Molecular cloning of estrogen receptor alpha (ERα; ESR1) of the Japanese giant salamander, Andrias japonicus

Molecular and Cellular Endocrinology 257–258 (2006) 84–94

Molecular cloning of estrogen receptor alpha (ER␣; ESR1) of the Japanese giant salamander, Andrias japonicus Yoshinao Katsu a,b , Satomi Kohno c , Tomohiro Oka d , Naoko Mitsui d , Osamu Tooi d , Noriaki Santo d , Hiroshi Urushitani a,d , Yukio Fukumoto e , Kazushi Kuwabara e , Kazuhide Ashikaga e , Shinji Minami e , Shigeaki Kato f , Yasuhiko Ohta g , Louis J. Guillette Jr. c , Taisen Iguchi a,b,∗ a

Okazaki Institute for Integrative Bioscience, National Institute for Basic Biology, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki 444-8787, Aichi, Japan b Department of Basic Biology, The Graduate University for Advanced Studies (SOKENDAI), Aichi, Japan c Department of Zoology, University of Florida, Gainesville, FL 32611, USA d Biotechnology Research Laboratory, Towakagaku Co. Ltd., Hiroshima, Japan e Hiroshima City Asa Zoological Park, Hiroshima, Japan f Institute of Molecular and Cellular Biosciences, University of Tokyo, Japan g Laboratory of Animal Science, Department of Veterinary Science, Faculty of Agriculture, Tottori University, Koyama, Tottori, Japan Received 6 April 2006; received in revised form 11 July 2006; accepted 13 July 2006

Abstract Estrogens are essential for normal reproductive activity in females and males and for ovarian differentiation during a critical developmental stage in many vertebrates. To understand the molecular mechanisms of estrogen action and to evaluate estrogen receptor ligand interactions in the Japanese giant salamander (Andrias japonicus), we isolated cDNA encoding the estrogen receptor (ER) from the liver. A full-length Japanese giant salamander ER cDNA (jgsER) was obtained using 5 and 3 rapid amplification cDNA ends (RACE). The deduced amino acid sequence of the jgsER showed high identity to the Xenopus ER␣ (ESR1) (77.7%). We have applied both the conventional ERE-luciferase reporter assay system and the GAL4-transactivation system to characterize this receptor. In two different transient transfection assay systems using mammalian cells, the jgsER protein displayed estrogen-dependent activation of transcription. The GAL4-transactivation system showed about 10-fold greater activity of the estrogen receptor by hormone when compared to the conventional ERE-luciferase reporter assay system. Tissue distribution of ER␣ mRNA was examined and kidney, ovary and liver exhibited expression. This is the first isolation of an estrogen receptor from a salamander and also is the first functional cDNA obtained from the Japanese giant salamander, an endangered species considered a special natural monument of Japan. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Japanese giant salamander; Estrogen receptor; Cloning; Transactivation; Receptor evolution

1. Introduction Estrogens play important roles in the reproductive biology of vertebrates including amphibians. The majority of actions of estrogens are mediated by specific receptors that are localized in the nucleus of target cells. Estrogen receptors (ERs) belong to a superfamily of nuclear transcription factors that include all other steroid hormone receptors such as progestogens, androgens, glucocorticoids, mineralocorticoids, Vitamin

∗

Corresponding author. Tel.: +81 564 59 5235; fax: +81 564 59 5236. E-mail address: [email protected] (T. Iguchi).

0303-7207/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2006.07.001

D receptor, and the retinoic acid receptor (Blumberg and Evans, 1998). Three distinct types of ER have been isolated to date in vertebrates. Teleost fish have ER␣, ER␤ and ER␥-form but the teleost ER␥-form appears to be closely related to the teleost ER␤, suggesting that it reflects the gene duplication event which occurred within the teleosts (see Hawkins et al., 2000). Thus, the ancestral condition for the jawed vertebrates (Gnathostomata) is considered to be the presence of two forms of ER, corresponding to ER␣ and ER␤ (Thornton, 2001). Indeed, these two forms of ER have been previously found in mammals, fish, birds, reptiles and amphibians. cDNAs encoding ER␣ have been cloned from several vertebrate species including mammals (Green et al., 1986; Koike et al., 1987; White et al., 1987), birds (Krust

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Fig. 1. Photograph of the Japanese giant salamander, Andrias japonicus.

