Steroidogenic Factor-1 Is an Essential Transcriptional Activator for Gonad-specific Expression of Promoter I of the Rat Prolactin Receptor Gene*

(Received for publication, February 19, 1997)

Zhangzhi Hu Dagger , Li Zhuang Dagger , Xinyuan Guan §, Jianping Meng Dagger and Maria L. Dufau Dagger

From the Dagger  Section on Molecular Endocrinology, Endocrinology and Reproduction Research Branch, NICHD and the § Laboratory of Cancer Genetics, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

The expression of the prolactin receptor is under the control of two putative tissue-specific (PI, gonads; PII, liver) and one common (PIII) promoters (Hu, Z. Z., Zhuang, L., and Dufau, M. L. (1996) J. Biol. Chem. 271, 10242-10246). The three promoter regions were co-localized to the rat chromosomal locus 2ql6, in the order 5'-PIII-PI-PII-3'. To investigate the mechanisms of gonad-specific utilization of PI, the promoter domain, regulatory cis-elements, and trans-factors were identified in gonadal cells. The promoter domain localized to the 152-base pair 5' of the transcriptional start site at -549 is highly active in gonadal cells but has minimal activity in hepatoma cells. It contains a steroidogenic factor 1 (SF-1) element (-668) that binds the SF-1 protein of nuclear extracts from gonadal cells and is essential for promoter activation. A CCAAT box (-623) contributes minimally to basal activity in the absence of the SF-1 element, and two adjacent TATA-like sequences act as inhibitory elements. Thus, PI belongs to a class of TATA-less/non-initiator gene promoters. These findings demonstrate an essential role for SF-1 in transcriptional activation of promoter I of the prolactin receptor gene, which may explain the tissue-specific expression of PI in the gonads but not in the liver and the mammary gland.


INTRODUCTION

The functional diversity of prolactin, involving lactation and reproduction, growth and metabolism, immune regulation, behavior, and homeostasis, has been well documented in the past several decades (2, 3). However, the mechanisms underlying the regulation of prolactin receptors present in specific target tissues have only recently been investigated. Prolactin receptors are widely expressed, and multiple mRNA transcripts corresponding to the long and short forms of the receptor are present with various proportions in different tissues (4, 5). The diverse actions of prolactin could be manifested by the expression of different receptor forms and signal transduction pathways and also by differential control of gene transcription and mRNA regulation of PRLR1 in target tissues.

Recently, the complexity of the mechanism by which the PRLR gene is controlled was revealed by the demonstration of multiple and tissue-specific promoters of the rat prolactin receptor gene (1). Three putative promoters PI, PII, and PIII that initiate the transcription of alternative first exons E11, E12, and E13, respectively, were identified in the rat gonads and the liver. The three alternate first exons are alternatively spliced to a common noncoding exon 2 that precedes the third exon containing the translation initiation codon of the prolactin receptor. E11 is expressed in ovaries and in Leydig cells, E12 in the liver, and E13 in all three tissues, indicating that PI is gonad-specific, PII is liver-specific, and PIII is a common promoter for the prolactin receptor in these tissues and is the sole promoter utilized in the rat mammary gland (our current study). The various prolactin receptor forms were found not to be dependent on the utilization of individual promoters, which may result from a post-transcriptionally regulated process. However, these accounted for the 5'-untranslated region heterogeneity of the first exons of the PRLR mRNA transcripts (E11, E12, and E13) (1). The heterogeneity of the 5'-untranslated region of the PRLR transcripts was also reported recently in the gonads and the liver by others (6, 7). Sequence analyses of 5'-flanking regions showed a lack of consensus TATA box sequences positioned within the expected distance from the transcriptional start site (TSS) in all three putative promoter regions (1). However, potentially functional non-canonical TATA box sequences are present in these regions, and in PI two adjacent TATA-like sequences reside 10 and 23 bp 5' from the TSS. In addition, consensus sequences for several transcription factors are also present in these promoters, specifically SF-1, C/EBP, and CAAT box in PI; GATA-1, ERE and AP-1 in PII; and GAGA, GATA-1 and AABS in PIII. Therefore, it is assumed that different transcription factors and other cellular and extracellular regulators may be involved in the control and regulation of the PRLR gene in diverse target tissues.

In the present study, we have determined the co-localization and the relative orientation of the three 5'-untranslated region/promoter regions in the rat chromosome, and we have investigated the underlying mechanisms of the tissue-specific promoter control of the PRLR gene in the gonads by the characterization of the gonad-specific promoter domain PI and its relevant cis-elements and trans-factors. These studies have demonstrated that steroidogenic factor-1 (SF-1) (8), also known as Ad4BP (9), is an essential transcriptional activator of PI in testicular and ovarian cells. Such regulation by SF-1 appears to determine the specific gonadal utilization of promoter I.


MATERIALS AND METHODS

Animals

Adult male, prepubertal female, and lactating Harlan Sprague Dawley rats (Charles River, Wilmington, MA) were housed in pathogen-free, temperature- and light-controlled conditions (20 °C; alternating light-dark cycle with 14 h of light and 10 h of darkness; lights on at 0600 h). The animals were given free access to tap water and standard Purina lab chow (Ralston-Purina, St. Louis, MO). All animals were killed between 1000 and 1100 h by asphyxiation with CO2. All animal studies were approved by the NICHD Animal Care and Use Committee (protocols 94-040 and 94-041). 40-day-old adult male rats were used for preparation of Leydig cells. 24-day-old immature female rats were injected subcutaneously daily for 3 days with 1.5 mg/day 17beta -estradiol (dissolved in propylene glycol) (Sigma). On the 4th day, the animals were sacrificed for preparation of ovarian granulosa cells. The mammary gland of lactating female rats were resected and freed of surrounding adipose tissue and used for mRNA preparation.

Culture of Cell Lines and Primary Rat Gonadal Cells

Cultures of mouse Leydig tumor cells (MLTC-1), a stable steroidogenic cell line that expresses prolactin and luteinizing hormone receptors (kindly provided by Dr. R. V. Rebois, National Institutes of Health, Bethesda, MD), were maintained in RPMI 1640 (BioWhittaker, Walkersville, MD) supplemented with 10% fetal bovine serum and 1 × antibiotic/antimycotic mixture (Life Technologies, Inc.), and the human hepatoma cell line (HepG2, American Type Culture Collection, Rockville, MD), which expresses prolactin receptors, was maintained in minimal essential medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum, non-essential amino acids, and sodium pyruvate.

