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Endocrinology, doi:10.1210/en.2003-1343
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Endocrinology Vol. 145, No. 5 2307-2318
Copyright © 2004 by The Endocrine Society

Cloning and Characterization of Granulosa Cell High-Mobility Group (HMG)-Box Protein-1, a Novel HMG-Box Transcriptional Regulator Strongly Expressed in Rat Ovarian Granulosa Cells

Takashi Kajitani, Tetsuya Mizutani, Kazuya Yamada, Takashi Yazawa, Toshio Sekiguchi, Miki Yoshino, Hiroko Kawata and Kaoru Miyamoto

Department of Biochemistry (T.K., T.M., K.Y., T.Y., T.S., M.Y., K.M.), Fukui Medical University, Matsuoka, Fukui, 910-1193, Japan; and Core Research for Evolutional Science and Technology (T.K., T.M., K.Y., T.Y., T.S., M.Y., H.K., K.M.), Japan Science and Technology Corporation, Kawaguchi, Saitama 332-0012, Japan

Address all correspondence and requests for reprints to: Kaoru Miyamoto, Department of Biochemistry, Fukui Medical University, Shimoaizuki, Matsuoka, Fukui 910-1193, Japan. E-mail: kmiyamot{at}fmsrsa.fukui-med.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Specific events in the ovary are dependent on gene expression in the tissue. By screening a rat ovarian granulosa cell cDNA library, a cDNA clone encoding a novel transcription factor-like protein containing a high-mobility group-box, referred to as granulosa cell high-mobility group-box protein-1 (GCX-1), was identified. The expression of GCX-1 is restricted to the hypothalamus, pituitary, testis, uterus, and ovary but was not detected in the adrenal gland. An in situ hybridization study revealed that the expression of GCX-1 was restricted to granulosa cell layers in early-stage follicles, and the expression was very low in large antral follicles and the corpus luteum, but localized expression in the testis or pituitary was not clear. Endogenous GCX-1 protein in the granulosa cells was identified by a Western blot analysis, and an analysis using the green fluorescence protein-GCX-1 fusion protein revealed that the GCX-1 protein was localized in the cell nucleus. GAL4 fusion protein-based assays demonstrated that GCX-1 is a potent transcriptional activator, and its putative transactivation domain was mapped to the region between amino acid residues 25 and 63 from the N terminus. These data strongly suggest that GCX-1 is likely a novel transcriptional activator that is exclusively expressed in reproductive tissues involving the hypothalamo-pituitary-gonadal axis, and functions as a specific regulator of follicular development, and may also participate in other specific events related to reproduction, particularly in the female.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SPECIFIC EVENTS IN the ovary, namely folliculogenesis, ovulation, or steroidogenesis, are strictly dependent on gene expression in the related tissues. Many studies have been carried out with the goal of establishing relationships between functional events and gene expression in the ovary (1, 2, 3, 4, 5). By focusing on the molecular mechanisms of follicular development, we have been able to identify a number of ovarian genes that are induced by gonadotropins (6, 7, 8, 9, 10). We recently reported on a novel transcriptional repressor that is strongly induced in the rat ovary by gonadotropin treatment, and designated it as gonadotropin-inducible ovarian transcription factor 1 (GIOT1) (11). In subsequent studies, we attempted to identify proteins that interact with GIOT1 in the ovary to reveal physiological functions of GIOT1, and reported that a transcriptional coregulator, transcriptional intermediary factor 1ß (12) and a transcriptional regulator, rat homologs of human I-mfa domain containing protein (13), interact with GIOT1. Simultaneously, from a rat ovarian granulosa cell cDNA library, we isolated a novel clone encoding a transcription factor-like protein, which contains a high-mobility group (HMG)-box domain. In this report, we report on the isolation, functional characterization, and tissue specificity of this novel gene, which is designated granulosa cell HMG-box protein-1 (GCX-1). Our findings show that GCX-1 gene expression occurred exclusively in reproductive tissues including the hypothalamo-pituitary-gonadal axis.

Transcription factors that are known to be expressed in gonadal tissues include adrenal 4 binding protein/steroidogenic factor-1 (Ad4BP/SF-1) and the dosage-sensitive sex reversal-adrenal hypoplasia congenita-critical region on the X chromosome-1 (DAX-1) (14, 15, 16). Ad4BP/SF-1 regulates many genes that are related to steroidogenesis, including steroidogenic enzymes, steroidogenic acute regulatory protein (StAR) (14) or GIOT1 (17). Ad4BP/SF-1 also plays important roles in the development of gonadal systems during embryogenesis. DAX-1 also participates in tissue development through antagonizing against the functions of Ad4BP/SF-1. However, these factors are also expressed in the adrenal gland and participate in its development and therefore are not specific to reproductive organs. On the other hand, unlike Ad4BP/SF-1 and DAX-1, GCX-1 is specific to reproductive tissues and may play significant roles in the hypothalamo-pituitary-gonadal axis.

GCX-1 contains an HMG-box motif. The HMG-box, a DNA-binding motif, is widely distributed throughout eukaryotic organisms from yeast to mammals (18, 19), and a number of HMG-box proteins are known to play important roles in various physiological events: for example, the upstream binding factor (UBF) (20, 21) as a rRNA transcriptional regulator, the sex determining region Y (SRY) (22, 23) as a protein essential for sex determination, the T cell factor (TCF) (24) and the thymus HMG-box (TOX) (25) as T cell differentiation regulators. Moreover, recent studies have reported that some HMG-box proteins function not as transcription regulators but rather as intercellular signal transducers (26, 27, 28). In addition, it has been reported that HMG-box proteins interact with nuclear steroid hormone receptors and function as transcriptional coregulators (29, 30). Therefore, HMG-box proteins may potentially participate in the biological functions of reproductive-endocrine systems.