et al., 1986), reptiles (Sumida et al., 2001; Katsu et al., 2004, 2006), amphibians (Weiler et al., 1987) and teleost fish (Pakdel et al., 1990). Estradiol-17␤ is the principal estrogen in circulation and appears essential for normal ovarian development in many vertebrates (Wallace, 1985). Embryonic exposure to inhibitors of aromatase, the enzyme responsible for the conversion of testosterone to estradiol-17␤, cause genetic females to become phenotypic males in chicken and turtle (Elbrecht and Smith, 1992; Dorizzi et al., 1994). Likewise, embryonic exposure of various fishes, amphibians or reptiles to estradiol-17␤ or estrogenic chemicals, pharmaceutical agents or environmental contaminants, can induce highly skewed sex ratios toward females (for reviews, see Guillette et al., 1996; Tyler et al., 1998; Iguchi et al., 2001; Milnes et al., 2006). In amphibians Xenopus laevis has been used as a model for investigating endocrine disruption by environmental chemicals (Oka et al., 2006; Huang et al., 2005; Sone et al., 2004). It is not clear whether pharmaceutical agents or environmental contaminants influence the endocrine system of the Japanese giant salamander, and the reciprocal actions of the environmental chemicals and ER of the salamander are not fully understood. A number of studies strongly suggest that endogenous estradiol-17␤ acts as a natural inducer of ovarian differentiation in non-mammalian vertebrates (Devlin and Nagahama, 2002; Sinclair et al., 2002). However, the mechanisms of estrogen action on ovarian differentiation in any non-mammalian vertebrates remain under study. There are three orders within the class Amphibia: the Anura or frogs, the Caudata or salamanders and the Gymnophiona or caecilians. Within the salamanders, only three extant species of giant salamander remain: the Japanese giant salamander (Andrias japonicus), the American giant salamander or Hellbender (Cryptobranchus alleganiensis) and the Chinese giant salamander (Andrias davidiaus). The Japanese giant salamander is one of the largest extant amphibians and is considered a special natural monument of Japan (Fig. 1). This giant salamander was first provided legal protection in 1952, and was recognized as an endangered species (IUCN, The IUCN Red List of Threatened Species). It has no natural predators, but is losing its habitat to deforestation and/or to shore protection works. The Japanese salamander can weigh 25 kg and obtain 1.5 m in total length. As with the other salamanders, the Japanese giant salaman-

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der inhabits cold mountain streams and rivers, primarily in the regions of Oita Prefecture in Kyushu Island and Western Honshu Island, Japan. Little is known concerning the reproductive biology of this species, but reproductive activity begins in August with spawning in early September (Kawamichi and Ueda, 1998; Kuwabara et al., 1980, 2005). Females lay between 400 and 500 eggs in the fall, and males aggressively guard the nests, which may contain eggs from several females, until they hatch in the early November. Almost nothing is known concerning the reproductive endocrinology of these species but it is assumed to be similar to that of other amphibians. Although frogs, such as the African clawed frogs (X. laevis), are major experimental animals for developmental biology and endocrinology much less is known concerning the other amphibian groups although extensive neuroendocrine work has been performed on newts (Urodela) (for review, see Moore et al., 2005). However, even with this extensive work, only two estrogen receptor sequences have been reported from amphibians, both are for frog species of the genus Xenopus. To begin to understand the endocrine system of the Japanese giant salamander, as well as provide some data on the evolution of the vertebrate estrogen receptors, we isolated cDNA clones encoding Japanese giant salamander homologs of ER␣ from the liver. We analyzed its phylogenic relationship with other known vertebrate ERs. Further, the transactivation function of the Japanese giant salamander ER␣ was determined by expressing the ER␣ in transiently transfected CHO-K1 cells using a general reporter gene assay with an ERE-luciferase construct and the GAL4 system. The ER␣ mRNA levels in various tissues from the Japanese giant salamander also were measured by reverse transcription-polymerase chain reaction (RT-PCR). 2. Materials and methods 2.1. Experimental animal The female Japanese giant salamander (registration no. 380, individual no. 12154) was born in 1989 and maintained under natural conditions in the Hiroshima City Asa Zoological Park. Just after death (28 August 2004) from natural causes, various tissues were collected and preserved for study. The body weight and total length were 2.44 kg and 670 mm, respectively.