Rat ovarian granulosa cells were prepared from estrogen-primed rat ovaries as described previously (10) with some modifications. Briefly, the ovaries were removed and cleared of the surrounding fat tissues. These were washed in Medium 199 (M199, BioWhittaker) containing 0.2% bovine serum albumin and 10 mM HEPES, pH 7.2 (Medium A), and punctured 20 times with a 23-gauge needle in Medium B (Medium A with additional 6.8 mM EGTA) followed by incubation in a CO2 incubator at 37 °C for 10 min and centrifugation at 700 rpm for 10 min. The ovaries were then incubated with Medium C (Medium A with additional 1.8 mM EGTA and 0.5 M sucrose) for 6 min in CO2 incubator at 37 °C and centrifuged as above. To disperse the granulosa cells, the ovaries were pressed against a 40-mesh metal sieve with a spatula, and the dispersed cells were collected. The cells were treated with trypsin (Sigma) (20 mg/ml) for 1 min at 37 °C followed by addition of soybean trypsin inhibitor (Sigma) (300 µg/ml) and DNase I (Sigma) (100 µg/ml) for 4 min at 37 °C. The enzymes were removed by centrifugation, and the cells were washed twice with Medium A. Cells were suspended in medium Dulbecco's modified Eagle's medium/F12 (Life Technologies, Inc.) supplemented with 1% fetal bovine serum, and with 15 ng/ml ovine follicle stimulating hormone (ovine FSH-20, National Pituitary Program, NIDDK), 10 ng/ml testosterone (Sigma) and plated at a density of 0.5 × 106 cells/cm2 in 24-well plates and cultured at a CO2 incubator for 3-5 days.

Rat Leydig cells were prepared from adult male rat testes by collagenase digestion and purified by centrifugal elutriation (11). The purified Leydig cells were suspended in M199 containing 0.1% bovine serum albumin and 1 × antibiotic/antimycotic and plated at the density of 0.5 × 106 cells/cm2 and cultured for up to 20 h.

Fluorescence in Situ Hybridization and Mapping of 5'-Flanking/Promoter Regions

Lambda phage DNA with genomic PRLR DNA inserts of 15 and 14.5 kb were used for fluorescence in situ hybridization analysis. The probes were labeled with biotin (lambda phage clone lambda 11-1, 15 kb, containing PRLR PI/PII regions) or spectrum orange (lambda phage clone lambda 3a, 14.5 kb, containing PIII region) by nick translation (Life Technologies, Inc.) and hybridized to rat metaphase chromosomes as described previously (12). Briefly, about 200 ng of each probe was used in 10 µl of hybridization mixture containing 55% formamide, 2 × SSC, and 1 µg of human CotI DNA (Life Technologies, Inc.) that was denatured at 75 °C for 5 min. Slides with rat metaphase chromosomal spreads were denatured in 70% formamide, 2 × SSC at 72 °C for 2 min, and hybridized with the specified probes at 37 °C in a moist chamber overnight. Slides were then washed three times in 50% formamide, 2 × SSC at 45 °C for 3 min each. The hybridization signal of the probe was detected by two layers of fluorescein isothiocyanate-conjugated avidin (Vector Laboratories, Inc, Burlingame, CA) and amplified with one layer of anti-avidin antibody (Vector). Slides were counterstained with 0.5 µg/ml 4,6-diamidino-2-phenylindole in an antifade solution (1 mg/ml p-phenylenediamine dihydrochloride, 10% phosphate-buffered saline (v/v), 90% glycerol (v/v), 4.2% sodium carbonate (w/v)) and was examined with a Zeiss Axiophot microscope equipped with a dual bandpass filter.

For mapping of the plasmid DNA isolated from a P1 genomic library (Genome Systems, Inc., St. Louis, MO) containing the 5'-flanking regions of the rat PRLR gene, the plasmid was digested with BamHI or in combination with NotI followed by Southern blot hybridization with oligonucleotide probes derived from E11, E12, and E13.

Construction of Reporter Plasmids and Recombinant Adenovirus Genes

DNA fragments of the 5'-flanking region containing promoter I were either generated by restriction enzyme digestion (KpnI/XbaI, -1566/-124) or by PCR amplification. For generation of plasmid constructs, the 5'-flanking DNA fragments of PI were ligated 5' to the luciferase gene (LUC) of the linearized plasmid pGL2 (Promega, Madison, WI). The pGL2 constructs were numbered relative to the translation initiation codon (PI(-#/-#)/LUC). The recombinant adenovirus (Adv) luciferase reporter constructs containing the putative promoter region (5'-flanking region of the PRLR) 5' to the luciferase gene [PI(-#/-#)/LUC/Adv] were prepared as described previously (13). Briefly, the 5'-flanking region of PRLR-luciferase (5'PRLR/LUC) minigenes were excised from pGL2 plasmid constructs using SmaI/BamHI and inserted to the EcoRV/BamHI site of pAC plasmid, which contains partial sequences of the adenovirus 5 genome. The resulting pAC/PI/LUC constructs were co-transfected with plasmid pJM17, which contains the remainder of the adenovirus genome into human embryonic 293 cells. In vivo homologous recombination of the plasmids yields recombinant viral genome (PI/LUC/Adv) and subsequent generation of infectious viral particles. Viral plaques were isolated and propagated to a titer of 109 ml-1 in the 293 cells and were used for infection of primary cultures of rat gonadal cells. Structure of the fusion genes was verified by PCR amplification of the insert and the subsequent DNA sequence analysis.