In this context, we determined the structure and localization of GCX-1, and analyzed the functions of the gene to clarify the roles of GCX-1 in the reproductive system, particularly in the ovary. The expression patterns of GCX-1 were examined, and we report here that GCX-1 is expressed only in tissues related to reproduction, i.e. the hypothalamus, pituitary, gonads, and uterus. We also show here that the GCX-1 protein is localized in the nucleus. Finally, we show that the GCX-1 is able to activate gene transcription, as evidenced by a GAL4-based heterologous transcription assay.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Immature female and male (8 and 21 d old, respectively), mature female and male (8 wk old) Kwl:Wistar rats were used in this study. The rats were housed in a photoperiod of 14 h light, 10 h dark, with food and water freely available. At all times, the animals were treated according to National Institutes of Health guidelines.

Rat granulosa cell culture
Immature, Kwl:Wistar female rats (21 d old) were used. The rats were treated with 2 mg diethylstilbestrol (Sigma Chemical Co., St. Louis, MO) in 0.1 ml sesame oil, once daily for 4 d, to stimulate the proliferation of ovarian granulosa cells. The ovaries were then excised, and granulosa cells were isolated by puncturing the follicles with a 26-gauge needle, and the released granulosa cells were collected. The cells were washed and collected by a brief centrifugation at 500 x g for 5 min at room temperature, and cell viability was determined by trypan blue staining. Cell viability was in excess of 90%. The granulosa cells were then cultured in Ham F-12:DMEM (1:1, vol:vol) supplemented with antibiotics and 0.1% BSA on collagen-coated plates in a humidified atmosphere containing 5% CO2-95% air at 37 C (31).

Yeast two-hybrid screening
A kit purchased from CLONTECH Laboratories, Inc. (Palo Alto, CA) was used for the yeast two-hybrid system. All procedures were performed as described by the manufacturer’s instructions unless otherwise stated. The pGBKT7 vector, a parent vector for the yeast two-hybrid system, expresses the GAL4 DNA binding domain (DBD) fusion protein in yeast. The pGBKT7-GIOT1 vector, a bait plasmid, was generated as described previously (11). AH109 cells were transformed with the indicated bait plasmid by means of a TE/lithium acetate-based high-efficiency transformation method (32). The construction of a plasmid cDNA library from rat granulosa cells for yeast two-hybrid screening was described (33). When a yeast strain harboring pGBKT7-GIOT1 was transformed with the library, approximately 7 x 106 primary transformants were obtained. All clones were then screened, and seven HIS3+/ADE2+/MEL1+ clones were subsequently obtained. We characterized all clones by nucleotide sequence analysis using a Big Dye terminator FS cycle sequencing kit and a 3100 Genetic Analyzer (Applied Biosystems Japan, Tokyo, Japan) and by a subsequent homology search on the GenBank DNA databases. A database search revealed that the nucleotide sequences of four clones showed a high similarity with the genes encoding known proteins, whereas the other three clones encoded novel proteins (11). From the three novel clones, a plasmid, referred to as pACT2-GCX-1, containing a 1219-bp insert with an HMG-box motif, was isolated. In a further analysis, a 758-bp EcoRI/BamHI fragment (nt –160/597) of the pACT2-GCX-1 was subcloned into the EcoRI/BamHI sites of the pBluescript II SK(+) vector (Stratagene, La Jolla, CA), and the resulting plasmid was designated as pBS-GCX-1.

Construction of {lambda}-phage cDNA library and cloning of full-length GCX-1 cDNA
Total RNA was prepared from granulosa cells that had been treated with 30 ng/ml rat FSH (National Hormone and Pituitary Distribution Program, Bethesda, MD) for 1.5 h, by using the acid-guanidium-thiocyanate extraction method (34). Poly (A)+-RNA was isolated with the oligo(deoxythymidilic acid)-latex beads (Takara BIOMEDICALS, Kyoto, Japan). Complementary DNA was synthesized from 5 µg poly (A)+-RNA with the cDNA synthesis system and Superscript II (Invitrogen, Groningen, Netherlands) using oligo(deoxythymidilic acid) as a primer. The EcoRI/NotI adaptor was then ligated to a double-stranded cDNA, and both ends were phosphorylated with a T4 polynucleotide kinase (New England Biolabs, Inc., Beverly, MA). The cDNA was ligated to {lambda}ZAP Express phage arms (Stratagene), followed by in vitro packaging using Gigapack III gold (Stratagene), to generate a cDNA library. The cDNA library contained 1 x 106 independent clones (8).

To isolate the full-length cDNA corresponding to GCX-1, the library was screened with a 758-base {alpha}-32P deoxy-CTP (Amersham Biosciences Co., Piscataway, NJ)-labeled EcoRI/BamHI fragment of the pBS-GCX-1, which was labeled with the BcaBest DNA labeling kit (Takara BIOMEDICALS). Eleven positive clones were isolated from approximately 60,000 cDNA clones. These clones were excised in vivo to recover the pBK-CMV plasmids, and the nucleotide sequences were determined from both ends.