2.2. Reagents Estradiol-17␤ (E2), estrone (E1), estriol (E3), 17␣-ethynylestradiol (EE2), testosterone (T), progesterone (P) were purchased from Sigma Chemical Co. (St. Louis, MO). As the representative endocrine disruptive chemicals, bisphenol A (BPA) and nonylphenol (NP) were purchased from Nacalai Tesque (Kyoto, Japan) and Tokyo Kasei (Tokyo, Japan), respectively. All chemicals were dissolved in dimethylsulfoxide (DMSO). The concentration of DMSO in the culture medium did not exceed 0.1%.

2.3. Molecular cloning of estrogen receptor Two conserved amino acid regions in the DNA-binding domain (CAVCNDY) and the ligand-binding domain (MKCKNVV) of the ER␣ were selected and their degenerate oligonucleotides were used as primers for polymerase chain reaction (PCR). As a template for PCR, first-strand cDNA was synthesized from 2.0 ␮g of total RNA isolated from the liver of Japanese giant salamander. After amplification, two additional primer sets, MCPATNQ and KCVEGMV were used for the second PCR reaction. The amplified DNA fragment was sub-

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cloned with the TA-cloning plasmid pGEM-T Easy (Promega, Madison, WI), sequenced using a BigDye terminator Cycle Sequencing-kit (PE Biosystems, Foster City, CA) with T7 and SP6 primers, and analyzed on the ABI PRISM 377 automatic sequencer (PE Biosystems). The 5 - and 3 -ends of the ER cDNA were amplified by rapid amplification of the cDNA end (RACE) using a SMART RACE cDNA Amplification kit (BD Biosciences Clontech., Palo Alto, CA).

2.4. RNA isolation and RT-PCR Total RNA was isolated from kidney, spleen, lung, stomach, skin, small intestine, large intestine, ovary, heart, pancreas and liver of adult female giant salamander using RNeasy (QIAGEN, Chatsworth, CA). For RT-PCR, 2.0 ␮g of total RNA was reverse transcribed using SuperScript III transcriptase (Invitrogen, Carlsbad, CA) and oligo (dT) primer. The following primer sets were used for RT-PCR: actin-1, 5 -CCTGAACCCCAAAGCCAACAGAG3 and actin-2, 5 -GCCAGGGCTGTAATCTCCTTCTG-3 for ␤-actin and ER-1, 5 -CGATCAGAAGCCAGAGGAGGCTG-3 and ER-2, 5 GGGGATTCCACGGTGTTAGACAG-3 for ER. Twenty-eight cycles of amplification were carried out under the following conditions: denaturation at 94 ◦ C for 30 s, annealing at 60 ◦ C for 30 s and extension at 72 ◦ C for 1 min. At completion of the PCR, fragments were resolved on 1.5% agarose gels.

2.5. Construction of plasmid vectors pcDNA3.1–jgsER␣ and pBIND–jgsER␣ were constructed by PCR amplification of the entire protein coding region (amino acids 1–560) of the jgsER␣, using primers (5 -ATGCCCCTTCACAGCAAGATC-3 and 5 TCATACGGCGCTCTTCAAACTCG-3 ) with KOD DNA polymerase (TOYOBO Biochemicals, Osaka, Japan). PCR product was gel-purified and ligated into pcDNA3.1(+) vector (Invitrogen) for pcDNA3.1–jgsER␣ or pBIND vector (Progema) for pBIND–jgsER␣. An estrogen-regulated reporter vector that has one estrogen-responsive element (ERE) (GGATCnnnAATCG), named pGL3Basic-1× ERE-tk-Luc, was construct as described previously (Kobayashi et al., 2000). The construct having four EREs, named pGL3-Basic-4× ERE-tkLuc, was construct by subcloning of oligonucleotides having 3× ERE into the KpnI–SacI site of pGL3-Basic-ERE-tk-Luc.