RT-PCR, 5'-RACE PCR, and Northern Blot

For RT-PCR analyses of RNA from MLTC, first strand cDNA was synthesized with random primers using Superscript reverse transcriptase (Life Technologies, Inc.) at 42 °C for 30 min. Primers used for PCR amplification of exons E11 and E13 were 5'-GTGGCCAGAGCCATGGACAG-3' (forward) and 5'-AAACTCTTTCCTCGGAGGTCACTAG-3' (reverse), 5'-TCTCAGAGACACGCGGCTG-3' (forward) and 5'-TTCTGCTGGAGAGAAAAGTCTG-3' (reverse), respectively. PCR fragments were resolved on 1.5% agarose gel. 5'-RACE PCR analyses of RNA from luciferase reporter plasmid PI(-1566/-124)/LUC transfected in MLTC were performed as described previously (1). First strand cDNA was synthesized using primer GS1 (5'-AGCGGTTCCATCCTCTAG-3') (+7 to +24 of the luciferase coding region) and 3'-end-tailed with dCTP using terminal deoxynucleotidyltransferase followed by PCR with primer GS2 (5'-CTTTATGTTTTTGGCGTCTTCCA-3') (+44 to +66 of the LUC coding region) and dG-adaptor primer (5'-GCGAATTCTCGAGATCTGGGIIGGGIIGGGIIG-3'), where I represents inosine. The PCR products were resolved on 1.5% agarose gel and subjected to Southern blot analyses using nested oligonucleotide probe GS3 (5'-TCTACCTAACCCGCCCACTGGTT-3') within E11. Primers used for 5'-RACE PCR analyses of rat mammary gland PRLR mRNA were same as described previously (1). Poly(A)+ RNAs from the rat ovary, Leydig cells, mammary gland, and MLTC were prepared and analyzed by Northern blot as described previously (1, 4).

Preparation of Nuclear Proteins

Nuclear proteins from MLTC, rat granulosa cells, and rat Leydig cells were extracted as described (14) with some modifications. Briefly, cells were suspended in buffer A containing 10 mM HEPES, pH 7.9, 10 mM KCl, 1.5 mM MgCl, 0.1 mM EDTA, 0.5 mM DTT, 0.4 mM Pefabloc SC (Boehringer Mannheim), 2 µg/ml leupeptin (Sigma), and 2 µg/ml pepstatin A (Sigma) with additional 0.3 M sucrose and 2% Nonidet P-40 and homogenized 20 strokes with type B pestle. The nuclei were recovered by centrifugation of the cell homogenate through a 1.5 M sucrose cushion contained in buffer A and lysed in buffer B (10 mM HEPES, pH 7.9, 420 mM KCl, 1.5 mM MgCl, 0.1 mM EDTA, 10% glycerol, 0.5 mM DTT, 0.4 mM pefabloc SC, 2 µg/ml leupeptin, and 2 µg/ml pepstatin A). The nuclear lysate was dialyzed against buffer D (20 mM HEPES, pH 7.9, 100 mM KCl, 0.1 mM EDTA, 20% glycerol, 0.5 mM DTT, 0.4 mM pefabloc SC, 2 µg/ml leupeptin and pepstatin A) overnight at 4 °C. The concentration of the nuclear proteins was measured by the protein assay kit (Bio-Rad).

DNase I Footprinting Assay

Double-stranded DNA fragment corresponding to the region of PI at -827 to -440 was used for DNase I footprinting analysis. One strand end-labeled DNA probes were generated by first labeling the 5' end with [gamma -32P]ATP (3000 mCi/mmol, DuPont NEN) of either forward primer (5'-TGTCTGCCTCATGAGAATAC-3') or reverse primer (5'-TCTACCTAACCCGCCCACTGGTT-3') followed by PCR amplification of the DNA fragment with one labeled and one unlabeled primer. For each footprinting reaction, 5 × 104 cpm of the probe was added to 10-20 µg of nuclear protein in 50 µl of mixture containing 20 mM HEPES, pH 7.5, 50 mM KCl, 0.5 mM EDTA, 1 mM DTT, 5% glycerol and incubated at room temperature for 15 min. DNase I digestion was performed by adding 1 × DNase I buffer (25 mM NaCl, 10 mM HEPES, 5 mM MgCl, and 1 mM CaCl2) containing 1 unit of DNase I (Promega) and incubation for 1 min at 22 °C. The reaction was terminated by adding 10 µl of stop buffer (200 mM NaCl, 30 mM EDTA, 1% SDS, and 100 µg/ml yeast RNA) and 110 µl of phenol/chloroform. The digested DNA fragments were recovered by ethanol precipitation and resolved on 6% polyacrylamide-urea gel electrophoresis.

Electrophoresis Mobility Shift Assay (EMSA)

Synthesized oligonucleotides were used for gel mobility shift assays unless otherwise indicated. The oligomers were annealed and 5'-end-labeled with [gamma -32P]ATP (3000 mCi/mmol, DuPont NEN). 1-3 µg of nuclear protein was added to 20 µl of reaction containing 12 mM HEPES, pH 7.6, 60 mM KCl, 4 mM Tris-HCl, 5% glycerol, 1 mM EDTA, 1 mM DTT, and 25 µg/ml polydeoxyinosinic deoxycytidylic acid on ice for 15-30 min followed by addition of 5 × 104 cpm of the probe for additional 15 min. For competition assay, unlabeled DNA sequences were added to the reaction 15 min prior to the addition of the probe. For supershift assay, SF-1 antibody 1 (a gift from Dr. Ken-ichirou Morohashi, Kyushu University, Japan) (15) or antibody 2 (8) (purchased from Upstate Biotechnology, Inc, Lake Placid, NY) was incubated with nuclear proteins for 30 min prior to the addition of the probe. DNA-protein complexes were resolved on 5% native polyacrylamide gel electrophoresis.

Transient Expression of Reporter Genes

MLTC cells were transfected at 50-70% confluency ~48 h after plating using N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (Boehringer Mannheim) as described previously (16). Cells were harvested 40-60 h after transfection, and whole cell lysates were assayed for luciferase activity (17). Since the primary rat gonadal cells were not amenable for transient expression of PRLR-PI/LUC reporter gene constructs by conventional liposome-mediated transfection in our experiments, the recombinant adenovirus infection method was applied in this study. Adenovirus reporter gene constructs (PI/LUC/Adv) were added at multiplicity of infection of 100 to the granulosa or Leydig cell culture and incubated for 20 h. Adenovirus construct containing the promoterless luciferase gene served as background control and a construct containing the SV40 promoter-luciferase gene as positive control. The granulosa cells were infected after 3 days of culture, and the infection was allowed to continue for 20 h before termination. The Leydig cells were infected 2 h after plating, and human chorionic gonadotropin was added to a final concentration of 20 ng/ml 6 h before termination. The infection was also allowed to continue for 20 h. Whole cell lysates were used for measurement of luciferase activity.