Northern blot analysis and RT-PCR
Twenty-one-day-old female rats were primed with pregnant mare’s serum gonadotropin (PMSG, Teikokuzouki, Inc., Tokyo, Japan) or with PMSG followed by human chorionic gonadotropin (hCG, Sankyo Co., Ltd., Tokyo, Japan) as described previously (35), and the ovaries were collected at the indicated times. Granulosa cells (5 x 106 cells) were cultured in 60-mm dishes in 5 ml medium, and 30 ng/ml rat FSH was added to the medium after a 24-h cell culture. When observing the effects of LH, hCG (30 ng/ml) was added to the medium 48 h after the addition of FSH. The cultures were stopped at various intervals. Total RNA was extracted from various tissues (hypothalamus, pituitary, cerebellum, adrenal gland, kidney, spleen, intestine, liver, uterus, and ovary) of immature female rats, from the testis of an immature male rat, and from primary cultured granulosa cells using the TRIzol reagent (Invitrogen). For Northern blot analysis, 10 µg total RNA was separated by electrophoresis on a 1% denaturing agarose gel, transferred to a nylon membrane (Biodyne, ICN Biomedicals, Inc., Glen Cove, NY), and cross-linked by UV irradiation. For prehybridization and hybridization, the ExressHyb hybridization solution (CLONTECH) was used. A 758-base radiolabeled EcoRI/BamHI fragment of the pBS-GCX1, which was mentioned above, or for the control, a 1.4-kb {alpha}-32P deoxy-CTP-labeled BamHI fragment of the rat upstream stimulatory factor 2 (USF2) (33), was used as a probe. Conditions for prehybridization, hybridization, and washing procedures were performed according to the protocol provided by the supplier. The blot was exposed to a FUJIX imaging plate (Fuji Photo Film, Kanagawa, Japan). Hybridization signals were detected with a FUJIX BAS-2000 image analyzing system. For RT-PCR, 5 µg total RNA was reverse-transcribed, and a portion (one hundredth) of the reaction mixture was subjected to the PCR. Primers for GCX-1 were 5'-CCCAATGAGCCACAGAAGCCA-3' [5'-primer: nucleotide (nt) 589/609] and 5'-GGAAAGCCTGCAGGTCGGAG-3' (3'-primer: nt 936/955), respectively. Primers for glyceraldehyde 3-phospate dehydrogenase (GAPDH) were 5'-GAACGGGAAGCTCACTGGCA-3' (5'-primer: nt 689/708) and 5'-TCCACCACCCTGTTGCTGTA-3' (3'-primer: nt 955/994), respectively. Primers for rat LH receptor were 5'-CCTTCGTCGTCATCTGTGCTT-3' (5'-primer: nt 1625/1645) and 5'-CTCTCGGTGGTATGGGCTGTT-3' (3'-primer: nt 2069/2089), respectively. Reaction conditions were 30 cycles for GCX-1 and 23 cycles for the GAPDH and LH receptors, respectively, by denaturing at 94 C for 20 sec, annealing at 55 C for 30 sec, and extending at 72 C for 45 sec using the FastStart Taq DNA Polymerase (Roche Molecular Biochemicals, Indianapolis, IN). Ten microliters of the PCR products were electrophoresed on a 1.5% agarose gel and subsequently visualized by ethidium bromide staining.

In situ hybridization
For the GCX-1 antisense cRNA probe, pBS-GCX-1, linearlized with EcoRI, was in vitro transcribed with T3 RNA polymerase (Roche Molecular Biochemicals) and {alpha}-35S CTP (NEN Life Science Products, Wilmington, DE). The sense probe for GCX-1 was transcribed from BamHI-digested pBS-GCX-1 with T7 RNA polymerase (Roche Molecular Biochemicals) and {alpha}-35S CTP. Rat LH-ß cDNA fragment (nt 1/475) was generated by RT-PCR using the same conditions that were used for the detection of GCX-1 (see above) using total RNA extracted from an 8-wk-old male rat pituitary and the primer pairs (5'-AAATGGAGAGGCTCCAGG-3' and 5'-TAGAACACCTGCTGGCTC-3'). The fragment was ligated to the pGEM-T EASY vector (Promega, Madison, WI), and its EcoRI fragment was subcloned to pBluescript II SK(+) vector. The resulting plasmid was linearized with BamHI and HindIII, and then in vitro transcribed with T7 and T3 RNA polymerase, generating LH-ß antisense and sense 35S-labeled cRNA probes, respectively. In situ hybridization was performed as described previously (10), with minor modifications in which paraffin-embedded tissues were used instead of frozen tissues. Briefly, rat ovaries, testes, and pituitaries were fixed in freshly prepared 4% paraformaldehyde (PFA) at 4 C, dehydrated with ethanol, cleared in xylene, and embedded in paraffin. Seven-micrometer-thick sections were mounted on (3-aminopropyl)triethoxysilane-coated glass slides for the in situ hybridization. The sections were deparaffinized, rehydrated, fixed in 4% PFA, treated with proteinase-K, postfixed in 4% PFA, and acetylated before hybridization. Hybridization with the 35S-labeled cRNA probes was performed at 60 C for 6 h, and the sections were then washed under conditions of high stringency and autoradiographed using a NTB2 emulsion (Eastman Kodak Co., Rochester, NY). After development, all slides were counterstained with hematoxylin, dehydrated, and mounted.

Antiserum and Western blotting
The GCX-1-specific rabbit polyclonal antiserum was generated using the peptide sequence NH2-SLLHLGDHEAGYHSLC-CO2H and purified IgG. Granulosa cells were cultured for 24 h under hormone-free conditions in 60-mm dishes containing 5 x 106 viable cells in 5 ml medium, and the cells were then collected by means of a scraper and washed with 10 ml PBS. The resulting cells were suspended in 1 ml PBS, transferred to an Eppendorf tube, and pelleted by centrifugation at 1,500 x g for 10 min at 4 C. Cell extracts from granulosa cells were prepared by the method of Schreiber et al. (36) with minor modifications. The cell pellet was resusupended in 600 µl cold buffer A (10 mM HEPES, pH 7.9; 10 mM KCl; 1 mM EDTA; 0.5 mM EGTA; 1 mM dithiothreitol; and 0.5 mM phenylmethylsulfonylfluoride) by gentle pipetting. The cells were allowed to swell on ice for 15 min; after which, 37.5 µl of a 10% solution of Nonidet P-40 was added and the tube vigorously vortexed for 10 sec. The homogenate was centrifuged at 17,000 x g for 5 min at 4 C. The supernatant served as a cytoplasmic fraction for protein analysis, and the nuclear pellet was resuspended in 40 µl ice-cold buffer C (20 mM HEPES, pH 7.9; 0.4 M NaCl; 1 mM EDTA; 1 mM EGTA; 1 mM dithiothreitol; and 1 mM phenylmethylsulfonylfluoride), and the tube was then vigorously shaken at 4 C for 15 min on a shaking-platform. The mixture was centrifuged at 17,000 x g for 15 min at 4 C, and the supernatant was recovered and used for protein analysis as a nuclear fraction. Nuclear extracts from HepG2 cells expressing green fluorescence protein (GFP)-GCX-1 fusion proteins were prepared by the same protocol. The cytoplasmic and nuclear extracts (100 µg each) were electrophoresed by SDS-PAGE on a 10% acrylamide gel under reducing conditions. Proteins were then electrophoretically transferred to an Immobilon-P polyvinylidene difluoride membrane (Millipore Co., Bedford, MA) followed by blocking in milk buffer [5% skim milk in PBS-T (PBS containing 0.1% of Tween-20)] and incubation with the IgG-purified GCX-1 antibody (0.19 µg/ml) in milk buffer for 1 h at room temperature. The membrane was washed using PBS-T, and immunoreactive GCX-1 protein was subsequently detected using the ECL-Plus kit (Amersham Biosciences) according to the manufacturer’s instructions. To confirm the specificity of the antibody, we conducted absorption experiments with the synthetic GCX-1 peptide used for producing the antibody. Staining of the Western blots was blocked by the addition of 400 nmol/ml of the peptide in all cases (detection of GFP-GCX-1 and endogenous GCX-1).

