with specificantibody at 4 ◦C overnight. The antibodies used were anti-SF-1, anti-PGC-1α or, as a negative control, IgG antibody.Immune complexes were collected with 50 μl of Protein G–SepharoseTM 4 Fast Flow beads. PCR analysis was performed withinput and immunoprecipitated DNA. The primers were designedto amplify the proximal region of the mouse SF-1 promotercontaining GSE or the distal region without GSE as a negativecontrol. The specific primers are shown in Supplementary TableS2 (at http://www.BiochemJ.org/bj/432/bj4320473add.htm).

RNA isolation and real-time PCR

The αT3-1, Y-1 and CV-1 cells were plated at a density of 60% in60-mm dishes and transfected and/or infected for 48 h. Total RNAwas extracted using TRIzol® reagent (Invitrogen) as specifiedby the manufacturer. RNA (4 μg) was reverse transcribed tocDNA using the Transcriptor High Fidelity cDNA SynthesisSample kit (Roche), and specific transcripts were quantified byreal-time PCR using an iQ5 Real-Time PCR Detection System(Bio-Rad). Quantitative real-time PCR was performed usingTransStart Green qPCR SuperMix (TransGen Biotech). Primersused in the study are shown in Supplementary Table S3 (athttp://www.BiochemJ.org/bj/432/bj4320473add.htm). RelativemRNA levels for the specific genes were normalized toβ-actin mRNA levels.

Western blot analysis

Adenoviruses were infected into αT3-1 or Y-1 cells in 60-mmdishes. Cells were incubated for 48 h after infection andsubsequently lysed in lysis buffer [50 mM Tris/HCl (pH 8.0),150 mM NaCl, 1 mM EDTA, 1% Nonidet P40, 1% SDS, 1 mMPMSF, 1 mM DTT (dithiothreitol) and protease inhibitors]. CV-1cells were transfected with plasmids expressing PGC-1α and/orSF-1, or with pcDNA3.1 (negative control) for 48 h. Whole-cellproteins (60 μg) were used for Western blot analysis.

GST pull-down assays

GST and GST–PGC-1α (amino acids 1–180) fusion proteins wereexpressed in Escherichia coli BL21 (DE3), induced with 0.5 mMIPTG (isopropyl β-D-thiogalactopyranoside) and immobilizedon glutathione–Sepharose 4B beads (GE Healthcare). Then,293ET cells (5 × 105) plated in 60-mm dishes were transfectedwith plasmids expressing HA (haemagglutinin)-tagged SF-1 full-length or truncated versions for 48 h. Cells were lysed in 1 ml oflysis buffer [50 mM Tris/HCl (pH 7.4), 1 % Nonidet P40, 0.25%sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF

Figure 1 PGC-1α induces LHβ and αGSU gene expression in αT3-1 cells

(A) αT3-1 cells were infected with adenovirus expressing PGC-1α (Ad-PGC-1α) or GFP(Ad-GFP) as a control, proteins were extracted and Western blot analysis was performed usingantibodies specific to PGC-1α and β-actin. (B) αT3-1 cells were treated as described in(A), and total RNA was extracted 48 h after infection. The mRNA levels of αGSU, LHβ andFSHβ were quantified by real-time PCR, normalized to β-actin and expressed relative to theGFP-expressing control cells. Values are the means +− S.D. of three independent experiments(*P < 0.05, compared with control Ad-GFP-infected cells).

and protease inhibitors] on ice for 1 h. The supernatants wereincubated with the glutathione–Sepharose 4B beads carrying theGST or GST-fusion protein overnight at 4 ◦C. The beads werethen washed four times for 10 min each with the lysis bufferbefore Western blot analysis with an anti-HA antibody (Sigma),as described above.

Co-immunoprecipitation assays

Plasmids expressing HA-tagged SF-1 and Myc-tagged PGC-1αwere co-transfected into αT3-1 cells using LipofectamineTM 2000.Total cell extracts were prepared with immunoprecipitation lysisbuffer [10 mM Tris/HCl (pH 7.4), 250 mM NaCl, 1% NonidetP40, 1 mM EDTA, 1 mM PMSF and protease inhibitors] on ice for2 h, followed by pre-clearing with Protein G–SepharoseTM 4 FastFlow beads (GE Healthcare) at 4 ◦C for 2 h. Equal amounts of totalcell extracts were incubated with an anti-HA or IgG antibody at4 ◦C overnight prior to adding Protein G–SepharoseTM 4 Fast Flowbeads at 4 ◦C for 4 h. Immunoprecipitates were washed five timeswith PBS supplemented with freshly prepared protease inhibitors.Immunocomplexes were resolved by SDS/PAGE and transferredon to PVDF membranes. Western blot analysis was performedusing an anti-Myc antibody (Santa Cruz Biotechnology) asdescribed above.


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Figure 2 PGC-1α-dependent activation of LHβ transcription is mediated by SF-1

(A) A series of LHβ promoters fused to luciferase reporter constructs (0.25 μg), shown in (C) were co-transfected into αT3-1 cells with plasmids expressing PGC-1α (0.5 μg) or pcDNA3.1 (control).At 24 h later, cells were harvested, and the RLA (relative luciferase activity) was corrected for the Renilla luciferase activity and normalized to the control activity (*P < 0.05, compared with controlactivity of each reporter). (B) Alignment of the SF-1-binding element (GSE) of the LHβ promoter region demonstrating a highly conserved sequence among these species. Variant nucleotides areunderlined. (C) Schematic representation of the 5′-deletion/mutation series of the LHβ promoter from − 948 bp, − 160 bp and − 100 bp to +264 bp. The constructs denoted as − 948mutLucand − 160mutLuc contained a mutated GSE from − 127 bp to − 119 bp. The +1 represents the transcription start site of LHβ . (D) A series of reporter constructs (0.25 μg) was co-transfected intoαT3-1 cells with plasmid expressing SF-1 (0.5 μg) in the presence or absence of PGC-1α (0.5 μg). Transfections were normalized to the Renilla luciferase activity of pRL-TK (20 ng). The results areexpressed as fold increases over those in control cells. Values are means +− S.D. of at least three independent experiments (*P < 0.05, compared with control activity of each reporter). ctrl, control.