RESULTS

Chromosomal Localization of PRLR Promoters and Their Order in the Rat Genome

The fluorescence in situ hybridization method was used to establish the chromosomal location of the rat PRLR gene and to determine whether the three PRLR gene promoter regions that direct transcription of alternative first exons reside at the same gene locus. The genomic probe (15 kb) containing the 5'-flanking region corresponding to both PI and PII promoter regions has located these regions at the chromosomal locus 2q16 (Fig. 1, above). The same finding was obtained using the genomic probe containing coding exons 4 and 5 of the rat PRLR (not shown). The PIII promoter region was also located at the identical locus on the same metaphase chromosome when probed with a genomic fragment (14.5 kb) containing the PIII region (Fig. 1, above). The order of the three promoters in the gene was determined by mapping overlapping genomic clones (Fig. 1, middle). The size of the DNA insert cloned from the rat P1 genomic library is ~96 kb as determined by restriction digestion and agarose gel electrophoresis (Fig. 1, middle left). A 55-kb BamHI fragment was hybridized by two oligomer probes derived from E11 (Fig. 1, middle right, lanes 1-3) but not from E12 (not shown) or E13 (lane 4). Lanes 2 and 3 show a 35-kb positive fragment generated from the 55-kb fragment with NotI digestion, which cleaves at a unique site of the 16-kb vector. Furthermore, a BamHI-digested fragment of 8 kb was specifically hybridized by the oligomer probe derived from E13 (lane 4). However, no hybridization of this genomic DNA was revealed by the oligomer probes derived from E12 (not shown). These results indicated that this 96-kb genomic fragment of the rat PRLR gene contained both regions corresponding to exons E11 and E13 as well as their putative promoter regions PI and PIII but not PII and exon E12. Since previously we have established that PI region is located 10 kb 5' of PII region (1), we could derive that the order of these 5'-flanking/promoter regions of the PRLR gene is 5'-PIII-PI-PII-3', which spans over 20 kb in the genome.


Fig. 1. Chromosomal localization and genomic order of rat PRLR 5'-flanking/promoter regions. Above, genomic probes containing 5'-flanking/promoter regions, PI and PII (15 kb), and PIII region (14.5 kb) were labeled with biotin and spectrum orange, respectively, by nick-translation and hybridized to the metaphase chromosomes of the rat fibroblast. The signals in green (PI/PII) and red (PIII) were co-localized to the long arm of chromosome 2 at the 2q16 locus as indicated by the G-banding method (left). Middle: left, schematic representation of the plasmid DNA insert restriction map by BamHI. This clone contains a PRLR gene insert of 96 kb with at least nine BamHI sites (indicated by cross-lines). The NotI site is unique to the vector (16 kb, closed bar). The positions of E11 and E13 in the plasmid were determined by Southern hybridization of the digested plasmid DNA by oligonucleotide probes derived from E11, E12, and E13 separately. Right, Southern blot of digestion of plasmid DNA with BamHI (lane 1) and with BamHI plus NotI (lanes 2-4). Lanes 1 and 2 were hybridized with sequence 5'-TCCTCTCTCACCAGGCGAAAC-3' (E11), lane 3 with 5'-ACAGTACTGGGAGAATGGCTCTAAG-3' (E11), and lane 4 with 5'-GCCCCGTGTAAAATC CAGAACGCAG-3' (E13). No hybridization signal was obtained with probe (5'-TCCC TAGAATCCAGCTCGCTTGAC-3') from E12 (not shown). *, NotI shows partial digestion, which yields a 35-kb fragment from the 55-kb BamHI fragment. Below, the schematic drawing shows the order of the three first exons and corresponding 5'-flanking regions of the rat PRLR gene.
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Expression of the Mouse Counterparts of Rat Tissue-specific and Common First Exons, E11 and E13, in MLTC

Due to the lack of appropriate rat gonadal stable cell lines, we have utilized a mouse Leydig tumor cell line (MLTC-1) (18) for investigation of the transcriptional control of the PRLR gene promoters. For these studies, it was necessary to initially characterize the endogenous expression of PRLR alternative exons in these cells. Northern blot analysis showed a major mRNA transcript of 9.5 kb comparable to the 9.7 kb of the rat species using 5'-coding region of the rat PRLR as the probe (Fig. 2A, left). In addition, minor transcripts of 4.2, 2.4, and 1.4 kb were also revealed in MLTC. Based on the high similarity between the rat and the mouse PRLR cDNAs, primers derived from the rat E11 and E13 were used for RT-PCR analyses of mRNA from rat ovaries and MLTC cells. PCR products of identical sizes for E11 (325 bp) (Fig. 2B, lanes 2-4) and for E13 (229 bp) (lanes 5-7) were observed for the rat ovary and MLTC (mouse), indicating analogous expression of alternative first exons E11 and E13 in both species. Furthermore, recent genomic cloning of the mouse E13 and its 5'-flanking region showed 94% sequence similarity between the rat and the mouse within the noncoding exon E13 and the proximal 5'-flanking region (not shown). These results indicate that as in rat gonads, the PRLR expression is also under the control of putative promoters PI and PIII in MLTC cells.