Plasmids
The pSG424 vector, which is used for the expression of GAL4 DBD fusion proteins in mammalian cells, was kindly provided by Dr. R. Stein (Venderbilt University, TN) (37). Oligonucleotides 5'-AATTCCCGGGATCCGTCGACGAGCT-3' and 5'-CTAGAGCTCGTCGACGGATCCCGGG-3' were annealed, phosphorylated, and subcloned into EcoRI/XbaI sites of the pSG424 to give pSG424NRB1 (38). Oligonucleotides 5'-AATTCCCGGGGGATCCG-3' and 5'-TCGACGGATCCCCCGGG-3' were annaled, phosphorylated, and subcloned into EcoRI/SalI sites of the pSG424NRB1 to give pSG424NRB2. pSG-GCX-1 (1–473) vector for the GAL4-based mammalian heterologous reporter assay system was prepared as follows. The pBK-CMV plasmid that contains full-length of GCX-1 cDNA (pBK-GCX-1) was digested with EcoRI, and the insert was ligated into the EcoRI site of the pSG424NRB2 vector. The pSG-GCX-1 (1–473) was then digested with BamHI/XbaI and was blunt-ended with the Klenow fragment of DNA-polymerase I (New England Biolabs). The blunt-ended DNA was self-ligated to obtain pSG-GCX-1 (1–201). Both the pSG-GCX-1 (1–473) and pSG-GCX-1 (1–201) plasmids contain short translatable 5'-noncoding sequences of GCX-1 between GAL4 DBD and the entire GCX-1 protein. pSG-GCX-1 (202–473) was generated by subcloning a 850-bp BamHI fragment of the pBK-GCX-1 into the BamHI site of the pSG424NRB2. For the preparation of other pSG-GCX-1 deletion mutants, PCRs were performed using the pSG-GCX-1 (1–201) as a template and combinations of primers, as shown in Table 1Go. After digestion with EcoRI and SalI, these fragments were subcloned into the EcoRI/SalI sites of the pSG424NRB2 to generate each deletion mutant. In contrast, fragments 1 and 2 were digested with SacI and then subcloned into the SacI site of the pUC118 vector (Takara BIOMEDICALS) to produce pUC118-GCX-1 (7–171) and pUC118-GCX-1 (7–110), respectively. Both constructs were digested with EcoRI/BamHI, and the resulting inserts were subcloned into the EcoRI/BamHI sites of the pSG424NRB2 to give pSG-GCX-1 (7–171) and pSG-GCX-1 (7–110). Fragment 3 was generated using pSG-GCX-1 (1–473) as a template and, after digestion with EcoRI/BamHI, the insert was subcloned to the EcoRI/BamHI sites of the pSG424NRB2 to produce pSG-GCX-1 (120–201).


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  		TABLE 1. Combinations of primers for generating deletion mutants of pSG-GCX-1

 
Site-directed mutagenesis was carried out using the QuikChange site-directed mutagenesis kit (Stratagene). The mutagenesis reactions were performed using pSG-GCX-1 (25–63) as a template DNA, and combinations of oligonucleotides, as indicated in Table 2Go as mutation primers. After confirming these nucleotide sequences, their EcoRI/SalI fragments were subcloned into the EcoRI/SalI sites of the pSG424NRB2 to produce each pSG-GCX-1 activation domain (AD) mut.


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  		TABLE 2. Combinations of primers for site-directed mutagenesis of pSG-GCX-1-AD

 
A luciferase reporter vector, 5 x GAL4-E1b/Luc, for the GAL4 fusion protein-based assay was constructed as described previously (39).

A mammalian expression vector for the GFP fusion protein, pEGFP-C1E1, was produced as follows. Oligonucleotides 5'-AGCTTCGGAATTCG-3' and 5'-TCGACGAATTCCGAAGCTT-3' were annealed, phosphorylated, and ligated into the HindIII/SalI sites of the pEGFP-C1 (CLONTECH) (40). The pSG-GCX-1 (1–473), pSG-GCX-1 (1–201), pSG-GCX-1 (202–473), pSG-GCX-1 (7–171), and pSG-GCX-1 (120–201) were digested with EcoRI and SalI and subcloned into the EcoRI/SalI sites of the pEGFP-C1E1 to give pEGFP-GCX-1 (1–473), pEGFP-GCX-1 (1–201), pEGFP-GCX-1 (202–473), pEGFP-GCX-1 (7–171), and pEGFP-GCX-1 (120–201), respectively.

Yeast expression vectors, pGBKT7-GCX-1 and pACT2-GIOT1, to examine interactions of GCX-1 with GIOT1, were prepared as follows. The pBK-GCX-1 was digested with EcoRI, and the insert was ligated into the EcoRI site of the pGBKT7 vector to produce pGBKT7-GCX-1. pACT2 vector, a plasmid expressing GAL4 AD fusion protein in yeast (CLONTECH) was digested with XhoI, blunt-ended by the Klenow reaction, and then digested with BamHI. pSG-GIOT1 (11) was digested with BamHI and SmaI, and the BamHI/SmaI fragment was subcloned into the BamHI/XhoI (blunt-ended) site of the pACT2 vector to obtain pACT2-GIOT1.

All plasmid structures were confirmed by nucleotide sequencing.

Cell culture and transient transfections
HepG2 cells, a human hepatocellular carcinoma cell line, were purchased from the American Type Culture Collection (Manassas, VA). Cells were cultured in DMEM supplemented with antibiotics and 10% fetal bovine serum in a humidified atmosphere containing 5% CO2-95% air at 37 C. Rat ovarian granulosa cells were cultured as described above.