ELISA

ELISAs were performed to determine mouse LH and ALDlevels in the medium of αT3-1 and Y-1 cells respectively. Mediawere harvested 24 h after transfection, and LH and ALD weredetermined using the Mouse LH ELISA kit (Uscn) and MouseALD ELISA kit (Uscn) according to the manufacturer’s protocol.The concentrations were calculated by adopting the absorptionvalue into standard curves and normalizing to the amount of cellsin each condition. Results are shown as the means +− S.D. andrepresent at least three independent experiments.

Statistical analysis

Results are means +− S.D. of at least three independentexperiments. One-way ANOVA was applied to data followed

by a Student’s t test, and P < 0.05 was considered statisticallysignificant.

RESULTS

PGC-1α induces LHβ and αGSU gene expression in αT3-1 cells

To date, few studies have been performed to define the rolesof PGC-1α in endocrine organs, such as adrenal, gonads andpituitary. Since PGC-1α can co-activate a number of transcriptionfactors to stimulate downstream target genes, we hypothesizedthat PGC-1α may affect the expression of some key genes inthese tissues. To validate this hypothesis, αT3-1 cells derived frompituitary gonadotropes were used as a model system to examinethe effect of PGC-1α on the expression of LHβ, FSHβ andαGSU. αT3-1 cells were infected with Ad-PGC-1α or GFP (green



Figure 3 PGC-1α interacts with SF-1 in vivo and co-localizes with SF-1 inthe proximal region of the LHβ promoter containing a GSE

(A) ChIP assays were performed in αT3-1 cells. αT3-1 cells were cultured in 100-mmdishes. Cross-linked DNA fragments were immunoprecipitated with an anti-SF-1, anti-PGC-1αor IgG antibody (as a control). The LHβ promoter region harbouring the GSE site(− 243/ − 2 bp, proximal region) was amplified specifically by PCR. However, the distal region(− 1965/ − 1722 bp), having no GSE and used as a negative control, could not be amplified.(B) Two-hybrid assays in mammalian cells. pG5luc (0.25 μg) was co-transfected into αT3-1cells with various combinations of plasmids. The results were normalized to Renilla luciferaseactivity and are expressed as the fold-induction of luciferase units over the value obtained fromthe empty pACT and pBIND vectors (*P < 0.05, compared with the activity of co-transfectionof pACT and pBIND-PGC-1α). (C) Co-immunoprecipitation assays were performed in αT3-1cells. pcDNA3.1-PGC-1α–Myc was co-transfected into αT3-1 cells with pCMV-HA-SF-1, celllysates were immunoprecipitated with anti-HA or IgG antibody (as a control), andimmunoprecipitates were analysed by Western blot with an anti-Myc antibody. Input was usedas positive control. IB, immunoblot; IP, immunoprecipitation; RLA, relative luciferase activity.

fluorescent protein) as a negative control (Figure 1A). Total RNAwas extracted from these cells and quantified by real-time PCR.Indeed, overexpression of PGC-1α led to marked inductions ofLHβ and αGSU, and a modest increase in FSHβ (Figure 1B). Thisresult indicates that PGC-1α functions as an upstream activatorof these genes in αT3-1 cells.

PGC-1α-dependent activation of LHβ transcription is mediated bySF-1

To determine whether PGC-1α-dependent induction of LHβgene expression occurs at the transcriptional level, we performed

Figure 4 Two domains located in the C-terminus of SF-1 are crucial for itsinteraction with the N-terminal region of PGC-1α

(A) Schematic representation of key functional domains of PGC-1α. The LXXLL motif (aminoacids 142–146) is responsible for its interaction with certain hormone nuclear receptors. TheN-terminal 180 amino acids of PGC-1α were fused to GST. (B) Schematic diagram depictingpivotal domains within SF-1 and the structures of various truncated versions of SF-1. Thefull-length and truncated SF-1 were HA-tagged. (C) Mapping of the domain of SF-1 interactingwith PGC-1α. Fusion protein GST–PGC-1α (amino acids 1–180, right-hand panel) or GST(used as a control, middle panel) were bound to beads and incubated with cell extracts from 293ETcells overexpressing HA-tagged full-length or truncated SF-1. Lanes 1, 6 and 11, full-lengthSF-1; lanes 2, 7 and 12, amino acids 187–451 of SF-1; lanes 3, 8 and 13, amino acids 187–462of SF-1; lanes 4, 9 and 14, amino acids 246–451 of SF-1; and lanes 5, 10 and 15, amino acids246–462 of SF-1. The bound SF-1–HA was detected by Western blot analysis with an anti-HAantibody. GST–PGC-1α could interact with full-length HA–SF-1 or HA–SF-1-(187–462), butnot with HA–SF-1-(187–451) or HA–SF-1-(246–462), which suggest that both the proximalactivation domain and the AF-2 domain are crucial for the SF-1 interaction with PGC-1α. DBD,DNA-binding domain; LBD, ligand-binding domain.

promoter–reporter transfections in αT3-1 cells. The first construct(− 948Luc) contained the ∼1.2-kb promoter region of LHβ (from− 948 bp to +264 bp) governing the expression of luciferase.Co-transfection of − 948Luc with a PGC-1α-expressing vectorresulted in a marked activation of the reporter gene (Figure 2A).