2.8. Phylogenic analysis of estrogen receptor sequences All sequences generated were searched for similarity using blastn and blastp at web servers of the National Center of Biotechnology Information. Deduced amino acid sequences were aligned by using the Clustal X computer program (Jeanmougin et al., 1998). The phylogenic tree was constructed using the Phylip computer program (Felsenstein, 2004) with the JTT matrix, neighbor-joining method and bootstrap re-sampling for 1000 times. Accession numbers of the ER␣ amino acid sequences included in the tree are: CAC37560 (Clarias gariepinus), Q9YHZ7 (Ictalurus punctatus 1), AAC69548 (I. punctatus 2), AAR17610 (Carassius auratus 1), AAL12298 (C. auratus 2), P57717 (Danio rerio 1), BAB16893 (D. rerio 2), CAD32175 (Candidia barbatus), BAD91035 (Rutilus rutilus), AAU87498 (Pimephales promelas), A37197 (Oncorhynchus mykiss 1), CAB45140 (O. mykiss 2), AAY25396 (Salmo salar), P16058 (O. mykiss 3), AAS92970 (Oncorhynchus masou), BAB85622 (Paralichthys olivaceus), BAC76957 (Fundulus heteroclitus 1), AAT72914 (F. heteroclitus 2), P50241 (Oryzias latipes), BAA25900 (Oryzias sp.), AAX13999 (Oryzias javanicus), AAR82891 (Astatotilapia burtoni), Q9YH33 (Oreochromis niloticus), P50240 (Oreochromis aureus), AAO66473 (Zoarces viviparus), AAL82743 (Acanthopagrus schlegelii), Q9PVZ9 (Sparus aurata), O42132 (Pagrus major), AAG44622 (Micropterus salmoides), CAD43599 (Dicentrarchus labrax), ABB96483 (Pseudolabrus japonicus), AAP72178 (Halichoeres tenuispinis), AAQ84783 (Xenopus laevis 1), AAQ84784 (X. laevis 2), P81559 (X. laevis 3), AAQ84782 (X. laevis 4), AAQ84780 (Xenopus tropicalis), BAB79437 (Cnemidophorus uniparens), Q91250 (Taeniopygia guttata), P06212 (Gallus gallus), AAN63674 (Coturnix japonica), BAE45626 (Crocodylus niloticus), BAD08348 (Alligator mississippiensis), BAB79436 (Caiman crocodiles), Q9QZJ5 (Mesocricetus auratus), P19785 (Mus musculus), CAA43411 (Rattus norvegicus 1), P06211 (R. norvegicus 2), XP 533454 (Canis familiaris), AAU11443 (Felis catus), 1204262A (Homo sapiens 1), P03372 (H. sapiens 2), Q9TV98 (Equus caballus), Q29040 (Sus scrofa) and P49884 (Bos taurus). Accession number for the ER␤ amino acid sequence of X. tropicalis or laevis is AAQ84781 or Q6W5G8, respectively.

3. Results 2.6. Transactivation assays CHO-K1 cells were seeded in 24-well plates at 5 × 104 cells/well in phenolred free Dulbecco’s modified Eagle’s medium and F12-Ham (Sigma–Aldrich Corp., St. Louis, MO) supplemented with 10% charcoal/dextran treated fetal bovine serum (Hyclone, South Logan, UT). After 24 h, the cells were transfected with 400 ng of pGL3-Basic-ERE-tk-Luc, 100 ng of pRL-TK (as an internal control to normalize for variation in transfection efficiency; contains the Renilla reniformis luciferase gene with the herpes simplex virus thymidine kinase promoter; Promega, Madison, WI), and 200 ng of pcDNA3.1–jgsER␣ using Fugene 6 transfection reagent (Roche Diagnostics, Basel, Switzerland) according to the manufacturer’s instructions. After 1 h of incubation, various steroid hormones were applied to the medium. After 48 h (Chang et al., 1999), the cells were collected, and the luciferase activities of the cells were measured by a chemiluminescence assay with Dual-Luciferase Reporter Assay System (Promega). Luminescence was measured using a Turner Designs Luminometer TD-20/20 (Promega). Promoter activity was calculated as firefly (Photinus pyralis)-luciferase activity/sea pansy (R. reniformis)-luciferase activity. All transfections were done at least three times, employing triplicate sample points in each experiment. For analysis of the transcriptional activation of ER using the GAL4 system, the cells were transfected with 100 ng of pG5-luc and 100 ng of pBIND–jgsER␣ (containing the R. reniformis luciferase gene for control to normalize the variation in transfection efficiency) using the Fugene 6 transfection reagent (Roche) according to the manufacturer’s protocols.

2.7. Data analysis Statistical analyses were carried out using t-tests or Welch’s tests. All data are reported as mean ± S.E.M. In all cases, means were considered significantly different at p < 0.05.