DNA transfections were carried out using the LIPOFECTAMINE PLUS reagent (Invitrogen). All plasmids in the transfections were prepared using a HiSpeed Plasmid Maxi Kit (QIAGEN, Hilden, Germany), followed by CsCl density gradient ultracentrifugation. Cells (1.25 x 104 for observation of GFP-GCX-1 fusion protein, and 5 x 104 for determination of transcriptional activity) per well were inoculated in a 24-well plate on the day before transfection. In experiments for observing GFP-GCX-1 fusion protein, 300 ng of each GFP plasmid was used. In experiments for determining transcriptional activity of GCX-1, 100 ng of the 5 x GAL4-E1b/Luc or the E1b/Luc firefly luciferase reporter vector, 1 ng of the pRL-CMV expression vector for sea pansy luciferase (Promega), and the indicated amount of GAL4 DBD-GCX-1 fusion protein expression plasmid, were added. Three hours after transfection, the medium was changed. After 48 h, the cells were observed with an IX-70 fluorescence microscope (Olympus, Tokyo, Japan) after staining the cell nuclei with Hoechst 33342 reagent (Sigma), or subjected to a luciferase assay. The firefly and sea pansy luciferase assays were performed with the dual luciferase assay system (Promega) according to the manufacturer’s recommended protocol. Luciferase activities were determined using a Lumat model LB 9501 (Berthold, Wildbad, Germany). Firefly luciferase activities (relative light units) were normalized by sea pansy luciferase activities.

Liquid ß-galactosidase assay
The interaction of GCX-1 with GIOT1 was examined by means of a liquid ß-galactosidase assay. Y187 cells harboring the pGBKT7 or the pGBKT7-GCX-1 were transformed with the pACT2-GIOT1 as well as with the control pACT2 vector. ß-galactosidase activity was measured for the resulting transformants, as described previously (41, 42). Permeabilized cells were used for the quantitative ß-galactosidase assays, with o-nitrophenyl-ß-D-galactopyranoside as the substrate.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
cDNA cloning of GCX-1
To identify gene products that interact with GIOT1, a GAL4-based yeast two-hybrid system was used to screen a cDNA library from immature rat ovarian granulosa cells (treated with FSH for 1.5 h in the primary culture). Along with several known transcription factors, such as transcriptional intermediary factor 1ß and rat homolog of human I-mfa domain containing protein (11), a cDNA clone encoding a novel protein with an HMG-box motif was isolated. To isolate a full-length cDNA clone encoding the protein, a {lambda}-phage cDNA library from immature rat ovarian granulosa cells (treated with FSH for 1.5 h in the primary culture) was screened using the isolated clone as a probe, and we obtained a full-length cDNA clone of the gene, which encodes a 473-amino-acid protein (Fig. 1AGo). As schematically drawn in Fig. 1BGo, the protein contains one HMG-box motif at the center of the protein. Therefore, we refer to it as the granulosa cell HMG-box protein-1 (GCX-1). It also contains a nuclear localization signal (NLS)-like motif at the N-terminal side of the HMG-box, and three domains consisting of serine-rich, lysine-rich, and proline-rich regions, respectively. These structural features are typical of transcription regulators.



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  		FIG. 1. Deduced amino acid sequence and a schematical drawing of GCX-1. A, Deduced amino acid sequence of GCX-1. The amino acids are numbered starting from the initiation codon. A putative NLS region is underlined. The putative HMG-box domain is indicated in bold type. B, A schematic illustration of the structure of GCX-1. Ser, Serine; Lys, lysine; Pro, proline.

 
We next checked the sequence similarity of GCX-1 to the other known HMG-box-containing proteins using the NCBI Blast program, and found that only mouse TOX (39% identity at the amino acid level) and human TOX (38%) showed a moderate similarity to GCX-1.

The specific interaction between GCX-1 and GIOT1 was confirmed using yeast two-hybrid assays (Table 3Go). The yeast Y187 strain harboring pGBKT7 or pGBKT7-GCX-1 was transformed with pACT2 or pACT2-GIOT1, respectively. As shown in Table 3Go, the transformants containing both pGBKT7-GCX-1 and pACT2-GIOT1 plasmids exhibited a much higher ß-galactosidase activity than the other transformants tested, indicating that GCX-1 and GIOT1 actually interact with each other in the yeast system in vivo.


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  		TABLE 3. ß-Galactosidase activities of yeast, harboring various combinations of GAL4 DBD and AD fusion plasmids

 
RT-PCR and Northern analysis of GCX-1 expression
To examine the tissue distribution of GCX-1, RT-PCR of various tissues from 21-d-old rats was performed. As shown in Fig. 2AGo, the expression of the GCX-1 gene was detected in several restricted tissues, the hypothalamus, pituitary gland, testis, uterus, and ovary. The expression of GCX-1 was the highest in the ovary, whereas no expression was observed in the adrenal gland (Fig. 2AGo) and placenta (data not shown). This strongly suggests that GCX-1 functions on the hypothalamo-pituitary-gonadal axis. We next examined the effects of gonadotropin stimulation on the ovarian expression of GCX-1. Immature female rats were treated with PMSG followed by hCG, and the gene expression of GCX-1 in ovaries was analyzed by Northern blotting. Figure 2BGo shows that the expression of GCX-1 mRNA was decreased moderately, 12 h after PMSG treatment, and the decreased levels persisted for at least 36 h. When hCG was given after treatment with PMSG for 48 h, the expression of GCX-1 mRNA was not altered significantly (Fig. 2BGo). To investigate whether gonadotropins have a direct effect on GCX-1 mRNA expression, the effects of FSH or hCG on cultured granulosa cells were examined. As shown in Fig. 2CGo, an RT-PCR analysis revealed that the expression of GCX-1 was not significantly affected by FSH alone or by the subsequent hCG treatment after FSH stimulation; though in the case of a typical gonadotropin-inducible gene, LH receptor, its mRNA expression was induced by the gonadotropin treatment (Fig. 2CGo).