To map the cis-acting region conferring the PGC-1α-dependentactivation of the luciferase reporter gene, we performed co-transfection experiments with reporter constructs harbouringserial deletions at the 5′-end of the LHβ promoter. Our resultsindicate that this activation still remained after truncation ofthe promoter region to begin at − 160 bp (Figure 2A). Todeduce the molecular mechanism, we searched for the sequencemotif in the PGC-1α-responsive nucleotide region. A screen of424 bp between − 160 bp and +264 bp revealed a clear GSE(TGACCTTGT), which is reported to be highly conserved inthe LHβ promoter from different species and mediates SF-1



activation of the LHβ gene (Figures 2B and 2C) [30–32]. Thefact that PGC-1α can interact directly with and co-activate anumber of nuclear receptors prompted us to speculate about theparticipation of SF-1 in PGC-1α-induced activation of LHβ genetranscription. To test this potential interaction, mutational studieswere performed. When GSE was mutated in a way that has beenshown previously to inactivate this element [15], we observedthat the PGC-1α co-activation of the LHβ reporter gene was lost(Figures 2A and 2D), suggesting that the GSE identified in theLHβ promoter is involved in PGC-1α action.

To further confirm the involvement of SF-1 in the PGC-1αco-activation of LHβ, a series of reporter gene transfectionexperiments was performed. In agreement with previous results[15,16], SF-1 activated the − 948Luc and − 160Luc reportergenes, which contained the wild-type SF-1-binding site (GSE),and co-transfection of SF-1 and PGC-1α led to a synergic effect onthese two reporter gene constructs (Figure 2D). However, a furthertruncated promoter reporter gene (− 100Luc) was not activatedby SF-1 in the absence or presence of PGC-1α. Moreover,mutations of the GSE in the LHβ promoter (− 948mutLuc and− 160mutLuc) abolished the responses to SF-1 and/or PGC-1α(Figures 2A and 2D). Thus these results indicate that SF-1 andPGC-1α activate the mouse LHβ gene promoter via a GSE.

PGC-1α interacts with SF-1 in vivo

The results described above imply that PGC-1α-dependentactivation of the LHβ promoter is mediated by GSE andpresumably requires direct interaction with SF-1. To furtherdemonstrate that the SF-1-binding site (GSE) may mediate thePGC-1α co-activation of the mouse LHβ promoter in vivo, ChIPassays were performed in αT3-1 cells. The immunoprecipitateswere subjected to PCR using a pair of primers designed to amplifythe proximal region of the mouse LHβ promoter (from − 243 bpto − 2 bp) containing the GSE. As shown in Figure 3(A), theproximal region of the LHβ promoter could be amplified fromprecipitates obtained using anti-SF-1 or anti-PGC-1α antibodies;however, no signal was observed in the immunocomplex withIgG. As the negative control, the distal promoter region withoutGSE could not be amplified from any of the immunoprecipitates.Taken together, these results suggest that endogenous SF-1 andPGC-1α occupy the promoter site of mouse LHβ. Consideringthat PGC-1α cannot bind DNA directly, the association of PGC-1α with this promoter occurs via binding to SF-1.

To confirm the interaction between PGC-1α and SF-1 in intactcells, we first performed mammalian two-hybrid assays in αT3-1cells. For this, full-length SF-1 was fused to the transactivationdomain of VP16 in pACT (pACT-SF-1), and full-length PGC-1αwas fused to the GAL4 DNA-binding domain in pBIND (pBIND-PGC-1α). The two vectors were co-expressed with the pG5lucreporter plasmid containing five GAL4-binding sites. The co-expression of both fusion proteins yielded an induction ofpromoter activity much stronger than that obtained by co-expressing pACT-SF-1 with pBIND or pBIND-PGC-1α withpACT (Figure 3B). This result indicates a direct interactionbetween PGC-1α and SF-1.

In addition, co-immunoprecipitation assays were performed toexamine the interaction between PGC-1α and SF-1 in vivo. Aplasmid encoding Myc-tagged PGC-1α was co-transfected intoαT3-1 cells with HA-tagged SF-1. Potential protein complexeswere immunoprecipitated using an anti-HA or IgG antibody andsubjected to Western blot using an anti-Myc antibody (Figure 3C).PGC-1α was efficiently precipitated using an anti-HA antibody,

Figure 5 PGC-1α induces the expression of genes involved in steroido-genesis in Y-1 cells

(A) Y-1 cells were infected with adenoviruses expressing either GFP (Ad-GFP, as a control)or PGC-1α (Ad-PGC-1α), proteins were extracted, and Western blot analysis was performedusing antibodies specific to PGC-1α and β-actin. (B) Y-1 cells were treated as described in (A),and total RNA was extracted 48 h after infection. The mRNA levels of Cyp11b1, Cyp11b2 andP450scc were determined by real-time PCR, normalized to β-actin and expressed relative to theGFP-expressing control cells. Values are the means +− S.D. of three independent experiments(*P < 0.05, compared with control Ad-GFP-infected cells). (C) ChIP assays were performedin Y-1 cells. Y-1 cells were cultured in 100-mm dishes, and cross-linked DNA fragments wereimmunoprecipitated with an anti-SF-1, anti-PGC-1α or IgG antibody (as a control). Cyp11b1,Cyp11b2 and P450scc promoter regions harbouring GSEs were amplified specifically by PCR.However, the distal regions without GSEs, used as negative controls, could not be amplified.

whereas it was not detectable with control IgG. These resultsclearly indicate that PGC-1α interacts with SF-1 in vivo.