3.1. Cloning and sequence of Japanese giant salamander ERα A DNA fragment was obtained and sequence analysis showed that the fragment had similarity to ER␣ (data not shown). Using the RACE technique, we were able to clone the full-length Japanese giant salamander ER␣ cDNA in the 5 and 3 directions including the ATG start site and the TGA termination signal (Fig. 2; GenBank accession no. AB252211, designated jgsER␣). The cDNA for ER␣ is composed of 2125 bases and predicts a protein of 584 amino acids with a calculated molecular mass of 65.7 kDa (Fig. 2). Fig. 3 shows the deduced amino acid sequence of the Japanese giant salamander ER␣ aligned with the reported ER␣ sequences of X. laevis (L20735), chicken (X03805), American alligator (AB115909), human (M12674) and zebrafish (AB037185). Comparison of the amino acid sequence of the Japanese giant salamander ER␣ with those of other vertebrates indicates that the Japanese giant salamander ER␣ sequence is more closely related to the ER␣ of Xenopus (77.7%). Intriguingly, the Nterminal sequence of the Japanese giant salamander is shorter than that observed in other vertebrate ER␣ except for fish ER␣ sequences. The ER␣ sequences from mammalian, birds and reptiles, obtained to date, exhibit an N-terminal sequence of MTM(P/T)L(P/H), whereas the N-terminal sequence for the

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Fig. 2. Nucleotide sequence of ER␣ cDNA and the deduced amino acid sequence for the Japanese giant salamander, Andrias japonicus. The numbers on the right refer to the position of the nucleotides and the amino acids.

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Fig. 3. Aligned amino acid sequences of Japanese giant salamander ER␣ with that of the Xenopus (L20735), chicken (X03805), American alligator (AB115909), human (M12674) and zebrafish (AB037185). Asterisks indicate identical amino acid residues. Gaps (-) are introduced to optimize the sequence alignment.

ER␣ of the Japanese giant salamander is MPLH, thus lacking the first MT sequence. Using the nomenclature of Krust et al. (1986), the Japanese giant salamander ER␣ sequence can be divided into five domains based on its sequence homology to other steroid hormone receptors. The five ER␣ (salamander, human, chicken, alligator, Xeno-

pus) sequences examined here shared 75.1–48.5% identity in the A/B domain. However, the similarities with the zebrafish ER␣ sequence are quite low (7.2%). In general, the A/B domain of fish ERs is different from other vertebrates. We found a 100–96.9% homology in the C domain (DNA-binding domain) sequences among these five species, a 70.1–16.4% homology in the D

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Fig. 4. Domain structure of ER␣ in Japanese giant salamander, and homology with ER␣ of the human (M12674), chicken (X03805), alligator (AB115909), Xenopus (L20735) and zebrafish (AB037185). The functional AB to E/F domains are schematically represented with the numbers of amino acid residues indicated. The figures within each box indicate the percent homology of the domain relative to Japanese giant salamander ER␣.

domain, and a 76.2–49.5% sequence similarity in the E/F domain (the ligand-binding domain) (Fig. 4). Thus, domains C (DNAbinding domain) and E/F (ligand-binding domain) are highly conserved among all vertebrate ERs, whereas the A/B and D domains show greater variability. Phylogenetic analyses of ER␣ sequences were generally consistent with existing phylogenetic hypotheses regarding vertebrate relationships (e.g., Carroll, 1988). To better understand the position of salamander ER␣ protein in the evolutionary history of the ER␣ protein and their reciprocal relationship, a phylogenetic tree was constructed using Phylip software using numerous ER␣ proteins from various vertebrates (Fig. 5). The result shows that the Japanese giant salamander ER␣ is similar to several other amphibians, the frogs X. laevis and X. tropicalis. However, amphibian ER␣ sequences are not very similar to reptilian ER␣ sequences (Fig. 5). To determine relative similarity within a vertebrate clade, we compared the amino acid sequence of human and mouse (mammalian) ER␣, chicken and alligator (avian and reptile) ER␣, and salamander and Xenopus (amphibian) ER␣. Chicken and alligator belong historically to different classes (bird versus reptile, respectively), but are currently considered the Archosauria; their the amino acid sequences of ER␣ show high similarity (over all homology is 89.6%) as previously reported (Katsu et al., 2004, 2006). The similarity in ER␣ sequence for the two amphibians, Japanese giant salamander and Xenopus is 77.7%. This value is lower than that generated when the ER␣ sequences of human and mouse are compared (Fig. 6). 3.2. Estrogen-dependent transactivation function of jgsERα expressed in mammalian cells The vertebrate ER recognizes a response element termed the estrogen response element (ERE) consisting of a palindromic repeat of the 5 -TGACCT-3 half-site (Zilliacus et al., 1995) and