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  		FIG. 2. Gene expression of GCX-1 in the rat. A, RT-PCR was performed using GCX-1-specific primers. Total RNA was isolated from various tissues of immature rats. Five micrograms of total, extracted RNA were reverse-transcribed, and a portion (one hundredth) was subjected to PCR for the specific amplification of GCX-1 and GAPDH, respectively. The reaction mixtures were separated on a 1.5% agarose gel and visualized by ethidium bromide staining. N.C., Negative control. B, Northern blot analysis of GCX-1 mRNAs in immature rat ovaries primed with PMSG/hCG. Total RNA was isolated at the indicated times as described in Materials and Methods. All samples contain 10 µg total RNA. The blots were hybridized with a GCX-1-specific probe and then rehybridized with a probe of rat USF2 as control. C, RT-PCR analysis was performed using total RNA isolated, at the indicated times, from primary cultured rat ovarian granulosa cells that had been treated with FSH/hCG as described in Materials and Methods. LH receptor (LH-R) gene products were indicated as controls of gonadotropin effects.

 
In situ hybridization study of GCX-1 in the ovary, testis, and pituitary
We next examined the expression of GCX-1 at various stages of follicular development by in situ hybridization. Sections of ovaries from female rats at various stages of growth were prepared, and in situ hybridization was performed. As shown in Fig. 3Go, strong signals were detected in follicles at very early preantral stages, and the expression was restricted to the granulosa cell layers (8 d after birth, Fig. 3BGo). Similar levels of expression were also observed in follicles at slightly advanced stages with two or more granulosa cell layers. As shown in Fig. 3BGo (21 d), signals were also detected in the follicles at large preantral and small antral stages, whereas much lower levels of expression were observed in large antral follicles at more advanced stages. Moreover, weak GCX-1 mRNA signals were actually detected in the granulosa cells of some follicles but not (if any) in the corpus luteum in ovaries from adult rats. These observations indicate that GCX-1 gene expression is restricted to undifferentiated granulosa cells in follicles of early developmental stages. We next examined the expression of GCX-1 in the testis using the same cRNA probes. In testes from 8-d-old, 21-d-old, and 8-wk-old rats, the GCX-1 mRNA signals were weak and scattered over the seminiferous tubule and interstitial region (data not shown). Similarly, GCX-1 mRNA signals were weak and scattered throughout anterior, intermediate, and posterior pituitary lobes (Fig. 4AGo), whereas signals of LH-ß mRNA were clearly detected on anterior pituitary, probably in gonadotrophs (Fig. 4DGo). The expression pattern of GCX-1 in pituitary was not influenced by age or gender (data not shown).



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  		FIG. 3. In situ hybridization of GCX-1 in the rat ovaries. Ovaries from 8-d-old, 21-d-old, or adult rats were sectioned and hybridized with 35S-labeled cRNA probes. A, Bright-field photomicrographs of hematoxylin-stained sections. B, Darkfield illumination with a GCX-1 antisense strand cRNA probe. C, Dark-field illumination with a GCX-1 sense strand cRNA probe. CL, Corpus luteum. Scale bars, 0.1 mm (8 d) or 0.2 mm (21 d, adult).

 


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  		FIG. 4. In situ hybridization of GCX-1 in the rat pituitary. Pituitaries from adult male rats were sectioned and hybridized with 35S-labeled cRNA probes. A, Bright-field photomicrographs of hematoxylin-stained sections. B, Dark-field illumination with a GCX-1 antisense strand cRNA probe. C, Dark-field illumination with a GCX-1 sense strand cRNA probe. D, Dark-field illumination with an LH-ß antisense strand cRNA probe as a positive control of hybridization. AL, Anterior lobe; IL, intermediate lobe; PL, posterior lobe. Scale bars, 0.2 mm.

 
Intracellular localization of GCX-1
To examine the intracellular localization of the GCX-1 product, we prepared a plasmid vector, which expresses the GFP-GCX-1 fusion protein. The plasmid was transfected into the primary cultured rat ovarian granulosa cells, and the cellular localization of the fusion protein was observed by fluorescence microscopy (Fig. 5Go). The GFP-GCX-1 fusion protein was solely localized in the nucleus, whereas the control GFP protein was spread over entire cell regions. To map the domain necessary for the nuclear localization of GCX-1, several GCX-1 deletion mutants were constructed, and the constructs were transfected into the granulosa cells. As shown in Fig. 5Go, C–F, a putative NLS region expected from the deduced amino acid sequence was demonstrated to be essential for the nuclear localization of the GCX-1 gene product. These observations clearly demonstrate that GCX-1 is a nuclear protein and that the NLS region is essential for its nuclear localization.



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  		FIG. 5. Determination of the subcellular localization of GCX-1 protein. Expression plasmids (300 ng), which encode GFP alone or truncated GCX-1 proteins fused to the C terminus of GFP, were transfected into primary cultured rat ovarian granulosa cells. Forty-eight hours after transfection, the cell nuclei were stained with Hoechst 33342, and then the subcellular localization of GFP-GCX-1 fusion protein was observed. A, pEGFP-C1E1; B, pEGFP-GCX-1 (1–473); C, pEGFP-GCX-1 (1–201); D, pEGFP-GCX-1 (202–473); E, pEGFP-GCX-1 (7–171); F, pEGFP-GCX-1 (120–201).

 
Identification of endogenous GCX-1 protein
To confirm the presence of endogenous GCX-1 protein in cells, an antibody against GCX-1 was prepared. A peptide fragment of 39–54 amino acid residues from the N terminus was immunized to rabbits to generate the antiserum, and the IgG fraction was collected. To verify the specificity of the antibody, nuclear extracts from HepG2 cells, expressing GFP-GCX-1 fusion protein, were used for the detection of GCX-1. As shown in Fig. 6AGo, the polyclonal antibody efficiently recognized the GFP-GCX-1 protein (lane 2). In a related experiment, we show that immunoreactive GCX-1 was detected only in the nuclear extracts of rat granulosa cells (Fig. 6BGo, lane 2) but not in the cytoplasmic fraction (Fig. 6BGo, lane 1). This clearly indicates that the GCX-1 gene product was actually present in the nuclear fraction of rat ovarian granulosa cells. Unfortunately, an immunohistochemical study using the same antibody could not detect endogenous GCX-1 protein in situ on tissue sections of ovary, testis, or pituitary (data not shown).