Interaction between PGC-1α and SF-1 involves the N-terminalregion of PGC-1α and two regions of SF-1

The activation domain of PGC-1α, containing the LXXLL motif(amino acids 142–146), is responsible for interaction with anumber of hormone nuclear receptors [5,6,33,34]. In addition,the AF-2 hexamer (LLIEML) at the C-terminus of SF-1, whichis conserved in several nuclear receptors, and the proximalactivation domain (amino acids 187–245) are required for theSF-1 transcriptional activity [22]. We hypothesized that thesedomains mediate the direct interaction between PGC-1α and SF-1.Thus GST pull-down assays were performed. First we obtained



Figure 6 PGC-1α synergizes with SF-1 to promote the synthesis of LH and ALD in αT3-1 cells and Y-1 cells respectively

(A) αT3-1 cells were transfected with plasmids expressing SF-1 and/or PGC-1α. At 48 h later, total RNA was extracted, and LHβ mRNA was measured by real-time PCR, normalized toβ-actin and expressed relative to the control cells transfected with pcDNA3.1. (B) Y-1 cells were treated as described in (A). Total RNA was isolated, and Cyp11b2 mRNA levels were analysed.Values are expressed as the fold-induction over control. Results are the average of three independent experiments. (C) αT3-1 cells were treated as described in (A), and LH secreted into themedium was assessed by ELISA. Results are expressed as μIU of LH/105 cells. (D) Y-1 cells were treated as described in (B), and ALD secreted into the medium was determined by ELISA.Results are expressed as ng of ALD/105 cells. Values are the means +− S.D. of three independent experiments (*P < 0.05, compared with control; **P < 0.01, compared with SF-1-transfectedcells).

a bacterial GST-fusion protein with the N-terminal 180 aminoacids of PGC-1α [GST–PGC-1α-(1–180)] (Figure 4A). SF-1 wasproduced either as a full-length protein or as truncated versions(Figure 4B), and all of them were HA-tagged. As shown inFigure 4(C) (right-hand panel), full-length SF-1 exhibited strongbinding to GST–PGC-1α-(1–180), but no binding to GST (middlepanel), suggesting that PGC-1α interacts with SF-1 through aminoacids 1–180 of PGC-1α. Meanwhile, we observed that PGC-1αstill bound to SF-1 with a deleted DNA-binding domain. However,a further deletion of the proximal activation domain or AF-2domain of SF-1 essentially eliminated its interaction with PGC-1α. As a control, no binding was observed between variousversions of SF-1 and GST alone. Our data suggest that PGC-1α interacts directly with SF-1 in vitro and that this interactioninvolves the N-terminal region of PGC-1α (amino acids1–180) and at least two regions of SF-1, the proximal activationdomain and the AF-2 domain.

PGC-1α induces genes involved in steroidogenesis inadrenal-cortex-derived Y-1 cells

Since SF-1 is also highly expressed in adrenal cortex andour results indicate that PGC-1α interacts directly with andco-activates SF-1 to induce LHβ gene expression, we nextdetermined whether PGC-1α stimulates target genes of SF-1in adrenal-cortex-derived cells. Y-1 cells were used as a modelsystem and infected with Ad-PGC-1α or GFP as a negativecontrol (Figure 5A). Total RNA was extracted from these cells

and estimated by real-time PCR. As anticipated, we observed thatoverexpression of PGC-1α led to an increase in the expressionof several SF-1 target genes, including Cyp11b2, Cyp11b1and P450scc (Figure 5B). A previous study showed that thereis a GSE from − 67 to − 55 bp in the P450scc promoter that ishighly conserved among different species, including bovine, rat,mouse and humans [35], and a proposed GSE from − 440 bpto − 219 bp in the Cyp11b2 promoter was also identified [20].In addition, there is a putative SF-1-binding site in the Cyp11b1promoter between − 265 and − 52 bp [20,36]. A ChIP assay wasperformed in Y-1 cells, and we found that PGC-1α was recruitedto the proximal regions of these genes containing GSEs in Y-1cells (Figure 5C).

PGC-1α synergizes with SF-1 to promote the synthesis of LH andALD

Since PGC-1α interacted with SF-1 and stimulated LHβ andCyp11b2 gene transcription, we decided to determine whetherthese two factors could synergize to influence downstream targetgenes. αT3-1 and Y-1 cells were transfected with PGC-1α and/orSF-1 expression plasmids. Total RNA was extracted, and LHβand Cyp11b2 were measured by real-time PCR. As shown inFigure 6(A), overexpression of PGC-1α or SF-1 alone in αT3-1cells led to a modest increase in LHβ gene expression; however,overexpression of PGC-1α and SF-1 dramatically induced LHβ.Additionally, we observed a similar effect of PGC-1α and SF-1 onCyp11b2 gene expression in Y-1 cells (Figure 6B). These results



Figure 7 Knockdown of endogenous SF-1 abolishes the PGC-1α induction of LHβ and Cyp11b2 gene expression

(A) αT3-1 cells and (B) Y-1 cells were transfected with pBabe-Dual/si-scramble (as a control) or pBabe-Dual/si-SF-1. At 72 h later, total RNA was extracted and SF-1 mRNA was determined by real-timePCR and normalized to β-actin. Ctrl, untransfected cells (used as a control) (*P < 0.05, compared with si-scramble). (C) αT3-1 cells and (D) Y-1 cells were transfected with pBabe-Dual/si-scrambleor pBabe-Dual/si-SF-1 in the presence of plasmids expressing PGC-1α or pcDNA3.1 (as a control). AT 72 h later, total RNA was extracted, and LHβ and Cyp11b2 mRNA were determined byreal-time PCR and normalized to β-actin. Values are the means +− S.D. of three independent experiments (*P < 0.05, compared with control pcDNA3.1-transfected cells in each RNAi condition).

indicate that PGC-1α and SF-1 have synergic effects on thesetarget genes.

Our ELISA results show that overexpression of PGC-1α andSF-1 led to a markedly increased level of LH in the medium ofαT3-1 cells, much more than that obtained by transfectionof PGC-1α or SF-1 alone (Figure 6C). Additionally,overexpression of PGC-1α or SF-1 in Y-1 cells induced thesynthesis of ALD, and co-transfection of these factors furtherincreased the ALD level in the medium (Figure 6D).