Fig. 2. Endogenous expression of mouse counterparts of the rat PRLR exons E11 and E13 in MLTC. A, Northern blot analyses of PRLR mRNAs from MLTC cells (left) and rat ovary (right). The 32P-labeled probe of 0.4 kb used contained the common coding sequences for the long and short form of the rat PRLR (4). Left lane shows major transcript at 9.5 kb and minor species at 4.4, 2.4, and 1.4 kb. This lane is to be compared with the right lane showing the 9.7, 2.1, and 1.8-kb species previously demonstrated in rat ovaries (4). B, RT-PCR analyses of RNAs from MLTC (lanes 3, 4, 6, and 7) and rat ovaries (lanes 2 and 5). DNA standards are shown in lanes 1 and 8. Primers were derived from E11 and E13 as indicated below the gel. The primers used were 5'-GTGGCCAGAGCCATGGACAG-3' (upper strand) and 5'-AAACTCTTTCCTCGGAGGTCACTAG-3' (lower strand) for E11, 5'-TCTCAGAGACACGCGGCTG-3' (upper strand) and 5'-TTCTGCTGGAGAGAAAAGTCTG-3' (lower strand) for E13. The expected band size is indicated by the arrow in base pairs (bp). C, Northern blot analyses of PRLR mRNA from rat mammary gland (left) and Leydig cells (right). The probe used was the same as in A. The 1.8 kb is the major species in mammary tissue, whereas the 9.7 and 1.8 kb are the major species in rat Leydig cells (1). D, 5'-RACE PCR analyses of PRLR mRNA from the rat mammary gland. Primers at different locations were selected to generate different sizes of PCR products. PCR products of identical sizes as from those of the rat gonads and liver were obtained and hybridized by oligomer probes derived from E13 (lanes 1 and 2) but not from E11 (lanes 3 and 4) or E12 (lanes 5 and 6).
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The promoter utilization of the PRLR gene in rat mammary gland was also examined. Northern blot analyses showed that the major mRNA transcript in this tissue is the 1.8-kb species (Fig. 2C, left), which is also seen in the Leydig cells (right) and the liver (1), and corresponds to the short form of the receptor (1, 4). 5'-RACE PCR analyses of mRNA from the rat mammary gland showed that only E13 sequences were present in this tissue by sequencing the PCR products (not shown) and by Southern blot analysis of the PCR products (Fig. 2D). Neither E11 nor E12 sequences were found to be present in the mammary tissue by Southern hybridization by oligonucleotide probes derived from E11 (lane 3 and 4) or E12 (lane 5 and 6). This result indicated that PIII is the sole promoter utilized in the rat mammary gland and further demonstrated that the usage of PI is restricted to the gonads among the tissues examined.

Transcriptional Activity of Promoter I in MLTC Cells

A luciferase reporter gene construct containing the 5'-flanking region with adjacent E11 sequence [PI(-1566/-124)/LUC] was examined for its promoter activity in the steroidogenic MLTC and the non-steroidogenic cell line HepG2. This genomic fragment exhibited strong promoter activity, which was 43-fold over that of the promoterless vector and about 60% that of the SV40 promoter in MLTC. In contrast, only minimal induction was observed in HepG2 cells, where the SV40 promoter displayed activities comparable to those observed in MLTC cells (Fig. 3, left). This finding indicated the specificity of activation of promoter I in MLTC and is consistent with the pattern of promoter utilization in vivo, where PI activity was found only in gonads but not in the liver (1) and the mammary gland (Fig. 2D). The transcription initiation site from the PI(-1566/-124)/LUC construct transfected in MLTC was determined by the 5'-RACE PCR method. An extended product of ~540 bp was revealed on Southern hybridization of the PCR products by a nested probe (GS3, Fig. 3, right, above), and the transcriptional initiation site derived from this experiment was consistent with that reported for the rat ovary (-549 relative to translation initiation codon at +1) (1) (Fig. 3, right, below). These results indicate that MLTC is an appropriate cell line for characterization of the regulatory components of this gonad-specific promoter (PI), since the PI/LUC construct is highly active and faithfully transcribed in MLTC.


Fig. 3. Promoter I activity in MLTC and HepG2 cells and determination of the transcription start site for the expressed PRLR-PI/LUC gene construct in MLTC cells. Left, the luciferase reporter construct PI(-1566/-124)/LUC containing the 5'-flanking region and partial exon 1 (E11) sequences was transiently expressed in MLTC and HepG2 cells. The luciferase activities are expressed in fold induction over that of the basic vector (lacking PRLR gene sequence). Right, 5'-RACE PCR analysis of reporter gene mRNA transcripts from PI(-1566/-124)/LUC transfected in MLTC. The two gene-specific primers (GS1 and GS2) used were derived from regions of the luciferase gene to avoid interference from the endogenous mouse PRLR mRNA. Specific extended products of ~540 bp were generated from both total RNA (lane 1) and poly(A+) RNA (lane 2). The scheme for the analysis is indicated below the Southern blot. The sequences for GS1 and GS2 were 5'-AGCGGTTCCATCCTCTAG-3' and 5'-CTTTATGTTTTTGGCGTCTTCCA-3' located at +24 and +66 of the luciferase coding region, respectively. The wavy line is a portion of the polylinker sequence from the vector (55 bp). CC in the lower right indicates dC-tailing of the first strand cDNA synthesized with GS1. The sequence of dG adaptor primer is 5'-GCGAATTCTCGAGATCTGGGIIGGGIIGGGIIG-3'. The calculated size from the reporter construct is 538 bp.
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Localization of Promoter Domain of PI by Deletion Analysis and Identification of Cis-element(s) and Specific Nuclear Protein Binding to the Promoter Region of PI in MLTC

The PRLR PI/LUC constructs with serial deletion of the 5'-flanking region were expressed in MLTC to localize the minimal promoter domain. Deletion of the 5'-flanking sequences beyond -700 had no significant effect on luciferase activity (Fig. 4). The region between -700 and -549 was required for promoter activity, whereas deletion of the downstream exon 1 sequences (-124 to -549) had no significant effect on promoter activity. Thus, the 152-bp region between -700 and -549 retained full promoter activity in transfected MLTC cells. In contrast, the PI(-1566/-124)/LUC construct (Fig. 3) and the deletion mutants of PI including the minimal promoter domain -700/-549 (not shown) exerted only minor promoter activities in HepG2 cells, further indicating the tissue specificity of promoter I for expression in gonadal cells. The regulatory cis-elements and transcriptional activators required for this promoter were further analyzed by the DNA-protein binding analyses and site-directed mutagenesis of regulatory elements within the promoter domain.