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  		FIG. 6. Identification of endogenous GCX-1 protein. A, Analysis of GCX-1-specific antiserum. Nuclear extracts from pEGFP-C1E1 (lane 1)- or pEGFP-GCX-1 (1–473) (lane 2)-transfected HepG2 cells were subjected to Western blot analysis with anti-GCX-1 antiserum (IgG purified). The specific band corresponding to the GFP-GCX-1 fusion protein is indicated. B, Detection of endogenous GCX-1 protein in rat ovarian granulosa cells. Cytoplasmic (lane 1) or nuclear (lane 2) extracts were prepared from cultured granulosa cells and then subjected to Western blot analysis with the same antiserum. A GCX-1-specific band is indicated.

 
Transcriptional activity of GCX-1
Because GCX-1 is a nuclear protein and contains an HMG-box motif, one of the motifs typical of transcription factors, we next examined the transcriptional activity of GCX-1. A GAL4-based heterologous luciferase reporter system was used to verify the transcriptional ability of GCX-1, because target genes of this protein are not known at this time. Then, 5 x GAL4-E1b/Luc firefly luciferase reporter plasmid and GAL4 DBD-GCX-1 fusion protein expression plasmid were cotransfected into HepG2 cells. As shown in Fig. 7Go, the expression of the GAL4 DBD-GCX-1 fusion protein greatly enhanced the reporter luciferase activity. The addition of 0.1 ng of GCX-1 plasmid resulted in about a 25-fold induction of luciferase activity, and 1 ng of the plasmid caused about a 100-fold induction. On the other hand, when a reporter plasmid that does not possess the GAL4 binding sequences was used instead of 5 x GAL4-E1b/Luc, no induction was observed by GCX-1 plasmid cotransfection (Fig. 7Go). These observations clearly indicate that GCX-1 is a very strong transcriptional activator.



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  		FIG. 7. GCX-1 is a transcriptional activator. HepG2 cells were cotransfected with 1 ng of pRL-CMV and 100 ng of 5 x GAL4-E1b/Luc or E1b/Luc reporter plasmid. Indicated amounts of pSG-GCX-1 (1–473) were simultaneously transfected into the cells. pSG424, which expresses GAL4 DBD alone, was added to some samples so that each samples contained the same amount of DNA. Each value represents the mean and SD of four independent transfection experiments.

 
Mapping of activation domain of GCX-1
We next examined the transactivation domain of GCX-1. Various plasmid constructs of GAL4 DBD-GCX-1 deletion mutants were prepared, and the constructs were cotransfected with the 5 x GAL4-E1b/Luc firefly luciferase reporter plasmid into HepG2 cells. As shown in Fig. 8Go, a region between 25 and 63 amino acid residues from the N terminus was proved to be essential for the transcriptional activity of GCX-1. The region contains no typical transcriptional activation motifs that have been reported to date. Therefore, we attempted to determine the specific amino acid residues involved in the transactivation ability of GCX-1 by introducing site-directed mutagenesis in this region. An alanine substitution method was used for the site-directed mutagenesis by preparing mutant constructs of the GCX-1 activation domain, in which each five or six consecutive amino acid residues were substituted with alanine residues. Such constructs were cotransfected with the 5 x GAL4-E1b/Luc firefly luciferase reporter plasmid into HepG2 cells, as described above. As shown in Fig. 9Go, the cotransfection of all mutant constructs caused a decrease in luciferase activity, and, as a result, it was not possible to specify the exact amino acid residues that are essential for activity. These observations suggest that the entire structure of the activation domain, but not any particular amino acid residue, may be necessary for the transcriptional ability of this region. In addition, nuclear extracts were prepared from the cells transfected with GAL4 fusion constructs and were subjected to electro mobility shift assay analyses to check the expression levels of the GAL4 fusion proteins and their specific binding abilities to the GAL4 recognition sequence. All the fusion proteins were able to bind to the GAL4 site and showed similar expression levels (data not shown), indicating that transfection efficiency was sufficient for further experiments.



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  		FIG. 8. Determination of the transactivation domain of GCX-1. HepG2 cells were cotransfected with 1 ng of pRL-CMV, 100 ng of 5 x GAL4-E1b/Luc reporter plasmid, and 1 ng of the various deletion mutants of pSG-GCX-1. Each value represents the mean and SD of four independent transfection experiments.

 


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  		FIG. 9. Identification of the amino acid residue(s) important for transactivating ability of GCX-1. HepG2 cells were cotransfected with 1 ng of pRL-CMV, 100 ng of 5 x GAL4-E1b/Luc reporter plasmid, and 1 ng of the various mutants of pSG-GCX-1 AD prepared by site-directed mutagenesis. Each value represents the mean and SD of four independent transfection experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, a novel gene, GCX-1, which is expressed predominantly in the ovary, was isolated from a cDNA library of rat ovarian granulosa cells. The GCX-1 gene encodes a protein containing an HMG-box motif, a well-known DNA-binding motif that is widely distributed to all eukaryotic organisms, from yeast to mammals. A protein family having an HMG-box motif is roughly divided into two groups with respect to the mode of DNA-binding. Proteins in one group, such as SRY and TCF, recognize specific DNA sequences on their target genes. Proteins belonging to the other group, such as HMGB1, UBF, and others, show nonspecific DNA-binding. The former proteins contain only one HMG-box motif, whereas the latter proteins contain two or more HMG-box motifs (18, 19). Because GCX-1 has only one HMG-box motif, similar to specific type HMG-box proteins, we attempted to confirm that GCX-1 is able to bind to known binding sequences for sequence-specific type HMG-box proteins, such as A/T A/T C A/T A/T A/T G for TCF and SRY transcription factors, but the specific GCX-1 binding to this sequence could not be detected, even after several trials (data not shown). On the other hand, the amino acid sequence within the HMG-box of GCX-1 shows a low, but significant, similarity to those of nonspecific type HMG-box proteins (20, 21) and shows no clear homology to specific type proteins. Therefore, GCX-1 may belong to a novel type of HMG-box proteins.