To determine the role of SF-1 in the PGC-1α co-activationof LHβ and Cyp11b2, siRNA against SF-1 was employed toknockdown endogenous SF-1 gene expression in αT3-1 cellsand Y-1 cells (Figures 7A and 7B). Knockdown of SF-1 bysiRNA abolished the PGC-1α induction of LHβ and Cyp11b2gene expression (Figures 7C and 7D), which suggests thatSF-1 mediates the PGC-1α action on target genes LHβ andCyp11b2.

PGC-1α induces SF-1 gene expression

Several transcription factors, including NRF-1 and ERRα, co-activated by PGC-1α can also be induced by PGC-1α at themRNA level [8,37]. Next, to study whether PGC-1α can increaseSF-1 gene expression, quantitative real-time PCR assays wereperformed in αT3-1 and Y-1 cells infected with Ad-PGC-1αor GFP as a control. We observed that the overexpression ofPGC-1α powerfully stimulated SF-1 gene expression in bothαT3-1 and Y-1 cells (Figures 8A and 8B). Our Western blotresults also confirmed that PGC-1α induced the expression of

SF-1 at the protein level in these two cell lines (Figures 8C and8D). To determine whether PGC-1α-dependent induction of SF-1gene expression occurs at the transcriptional level, we performedpromoter–reporter transfections in 293ET cells. Overexpressionof PGC-1α caused a modest activation of an SF-1 promoterluciferase gene (Figure 8E). Inspecting the sequence of the5′-flanking region of mouse SF-1 (− 1892 to +1 bp), severalpotential transcription-factor-binding elements were identified,such as those recognized by HNF4 (hepatocyte nuclear factor4), SOX9 [SRY (sex-determining region Y)-box 9], PPARsand ERRs. Thus we tested these candidate transcription factorsmediating PGC-1α action on SF-1 transcription. As shown inFigure 8(F), transfection of ERRγ increased SF-1 gene promoteractivity (2.0 +− 0.7-fold), and overexpression of ERRα alone didnot change the luciferase activity. However, co-transfection ofPGC-1α and ERRα or ERRγ led to the dramatic activation of SF-1 promoter activity. Meanwhile, the other factors did not influenceSF-1 promoter activity (results not shown). In addition, increasingamounts of PGC-1α expression plasmids were co-transfected into293ET cells with ERRα, and a dose-dependent activation of theSF-1 promoter was observed (Figure 8G). These results indicatethat PGC-1α can co-activate ERRα to activate SF-1 promoteractivity.

These data allow us to propose a ‘dual-regulation model’mechanism of PGC-1α action on the production of hormones,including LH and FSH, in pituitary-derived cells andsteroidogenesis in adrenal-cortex-derived cells (Figure 9). In thismodel, PGC-1α co-activates ERRs or other factors to induce SF-1gene expression; meanwhile, PGC-1α interacts directly with andco-activates SF-1 to stimulate SF-1 downstream target genes,



Figure 8 PGC-1α stimulates SF-1 gene expression

(A) αT3-1 cells were infected with adenoviruses expressing PGC-1α or GFP. At 48 h later,total RNA was isolated. SF-1 mRNA was measured by real-time PCR and normalized toβ-actin (*P < 0.05, compared with control Ad-GFP-infected cells). (B) Y-1 cells were treatedas described in (A). Values are expressed as the fold-induction of SF-1 mRNA in cells infectedwith PGC-1α compared with GFP (*P < 0.05, compared with control Ad-GFP-infected cells).(C) αT3-1 cells and (D) Y-1 cells were treated as described in (A). Total cell lysate (60 μg) wassubjected to Western blot analysis with anti-PGC-1α, anti-SF-1 and anti-β-actin antibodies.(E) Mouse SF-1 promoter luciferase fusion constructs (0.25 μg) were transfected into 293ETcells with plasmids expressing PGC-1α (0.5 μg) or pcDNA3.1 (control). At 24 h later, fireflyluciferase levels were determined and normalized to Renilla luciferase activity (*P < 0.05). (F)SF-1 promoter luciferase fusion constructs (0.25 μg) were co-transfected into 293ET cellswith a plasmid expressing ERRα (0.5 μg) or ERRγ (0.5 μg) in the presence or the absence ofPGC-1α for 24 h. Results are expressed as normalized levels over control (*P < 0.05, comparedwith ERRα- or ERRγ -transfected cells respectively; **P < 0.01, compared with control). (G)The activation of SF-1 promoter activity is PGC-1α dose-dependent. Increasing amounts(0.08 μg, 0.2 μg, 0.4 μg and 0.6 μg) of PGC-1α-expression plasmids were co-transfectedinto 293ET cells with SF-1 promoter reporter gene (0.2 μg) in the presence of ERRα expressionplasmid (0.4 μg). Firefly luciferase levels were measured and normalized to Renilla luciferaseactivities. Values are the means +− S.D. of three independent experiments (*P < 0.05, comparedwith control cells transfected with ERRα alone). ctrl, control.

such as αGSU and LHβ in αT3-1 cells and Cyp11b2 in Y-1cells, subsequently leading to the increased synthesis of LH andALD.

DISCUSSION

To date, PGC-1α has been extensively studied in various tissues,including brown adipose tissue, skeletal muscle, heart, brain andliver. However, the physiological function of PGC-1α is lessknown in other tissues. We decided to determine its role in cells ofendocrine tissues, such as adrenal, gonads and pituitary. Thus we

Figure 9 Proposed model of PGC-1α regulation of SF-1 gene expressionand transcriptional activity

PGC-1α co-activates ERRs or other factors to induce SF-1 gene expression; meanwhile, PGC-1αinteracts directly with and co-activates SF-1 to influence its downstream target genes, such asαGSU, LHβ and Cyp11b2, subsequently leading to increased synthesis of LH in αT3-1 cellsand ALD in Y-1 cells respectively.

first performed studies using αT3-1 cells derived from pituitarygonadotropes as a model system. We examined mRNA levels ofseveral genes, including LHβ, FSHβ and αGSU, all of whichencode secreted hormones affecting reproductive developmentand function. We observed that LHβ and αGSU were significantlyinduced by PGC-1α.