Fig. 4. Basal activities of deletion constructs of the 5'-flanking region PI-exon E11 in MLTC. Left, schematic representation of deletion constructs expressed in MLTC cells. Luciferase reporter gene constructs with progressive 5' deletions of the 5'-flanking region as well as the 3' deletions of the adjacent first exon E11 are shown. Plasmid constructs are identified by nucleotide position calculated from the first base of translation initiation codon (ATG, +1). The transcription initiation site is indicated by arrowhead (-549). Right, the activities of the constructs were expressed as fold induction of luciferase activity over that of the basic vector. Results are expressed as mean ± S.E. of four to six independent experiments in triplicate.
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DNase I footprinting analysis using nuclear extracts from MLTC was employed to examine the specific nuclear protein binding activities within the promoter domain. DNA probes corresponding to the region of -440 to -827 labeled at 5' ends of either upper or lower strands were used in the DNase I footprinting assay (Fig. 5). Regions at -679 to -661 and -681 to -663 were protected for upper (Fig. 5, lanes 3-5) and lower strands (lanes 13-15), respectively, indicating a single footprint with asymmetrical binding of the protein complex to the double helixes of this region (Fig. 5, middle). Within this footprint resides the consensus binding element for the steroidogenic factor 1 CCAAGGTCA. Preincubation with unlabeled DNA sequences containing the SF-1 element (see also Fig. 6) can effectively prevent formation of the footprint (Fig. 5, lanes 6, 7, 16, and 17), whereas the mutated SF-1 sequence (m1, see also Fig. 6) had no effect (lanes 8 and 18). Although the region -628/-648 downstream of the putative SF-1 element contains a consensus C/EBP binding site (TTGTGTAA), sequences containing either wild type (lanes 9 and 19) or mutation of the C/EBP binding site (lanes 10 and 20) did not affect the footprinting pattern, indicating that the nuclear protein complex binds specifically to a single region containing the SF-1 element.


Fig. 5. DNase I footprinting analysis of promoter I protein binding domain using MLTC nuclear extracts. Both upper strand end-labeled probe (left) and lower strand end-labeled probe (right) were used in the assay. Probes were incubated with increasing concentrations of nuclear proteins (lanes 3-5 and 13-15), or bovine serum albumin (BSA) (20 µg, lanes 2 and 12) as control. The probes were also incubated with nuclear proteins in the presence of unlabeled DNA competitors as indicated (lanes 6-10 and 16-20). The molecular ladders are from sequencing reactions using primers derived from the labeled ends of either probes (see "Materials and Methods"). The protected region on either strand was indicated by shaded boxes. The sequences protected on both strands and their positions in relation to the transcription initiation site (-549) are indicated (between gels).
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Fig. 6. Electrophoresis mobility shift assay (EMSA) of binding of DNA footprinting sequences by nuclear proteins from MLTC cells. Double-stranded DNA probes A (-689/-650) and B (-689/-628) containing sequences corresponding to the footprint (sequences in bold, below) were used for the EMSA. Probe A contains the consensus SF-1 element (underlined sequences shown below) and probe B (DNA fragment generated by PCR) contains additional consensus sequence for C/EBP element (TTGTGTAA). Wild-type and mutated sequences of the probe were shown below. Probes were incubated with the nuclear protein in the absence (lanes 1, 10, and 15) or presence of excess molar of unlabeled DNA competitors, wild-type DNA (lanes 2-4, 16, 17, and 24), or mutated DNA (lanes 5-9 and 18-20), or in the presence of normal rabbit serum (NRS, lanes 11 and 21) or SF-1 antibodies (Ab1, lanes 12 and 22; Ab2, lanes 13, 14, and 23). The specific DNA-protein complex (SF-1) and free probes are indicated by arrows. SF-1 is defined as the protein from ovarian granulosa and Leydig cells that is responsible for DNA-specific retardation of the designated band.
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Electrophoretic mobility shift assay of MLTC nuclear extract using oligonucleotide probes (probes A and B, Fig. 6) containing the footprint sequence showed a major protein complex (Fig. 6, lanes 1, 10, and 15) that was specifically competed by unlabeled wild-type sequence even at a molar excess of 10 (lanes 2-4, 16, and 17) and was either supershifted (lanes 12 and 22) or inhibited (lanes 13, 14, and 23) by specific SF-1 polyclonal antibodies but not by normal rabbit serum (NRS, lanes 11 and 21). Mutation of the SF-1 element (m1, m2, and m3, Fig. 6, lanes 5-7, 18-20, and below) did not compete for the SF-1 protein binding, indicating that CCAAGGTCA (-676 to -668) is required for the SF-1 binding, conforming to the sequence requirement for SF-1 in other genes (19). It was noted that a sequence (AACAGGCCA, -682 to -674) resembling the SF-1 consensus sequence is overlapping (underlined) at 5' with the SF-1 binding site (in bold) (CGAAACAGGCCAAGGTCAAAC). To more accurately define the SF-1 binding site, the adjacent nucleotides were mutated and the DNA binding activity was evaluated. When the upstream AGG (-677) was mutated to cta (Fig. 6, m4), or cat, or tta (not shown), the SF1 binding was completely competed (Fig. 6, lane 8) by unlabeled mutant sequences. Furthermore, mutation of nucleotides AAC at -665 (m5) and at further upstream sites (-680, not shown) to ctg also competed for the binding, indicating that these flanking nucleotides are not required for SF-1 binding activity. These results demonstrate that the SF-1 binding site resides at CCAAGGTCA (-676/-668) but not at AACAGGCCA (-682/-674). Moreover, the longer probe B (62 bp) containing the consensus sequence for C/EBP (TTGTGTAA) in addition to the SF-1 element (Fig. 6, lower right) was shown to bind to the same protein complex(es) as the shorter probe A (30 bp) lacking the C/EBP sequence (lanes 15-23). This DNA-protein complex was not competed by the sequence -648/-628 (lane 24), further indicating that the consensus C/EBP site did not have significant binding activity at the given condition (Fig. 6). These findings are consistent with those derived from footprinting analysis that revealed a single protected region corresponding to the SF-1 element.

The trans-factors for promoter I present in the nuclei of the rat gonadal cells were also evaluated using EMSA. A single protein complex from granulosa cell nuclear extracts was shown to bind CCAAGGTCA (Fig. 7, lanes 1-10) as was found in MLTC cells. The retarded band was supershifted (lane 12) or inhibited (lane 14) by SF-1 antibodies but not by normal rabbit serum (lane 11). Similar results were obtained with rat Leydig cell nuclear protein extracts (lanes 15-23). However, the SF-1 binding complex was resolved as a doublet, and these bands may represent different states of the SF-1 protein.