The gene expression of GCX-1 is limited to the ovary, testis, uterus, pituitary, and hypothalamus (Fig. 2AGo), suggesting that GCX-1 functions at the hypothalamo-pituitary-gonadal axis. It is well known that transcription factors, Ad4BP/SF-1 and DAX-1, are essential for steroidogenesis and the embryonic development of endocrine tissues, and show gene expression patterns similar to that of GCX-1 (14, 15, 16). However, these genes are also expressed in the adrenal gland, where GCX-1 is not expressed. On the other hand, genes encoding P450 aromatase (43) and 17ß-hydoroxy-steroid dehydrogenase type 1 (44, 45) are known to be expressed only in reproductive tissues, but they are not expressed in the pituitary gland. Therefore, GCX-1 may play an important role in the hypothalamo-pituitary-gonadal system.

An in situ hybridization study revealed that the strong gene expression of GCX-1 was limited to granulosa cells at the early follicle stages; and in the larger antral follicles and the corpus luteum, the expression levels were lower than those in the small follicles (Fig. 3Go). These observations were in good agreement with the Northern analysis results using PMSG- and hCG-treated ovaries (Fig. 2BGo), because the population of early-stage follicles, which were the main source of GCX-1 mRNA, was decreased after gonadotropin treatment. However, GCX-1 mRNA expression levels were not significantly altered by gonadotropin treatment in the in vitro culture system (Fig. 2CGo). Therefore, GCX-1 gene expression does not appear to be directly controlled by gonadotropin stimulation, and the possibility that there may be complicated regulation systems for GCX-1 gene expression in vivo, such as cell-cell adhesion signaling, and autocrine or paracrine regulatory factors produced locally, cannot be excluded.

Because the gene expression of GCX-1 was strong in early-stage follicles, where steroidogenic activities are still very low, and was not observed in some other steroidogenic tissues, such as the adrenal gland or placenta, GCX-1 may play less important roles in the biosynthesis of steroid hormones. It is possible that GCX-1 may play some roles in embryonic development of reproductive tissues or in the growth and development of early-stage follicles. Events in early-stage follicles are thought to be independent of gonadotropin stimulation (3, 4). The observation that the expression of GCX-1 was not significantly changed by gonadotropin in cultured granulosa cells may be consistent with the above speculation.

Because a weak expression of GCX-1 mRNA was detected in the testis and pituitary, we conducted an in situ hybridization study of these tissues. However, only broad and scattered signals were observed in both tissues. We hypothesize that GCX-1 plays a more important regulatory role in the female reproductive system than in the male system, as in the case of the progesterone receptor, of which physiological roles have not yet been established in the male system (46, 47).

Western blotting analysis of GCX-1 revealed that the GCX-1 gene product was actually present in the nucleus of ovarian granulosa cells (Fig. 6Go), and a GFP fusion protein analysis indicated that a putative NLS motif was essential for the nuclear translocation of GCX-1 (Fig. 5Go). In addition, a GAL4 fusion protein-based heterologous reporter assay revealed that GCX-1 functions as a strong transcriptional activator (Fig. 7Go). These data suggest that GCX-1 functions in reproductive tissues as a transcriptional activator. The activation domain of GCX-1 was mapped to a region between 25 and 63 residues from the N terminus (Fig. 8Go). The GCX-1 also contains regions that are rich in serine, lysine, or proline residues, respectively, but these regions were not essential for its transcriptional activity. Although the activation domain does not contain typical transcriptional activation motifs, such as the LXXLL motif (48, 49, 50), the region is rich in hydrophobic amino acid residues, and contains LXXLL-like sequences [e.g. LLHLG (between 40 and 44 residues), LPEPSLL (between 35 and 41 residues)]. The LXXLL motif is known to be necessary for interactions with nuclear steroid hormone receptors or nuclear coactivators (48, 49, 50). However, we were not able to confirm interactions between the GCX-1 activation domain and several known coactivators (data not shown). From the site-directed mutagenesis analyses (Fig. 9Go), we conclude that the entire structure of the GCX-1 activation domain might be necessary for its full activity to be exerted. The above observations suggest that the GCX-1 activation domain may represent a novel type of transactivation domain, although little information is available concerning its secondary or tertiary structure.

Our preliminary experiments revealed the specific induction of StAR gene transcription (data not shown). This observation may provide clues to the physiological target gene of GCX-1, and a more detailed analysis is currently under way.

In conclusion, we report on the isolation and characterization of a novel transcriptional activator and have designated it as GCX-1. The gene expression of GCX-1 was exclusively observed in reproductive tissues, including the hypothalamo-pituitary-gonadal axis. GCX-1 may participate in controlling gene expression in the ovary during follicular growth and development. Detailed mechanisms of actions of GCX-1 will need to be examined further for a complete understanding of this process.


   Acknowledgments
 
We thank the National Pituitary and Hormone Distribution Program of the National Institute of Diabetes and Digestive and Kidney Diseases for rat FSH. We are also grateful to Dr. R. Stein for providing the plasmid and to Drs. Zhangfei Shou and Satoko Hirano and Ms. Yoshiko Inoue, Miyuki Nakagawa, Kazuyo Yoshida, and Kaoru Matsuura for technical assistance.


   Footnotes
 
This work was supported by a grant from the Ministry of Education, Science, Sports and Culture of Japan, and a grant from the Smoking Research Foundation.

The rat GCX-1 cDNA sequence (accession number AB096685) has been deposited in the DDBJ/EMBL/GenBank database.

Abbreviations: AD, Activation domain; Ad4BP/SF-1, adrenal 4 binding protein/steroidogenic factor-1; DAX-1, dosage sensitive sex reversal-adrenal hypoplasia congenita-critical region on the X chromosome-1; DBD, DNA-binding domain; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GCX-1, granulosa cell HMG-box protein-1; GFP, green fluorescence protein; GIOT1, gonadotropin-inducible ovarian transcription factor 1; hCG, human chorionic gonadotropin; HMG, high-mobility group; NLS, nuclear localization signal; nt, nucleotide; PFA, paraformaldehyde; PMSG, pregnant mare’s serum gonadotropin; SRY, sex determining region Y; StAR, steroidogenic acute regulatory protein; TCF, T cell factor; TOX, thymus HMG-box; UBF, upstream binding factor; USF2, upstream stimulatory factor 2.

Received October 7, 2003.

Accepted for publication January 27, 2004.


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