We speculated that PGC-1α may co-activate one or moretranscription factors to activate LHβ and αGSU gene expression.Several transcription-factor-binding sites exist in the LHβpromoter, including Egr1 (early growth response protein1), SF-1, Pitx1 (pituitary homeobox 1) and NF-Y (nuclearfactor-Y) sites [38]. In addition, USF (upstream stimulatoryfactor), CREB (CRE-binding protein), GATA2 (GATA-bindingprotein 2), SF-1, LH-2 and Mst-1 (macrophage stimulating 1)can bind to and activate the αGSU promoter [14]. The mutationof the SF-1-binding element abolished PGC-1α-activating LHβ,suggesting that SF-1 mediates PGC-1α-dependent inductionof LHβ gene expression. Furthermore, the synergy betweenPGC-1α and SF-1 was demonstrated in SF-1-deficient CV-1 cells (Supplementary Figure S1 at http://www.BiochemJ.org/bj/432/bj4320473add.htm).

The data in the present study provide substantial evidence thatPGC-1α can interact directly with SF-1. First, overexpressionof PGC-1α or SF-1 alone led to activation of the LHβ promoter,whereas co-transfection of PGC-1α and SF-1 caused a synergiceffect. Secondly, mammalian two-hybrid assays indicated thatactivation of the pG5luc reporter gene by co-transfection ofPGC-1α–BIND and SF-1–ACT was much stronger than PGC-1α–BIND or SF-1–ACT alone. Thirdly, co-immunoprecipitationassays indicated that PGC-1α interacts with SF-1 in vivo. Fourthly,GST pull-down assays revealed a direct interaction between PGC-1α and SF-1.

As a co-activator, PGC-1α is recruited to particular DNAsequences in gene promoters through direct interaction withtranscription factors. Embedded in the N-terminal 200 aminoacids of PGC-1α is an LXXLL sequence (amino acids 142–146),which is absolutely required for the ligand-dependent interactionwith ER (oestrogen receptor) [34], PPARα [6], RXRα (retinoidX receptor α) [39] and GR (glucocorticoid receptor) [40]. Ourresults clearly demonstrate that PGC-1α binds intensively to SF-1through the N-terminal region of PGC-1α containing this LXXLL





motif (amino acids 1–180). GST pull-down assays confirmed thatSF-1 interacts with PGC-1α through its C-terminus (amino acids187–462). Deletion of the SF-1 proximal activation domain or AF-2 domain abolished this interaction, suggesting that both domainsare required for the SF-1 interaction with PGC-1α.

Although a number of SF-1 target genes are induced by PGC-1αin Y-1 cells, we observed that several target genes are not influ-enced, including Cyp19a1 and Cyp21a1 (results not shown). Astriking feature of co-activation by PGC-1α is that it is promoter-selective.

The results of the present study clearly demonstrate thatoverexpression of PGC-1α and SF-1 leads to an increase inCyp11b2 transcription and subsequently promotes the synthesisof ALD in Y-1 cells. A previous study showed that Cyp11b2transcription is stimulated by angiotensin II, potassium ionsand cAMP signalling pathways using the SF-1 and CRE-like cis-elements [20], so PGC-1α may induce Cyp11b2 geneexpression through co-activating SF-1. In addition, recent studiessuggest that ALD contributes to blood pressure homoeostasis[41]. Overproduction of ALD causes cardiovascular disease.Since PGC-1α co-activates SF-1 to stimulate ALD productionin Y-1 cells, PGC-1α may be involved in cardiovascularpathophysiology.

PGC-1α interacts directly with ERRα [4]. Additionally, PGC-1α can induce ERRα gene expression [37,42]. Similar phenomenaoccur with PGC-1α and NRF-1 [5,8]. In the present study,we showed that PGC-1α not only interacts directly with SF-1,but also induces SF-1 gene expression in gonadotrope-derivedαT3-1 cells and adrenocortical Y-1 cells. Previous reports haveshown that SOX9 activates the SF-1 promoter [43] and PGC-1α can co-activate SOX9 to regulate chondrogenesis [44], so wehypothesized that PGC-1α co-activates SOX9 to activate the SF-1promoter; however, we did not observe this.

We did identify a potential ERR-binding element (− 227to − 219 bp) in the mouse SF-1 promoter. Our data suggestthat PGC-1α co-activates ERRα or ERRγ to activate SF-1promoter activity, implying that ERRs may mediate PGC-1αaction on SF-1 gene expression. However, we cannot rule outthe possibility that other factors also mediate PGC-1α action onSF-1 gene expression. Further studies are required to determinewhether ERRs binds to the SF-1 gene promoter and act as directtranscription factors for SF-1.

When our present manuscript was in preparation, Yazawaet al. [45] defined the role of PGC-1α in the differentiationof UCB (umbilical cord)-derived MSCs (mesenchymal stemcells) and demonstrated that PGC-1α regulates progesteroneproduction in granulose cells through SF-1 and LRH-1 (liverreceptor homologue 1). In the present study, we showed thatPGC-1α regulates LH and ALD production in αT3-1 cells and Y-1cells respectively through SF-1 and we provided more convincingevidence to demonstrate that PGC-1α interacts with SF-1 in vivoand in vitro, including promoter–luciferase reporter gene assays,mammalian two-hybrid, co-immunoprecipitation and ChIP assaysand GST pull-down experiments. We also demonstrated that SF-1mediates PGC-1α regulation of LH and ALD production in αT3-1cells and Y-1 cells respectively by SF-1-knockdown experiments.