Fig. 7. EMSA of binding of DNA containing SF-1 binding sequence by nuclear proteins from rat ovarian granulosa and testicular Leydig cells. Probe A (-689/-650) was incubated with the ovarian (lane 1-14) or testicular nuclear extracts (lanes 15-23) in the absence (lanes 1, 10, and 15) or the presence of designated molar excess of unlabeled competitors (lanes 2-4 and 16, wild type; lanes 5-9 and 17-19, SF-1 mutated). The specificity of SF-1 binding is confirmed by the use of two SF-1 antibodies Ab1 and Ab2 (lanes 12-14 and 21-23) to be compared with the negative control of normal rabbit serum (NRS, lanes 11 and 20). The two antibodies used are presumed to act as follows. Ab1 supershifts the SF-1·DNA complex after binding to the exposed surface of SF-1. In contrast, Ab2 inhibits the binding of SF-1, probably by interference or blockade of the SF-1 protein site that is required for interaction with the SF-1 DNA element. Specific DNA-protein complexes are indicated by arrowhead. Free DNA probes are also indicated (Free).
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Transcriptional Activation of Promoter I by SF-1 in MLTC and in Rat Gonadal Cells

Mutation of the SF-1 binding site not only abolished the SF-1 protein binding activity (Figs. 6 and 7) but also markedly decreased the promoter activity (by 8-fold) in MLTC (Fig. 8, construct 2). Similarly, deletion of SF-1 element between -660 and -700 decreased the promoter activity by 10-fold (construct 5). This indicates a crucial role for SF-1 in the transcriptional activation of promoter I in MLTC. A further reduction to basic level in promoter activity was observed when the deletion was extended to the position -627 (construct 6), which disrupted the consensus CCAAT box sequences (CCAATTA, -629/-623), suggesting that a sequence element downstream of -660 may also contribute to the basal promoter activity. Mutation of the CCAAT box caused a small but significant reduction (30%) in promoter activity (Fig. 8, construct 3). However, the activity attributable to the CCAAT element was only 10% in the absence of SF-1 sequence (mutation or deletion). Therefore, the CCAAT box element may participate minimally in the basal transcriptional activation of PI. In contrast, mutation of the two adjacent TATA-like sequences positioned at 10 and 23 bp 5' from the transcriptional initiation site caused marked activation of promoter I (by 113%).


Fig. 8. Functional analysis in MLTC of PI promoter domain by mutation of putative regulatory cis-elements. The minimal promoter region of PI (-700/-549) (construct 1) and its mutants (constructs 2-4) as well as deletion mutants (constructs 5 and 6) were transiently expressed in MLTC. The relative promoter activities are indicated as percentages of the wild-type activity. Data were collected from 3 to 6 independent transfections of triplicate wells and expressed as mean ± S.E. Arrows indicate the transcriptional initiation site. Numbers indicate the nucleotide position relative to the translation initiation site.
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The promoter domain -700/-549 was also evaluated for its transcriptional activation in primary cultures of rat granulosa cells and Leydig cells using recombinant adenovirus-reporter gene infection method. In granulosa cells, the wild-type PI promoter domain (-700/-549) showed 7.6-fold induction of luciferase activity over that of the promoterless adenovirus-reporter gene construct (Basic) (Fig. 9, left), an induction that is about 28% that observed for the control SV40 promoter (SV40). Mutation of the SF-1 element reduced the activity to 25% wild type (SF1X), indicating that this factor is also a major transcriptional activator of this promoter in the granulosa cells. In rat Leydig cell, PI promoter domain caused a significant induction (5.9-fold) over the basic construct, and the SF-1 mutated construct displayed reduced promoter activity to 33% wild type (Fig. 9, right) but significantly higher than that of the basic. Thus, the findings in the Leydig cells are consistent with those observed in MLTC and the rat granulosa cells.


Fig. 9. Transient expression of adenovirus constructs of promoter I and its mutants in rat granulosa and Leydig cells. Rat granulosa cells (left) and Leydig cells (right) were transiently infected with the adenovirus constructs containing the minimal promoter domain of PI(-700/-549)/LUC/Adv (wild type) or its SF-1 mutant adenovirus construct (SF1X). Promoterless (Basic) and SV40 promoter-driven (SV40) adenovirus constructs were also used for infection of both cell types. The promoter activities are indicated as percentages of the wild-type activity (100%). The data are expressed as mean ± S.E. of three independent infections in quadruplicate.
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DISCUSSION

Previous studies have identified three putative PRLR gene promoters (PI, PII, and PIII) that are alternatively utilized in a tissue-specific manner and suggested that the expression of PRLR in different tissues may be subject to differential regulation by different regulators at various levels (1). In this study, we have demonstrated that the three alternative promoters are co-localized in the rat chromosome 2q16 in the order of 5'-PIII-PI-PII-3', indicating that multiple PRLR mRNA species are transcribed from the same PRLR gene through usage of alternative promoters.

Our current study has predominantly focused on the elucidation of the mechanism controlling the gonad-specific expression of E11 by promoter I. The mouse Leydig tumor cell line (MLTC) has been used for characterization of cis-elements and transcription factor(s) required for promoter I activity. Analyses of endogenous PRLR mRNA transcripts in MLTC indicated that the mouse gonadal cell line expresses the counterparts of the rat first exons E11 and E13 and therefore possess promoters I and III activities. The expression of the PI(-1564/-124)/LUC construct in MLTC and HepG2 cells has not only demonstrated faithful transcription initiation from this transfected construct but also revealed preferential activation of PI in MLTC. This is in agreement with the in vivo expression pattern (1), where PI is utilized in the rat gonads but not in the rat liver.