In summary, in the present study, we have shown that PGC-1α can interact with and co-activate SF-1 to induce LHβand αGSU gene expression, leading to increased secretion ofLH in pituitary gonadotrope-derived αT3-1 cells. PGC-1α alsostimulates Cyp11b2 expression and ALD synthesis in adrenal-cortex-derived Y-1 cells. SF-1 is a key regulator of endocrinefunction within the hypothalamic–pituitary–gonadal reproductiveaxis and adrenal cortex. It is reasonable to propose that PGC-1αmay play important roles in steroidogenesis, gonad development

and sex differentiation in these tissues through its interaction withand co-activation of SF-1.

AUTHOR CONTRIBUTION

Liuluan Zhu and Yongshen Chang designed the experiments and prepared the manuscript.Liuluan Zhu performed the experiments with the assistance of Yaojun Ke, Di Shao, Ying Cuiand Aijun Qiao. Liuluan Zhu and Xiaojun Liu analysed the data. Fude Fang and YongshenChang supervised the project.

ACKNOWLEDGEMENTS

We thank Dr Teresa C. Leone (Center for Cardiovascular Research, Washington UniversitySchool of Medicine, St Louis, MO, U.S.A.) for providing us with the PGC-1α–Mycexpression plasmid, Dr Ye Zhang (Institute of Basic Medical Sciences, Chinese Academyof Medical Sciences and Peking Union Medical College, Beijing, China) for the pBabe-Dual plasmid, and Dr Bruce Spiegelman (Dana Farber Cancer Institute, Harvard MedicalSchool, Boston, MA, U.S.A.) for the pGEX-PGC-1α plasmid.

FUNDING

This work was supported by the Major State Basic Research Development Program of China[973 Program grant number 2011CB504004]; the National High Technology Researchand Development Program of China [863 Program, grant numbers 2006AA02A409,2006AA02Z192); the National Natural Science Foundation of China [grant numbers30700386, 30721063, 30800389, 90919019]; The Project for Extramural Scientists ofState Key Laboratory of Agrobiotechnology [grant number 2010SKLAB07–11]; and theNatural Science Foundation of Beijing [grant number 7082056].

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Received 30 March 2010/27 September 2010; accepted 5 October 2010Published on the Internet 25 November 2010, doi:10.1042/BJ20100460


Biochem. J. (2010) 432, 473–483 (Printed in Great Britain) doi:10.1042/BJ20100460

SUPPLEMENTARY ONLINE DATAPPARγ co-activator-1α co-activates steroidogenic factor 1 to stimulate thesynthesis of luteinizing hormone and aldosteroneLiuluan ZHU*†1, Yaojun KE‡1, Di SHAO*, Ying CUI*, Aijun QIAO*, Xiaojun LIU*, Fude FANG*2 and Yongsheng CHANG*2

*National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100005,China, †Institute of Infectious Diseases, Beijing Ditan Hospital, Capital Medical University, Beijing 100015, China, and ‡Department of Interventional Radiology, Tianyou Hospital,Wuhan University of Science and Technology, Wuhan 430064, China

Figure S1 PGC-1α synergizes SF-1 to activate LHβ transcription in SF-1-deficient CV-1 cells

(A) CV-1 cells were transfected with plasmids expressing SF-1, PGC-1α (0.5 μg) or pcDNA3.1 (control). At 48 h later, proteins were extracted and Western blot analysis was performedusing anti-SF-1 and β-actin. (B) Mouse LHβ promoter luciferase constructs (0.25 μg) weretransfected into CV-1 cells with plasmids expressing SF-1, PGC-1α (0.5 μg) or pcDNA 3.1(control). At 24 h later, firefly luciferase levels were determined and normalized to Renilla.Results are expressed as normalized levels over control. ctrl, control; RLA, relative luciferaseactivity.

1 These authors contributed equally to this work.2 Correspondence may be addressed to either of these authors (email [email protected] or [email protected]).


L. Zhu and others

Table S1 Primer sequences used in plasmid construction

Gene Forward/reverse Sequence Vector

SF-1 Forward 5′-CACTGGAATTCCGGGCATGGACTATTCGTAC-3′ pCMV-HAReverse 5′-GTGCAAGATCTCTCAAGTCTGCTTGGCCTGC-3′

SF-1-(187–451) Forward 5′-CTGGAATTCAGACCATGGCCTTCTCTAACCGCACCATC-3′ pCMV-HAReverse 5′-TGCATAGATCTTCAGTTGTTGCGGGGCATCTCG-3′

SF-1-(187–462) Forward 5′-CTGGAATTCAGACCATGGCCTTCTCTAACCGCACCATC-3′ pCMV-HAReverse 5′-TGCATAGATCTTCAAGTCTGCTTGGCCTGCAG-3′

SF-1-(246–451) Forward 5′-CTGGAATTCAGACCATGGTGGGCTGTCTGCAGGAGC-3′ pCMV-HAReverse 5′-TGCATAGATCTTCAGTTGTTGCGGGGCATCTCG-3′

SF-1-(246–462) Forward 5′-CTGGAATTCAGACCATGGTGGGCTGTCTGCAGGAGC-3′ pCMV-HAReverse 5′-TGCATAGATCTTCAAGTCTGCTTGGCCTGCAG-3′

SF-1 Forward 5′-TCACGTACGGATCCCGGGCATGGACTATTCGTAC-3′ pACTReverse 5′-TGAGTAGCTCTAGATCAAGTCTGCTTGGCCTGC-3′

PGC-1α Forward 5′-TACGAGTCGACGGATGGCTTGGGACATGTGCAG-3′ pBINDReverse 5′-ATCAGGATATCCCTGCGCAAGCTTCTCTGAGC-3′

ERRγ Forward 5′-AATCCAGAATTCGGGATTCGGTAGAACTTTGCCTT-3′ pCMV-HAReverse 5′-TAGCAACACCTCGAGTCAGACCTTGGCCTCCAACAT-3′

SF-1 promoter Forward 5′-AGCTGACTGAGCTCTCCAAGAAGGGGTACCTCTC-3′ pGL3-basicReverse 5′-TCAGCTGTCCCGGGGAAGGAGAATTCTCAGCCGG-3′