From deletion analyses, we have identified a 152-bp region (-700 to -549) as the minimal promoter I domain of the PRLR gene (Fig. 4), whereas sequences upstream of -700 do not influence the transcriptional activation of this promoter in transfected MLTC cells. We demonstrated that steroidogenic factor-1 binds with high affinity to the sequence CCAAGGTCA (-676/-668) positioned 119 bp upstream of the transcription initiation site, which is essential for the activity of this promoter in MLTC cultures as well as in primary cultures of rat ovarian granulosa and Leydig cells. The position of the SF-1 element in this promoter domain is consistent with its location in SF-1-dependent steroidogenic P450 genes (9, 19). SF-1 is a zinc finger DNA binding protein and is a member of the orphan nuclear receptor superfamily (9, 20). SF-1 was first identified as a transcriptional activator for steroidogenic P450 genes (9, 19) and subsequently was found to be important for a number of other genes including inhibin, müllerian inhibiting substance, oxytocin, and gonadotropin alpha and beta  subunits (21-25). The targeted disruption of the Ftz/F1 gene in the mouse has demonstrated that SF-1 is not only essential for steroidogenesis but is also crucial for gonadal and adrenal development and sexual differentiation (8). The absence of an SF-1 binding element in the promoter region of PII and PIII (1) may explain the lack of expression of the liver-specific promoter II in the gonads and suggests that a different transcriptional mechanism is involved in the activation of promoter PIII. Conversely, SF-1 protein is not expressed in the liver or mammary gland, which may explain the lack of PI activity in both tissues. In contrast, hepatocyte nuclear factor 4, an abundant transcription factor in the liver, has been reported to be a transcriptional activator of the promoter II that is functional in the liver (6). In our study, only PIII but neither PI nor PII activities was found in the rat mammary gland as revealed by Northern blot and 5'-RACE PCR analyses of mRNA transcripts, consistent with the notion that PI and PII are utilized specifically in the gonads and liver, respectively.

To our knowledge, this is the first report of a receptor gene that requires SF-1 as the major transcriptional activator and for the tissue-specific utilization of its alternative promoters. Although the gonadotropin releasing hormone receptor gene was recently shown to require a functional SF-1 element for its specific expression in alpha T3-1 cells, mutation of an upstream element in the presence of the downstream SF-1 element caused 63% reduction in promoter activity, indicating that SF-1 is not a major transactivator but a co-activator of the gonadotropin releasing hormone receptor gene promoter (26). Although the rat luteinizing hormone receptor gene contains an SF-1 consensus sequence located 5' adjacent to its promoter domain, it was not found to be functional in the rat.2 Furthermore, different mechanisms are involved in the promoter activation of all other pituitary hormone receptors cloned to date. Therefore, the gene for the PRLR can be viewed as one of a class of genes regulated by SF-1, unique among those of the receptors for pituitary trophic hormones. This may imply important functional correlation between PRLR and other SF-1 regulated gonadal-specific genes in the regulation of reproductive functions.

Additional factors may be required for full activation of this promoter, since mutation or deletion of SF-1 motif (-676/-668) greatly (70-90%) but not completely abolished the promoter activity. Although our current study using DNA-protein binding analyses has not identified the existence of an additional nuclear protein binding site within the promoter region that may contribute to the basal promoter activity, this could not be ruled out. It is probable that the presence of low affinity or unstable nuclear protein binding may not be readily detected by these approaches. Among other potential functional elements present within this promoter, the consensus C/EBP element (-643/-636) did not bind to C/EBP or participate in the activation of the PI promoter (not shown). The CCAAT element and the two adjacent TATA-like sequences present within this promoter located 75 and 10 base pairs, respectively, from the TSS, appeared to affect the basal promoter activation (Fig. 8). The disruption of the CCAAT box sequence caused a minor reduction (30%) in the promoter activity. However, in the absence of a functional SF-1 element (mutation or deletion), the region containing the CCAAT box showed only ~10% of the wild-type activity. This activity was comparable to that observed in HepG2 cells transfected with PI(-1566/-124)/LUC. However, the minimal activity presumably induced by the CCAAT element in transfected HepG2 cells was not effective for transcriptional activation in vivo since expression of the exon E11 was not observed in liver or mammary gland. The pentanucleotide CCAAT sequence is commonly found 60-80 bp upstream of the TSS (27). A number of nuclear proteins that can specifically bind to the CCAAT element have been identified, including human CCAAT-binding proteins CP1, CP2, and NF-1 (28) and the family of CCAAT/enhancer-binding protein (C/EBP alpha , beta , gamma , delta , epsilon , and CHOP10) (29). However, the possibility that member(s) of this large family of proteins may interact with the CCAAT element in PI promoter and affect its basal activity remains speculative. In addition, the TATA-like elements and presumably their associated proteins may exert constitutively down-regulatory influence(s) in promoter I function, conceivably through interaction with SF-1 or the preinitiation complex, whose formation should be presumably independent of the TATA boxes in this promoter. Therefore, the PRLR promoter I belongs to the class of TATA-less/non-initiator, GC box-less gene promoters.

Our studies have demonstrated that the SF-1 protein binds to its cognate regulatory element within the PI promoter domain and exerts a dominant effect on the activation of this promoter. In addition, a CCAAT element may have minor contribution to the basal promoter activity, and a TATA-like region may provide a constitutive inhibitory site for this promoter. These important functional gene structures are amenable to multifunctional regulation during the development of testicular Leydig cells from fetal to adult life and at the different stages of the ovarian cycle.


FOOTNOTES

*   The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
   To whom correspondence should be addressed: Bldg. 49, Rm. 6A 36, 49 Convent Dr. MSC 4510, National Institutes of Health, Bethesda, MD 20892-4510. Tel.: 301-496-2021; Fax: 301-480-8010.
1   The abbreviations used are: PRLR, prolactin receptor; SF-1, steroidogenic factor-1; 5'-RACE, rapid amplification of cDNA 5' ends; RT-PCR, reverse transcriptase-polymerase chain reaction; bp, base pairs; kb, kilobase(s); DTT, dithiothreitol; LUC, luciferase; MLTC, mouse Leydig tumor cells; EMSA, electrophoresis mobility shift assay; C/EBP, CCAAT/enhancer-binding protein; TSS, transcriptional start site; Adv, adenovirus.
2   C.-H. Tsai-Morris and M. L. Dufau, unpublished observation.

ACKNOWLEDGEMENTS

We thank Drs. Joseph L. Alcorn and Carole A. Mendelson (University of Texas Southwestern Medical Center, Dallas, TX) for providing plasmids pAC and pJM17 (13) and Dr. Ken-ichirou Morohashi (Kyushu University, Kyushu, Japan) for providing Ad4BP antibody (15).


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