LHβ promoter ( − 948Luc) Forward 5′-CATACGCGTTGAACTCTGCTGCACTTGGA-3′ pGL3-basicReverse 5′-GCTCAGATCTATAGCCAGTGTCTCACTCTACCC-3′

LHβ promoter ( − 160Luc) Forward 5′-CATACGCGTCACTGGGACACTGGAGCTAGTCC-3′ pGL3-basicReverse 5′-GCTCAGATCTATAGCCAGTGTCTCACTCTACCC-3′

LHβ promoter ( − 100Luc) Forward 5′-CATACGCGTGAGATTAGTGTCTAGGTTACCCAAGCC-3′ pGL3-basicReverse 5′-GCTCAGATCTATAGCCAGTGTCTCACTCTACCC-3′

LHβ promoter ( − 948mutLuc) Forward 5′-CATACGCGTTGAACTCTGCTGCACTTGGA-3′ pGL3-basicReverse 5′-GAGACACAGACCCTTTCAGGGAAGCC-3′

Forward 5′-GGCTTCCCTGAAAGGGTCTGTGTCTC-3′

Reverse 5′-GCTCAGATCTATAGCCAGTGTCTCACTCTACCC-3′

LHβ promoter ( − 160mutLuc) Forward 5′-CATACGCGTCACTGGGACACTGGAGCTAGTCC-3′ pGL3-basicReverse 5′-GAGACACAGACCCTTTCAGGGAAGCC-3′

Forward 5′-GGCTTCCCTGAAAGGGTCTGTGTCTC-3′

Reverse 5′-GCTCAGATCTATAGCCAGTGTCTCACTCTACCC-3′

si-scramble Forward 5′-AAAGGGCAAACCTAAACGATACTAA-3′ pBabe-DualReverse 5′-AAAATTAGTATCGTTTAGGTTTGCC-3′

si-SF-1 (321/341) Forward 5′-AAAGAGTCCAGAACAACAAGCATTA-3′ pBabe-DualReverse 5′-AAAATAATGCTTGTTGTTCTGGACT-3′


PGC-1α interacts with and co-activates SF-1

Table S2 Primer sequences used in ChIP assays

Promoter Forward/reverse Sequence Position (bp)

LHβ Forward 5′-TGCAGTGGCCTCCCCTTTAC-3′ − 243/ − 2Reverse 5′-TTCCCTACCTTGGGCACCTG-3′

LHβ Forward 5′-CTAAGAAGACATGAGGGACGG-3′ − 1965/ − 1722Reverse 5′-CCTCCCTAACCTAGACAGCC-3′

Cyp11b1 Forward 5′-GAGGAGAAAGAAGGCTCAAAC-3′ − 265/ − 52Reverse 5′-GCTGACTGATAATGTCACTGGAG-3′

Cyp11b1 Forward 5′-AGTGGGAATGGGAAATAGCA-3′ − 1305/ − 1087Reverse 5′-ATTCAAAGCTCCACCAGAAG-3′

Cyp11b2 Forward 5′-AACTGAATTCAAGCTTGTTTGG-3′ − 440/ − 219Reverse 5′-TTGGGTTGCCTGAGGTAGAG-3′

Cyp11b2 Forward 5′-GAGGTCTGCACCCAGCTGG-3′ − 1083/ − 837Reverse 5′-CCACTGCATGCCACTCTTCC-3′

P450scc Forward 5′-AAGTTCAGAAGTTCTTTCTCTGAG-3′ − 164/+37Reverse 5′-TCCTTTAGCCAGCATACTGTC-3′

P450scc Forward 5′-GTGGTGAATGCATATAATCCC-3′ − 1008/ − 815Reverse 5′-CTTACAGATAGTTGAGCTGCC-3′

Table S3 Primer sequences used in real-time PCR

Gene Forward/reverse Sequence Nucleotides

SF-1 Forward 5′-ACGAAGGTGCATGGTCTTTA-3′ 843/969Reverse 5′-GCTGTCTTCCTTGCCGTACT-3′

Cyp11b1 Forward 5′-TAGAGAGTACGCACCCTTGC-3′ 314/437Reverse 5′-AGCTGCAGTCGGTTGAAGTA-3′

Cyp11b2 Forward 5′-ATGCTGAGAAGTTGCACCAG-3′ 293/415Reverse 5′-ATTCTGGCCCATTTAGCAAG-3′

P450scc Forward 5′-CCTGGAAGAAAGACCGAATC-3′ 428/554Reverse 5′-TGCTTGATGCGTCTGTGTAA-3′

αGSU Forward 5′-GTATGGGCTGTTGCTTCTCC-3′ 167/290Reverse 5′-GTGGCCTTAGTAAATGCTTTGG-3′

LHβ Forward 5′-CTGAGCCCAAGTGTGGTGT-3′ 37/196Reverse 5′-GCAGTACTCGGACCATGCTA-3′

FSHβ Forward 5′-CTGACCAACATCACCATTGC-3′ 67/163Reverse 5′-ACCAGATCCCTGGTGTAGCA-3′

β-Actin Forward 5′-GACATGGAGAAGATCTGGCA-3′ 241/378Reverse 5′-GGTCTCAAACATGATCTGGGT-3′

Received 30 March 2010/27 September 2010; accepted 5 October 2010Published on the Internet 25 November 2010, doi:10.1042/BJ20100460


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PPARγ co-activator-1α co-activates steroidogenic factor 1 to stimulate the synthesis of luteinizing hormone and aldosterone