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induces transcription down stream from this region (Gruber et al., 2004). We examined whether jgsER␣ could induce estrogenregulated reporter activity. About 1.5-fold induction was found in the reporter construct containing 1× ERE (Fig. 7A). However, jgsER␣ was effective in inducing luciferase activity using the 4× ERE reporter construct (Fig. 7A), and this induction was specific for jgsER␣ (Fig. 7B). These results show that the cloned jgsER␣ encodes a functional ER of the Japanese giant salamander. We found that estradiol-17␤ (E2) added to cells transfected with the jgsER␣ clone stimulated luciferase activity in a dose-dependent manner. The minimum stimulatory dose of E2 was 0.1 nM, and maximum stimulation was found at 10 nM (Fig. 8A). The natural concentration of E2 in the serum of the Japanese giant salamander has not been reported. Ligand specificity for the induction of estrogen-regulated reporter activity was examined by incubation with 100 nM of various steroids (Fig. 8B). Estrogens were effective in inducing luciferase activity. No induction was found even in the presence of testosterone and progesterone when pcDNA–jgsER␣ was transfected into the culture cells. Environmental chemicals such as bisphenolA and nonylphenol have been found to induce disruption of endocrine systems of medaka (Kang et al., 2003; Ministry of the Environment Japan, 2004). We analyzed the induction of luciferase activities by the typical environmental chemicals, bisphenol-A and nonylphenol. Bisphenol-A-induced almost no luciferase activity of ERE-containing construct by jgsER␣. However, about two- to three-fold induction of luciferase activity was observed following exposure of transfected cells to 10 ␮M nonylphenol. These results suggest that high concentrations of the ubiquitous environmental pollutant nonylphenol could influence ER activity. We also established an assay for ER transactivation using the GAL4 system. We modified the two-hybrid system using mammalian cells that can be used to study protein–protein interactions (Dang et al., 1991; Fearon et al., 1992). The BIND vector (Promega) contains the yeast GAL4 DNA-binding domain upstream of a multiple coding region, and expresses R. reniformis luciferase for normalization of transfection efficiency. The pG5-luc vector (Promega) contains five GAL4 binding sites upstream of a minimal TATA-box, which in turn, is upstream of the firefly luciferase gene. Using these two vectors, we examined the luciferase expression in the cell line, CHO-K1. When the GAL4 DNA-binding domain, which was fused with jgsER␣ and the pG5-luc vector, was introduced into CHO-K1 cells, luciferase expression did not change when E2 was absent (Fig. 9). However, E2 treatment induced significant expression of luciferase in cells containing the GAL4 DNA-binding domain fused to jgsER␣ (Fig. 9A). These data suggest that the GAL4 DNA-binding domain fused with jgsER␣ can bind to a GAL4 binding site in pG5-luc vector following an estrogen-induced conformation change of the ER, resulting in an increase in the transcription of the firefly luciferase gene. We examined dose–response activation of the Japanese giant salamander ER␣ using this system and found that E2 stimulated luciferase activity in a dose-dependent manner similar to that reported above for the ERE-luciferase system (Fig. 9B). Progesterone did not induce activity of the jgsER␣. In addition, we found that E2-

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˜ The phylogenic tree was constructed using the Phylip computer Fig. 5. Phylogenic tree of vertebrate ER␣ using deduced amino acid sequences of vertebrate ERα. program with the JTT matrix, neighbor-joining method and bootstrap re-sampling for 1000 times. The number indicates the bootstrap value (%), and the width of branch reflects the support calculated by the bootstrap re-sampling. The length of branch reflects estimated numbers of substitutions along each branch. The scale bar indicates 0.1 EAASS (expected amino acid substitutions per site).

induced luciferase activity over 50-fold. This system appears to be more sensitive than the ERE-luciferase system and may be more suitable for assays of ER-transactivation in unique species, such as the Japanese giant salamander. 3.3. Tissue distribution pattern of ERα mRNA The relative expression of ER␣ mRNA in various tissues from the female Japanese giant salamander was determined by RT-PCR. High expression levels of ER␣ mRNA were found in kidney, ovary and liver. Weaker signals were detected in skin and pancreas (Fig. 10). We confirmed that the same amount of total RNA was used in Fig. 9 by ␤-actin detection. 4. Discussion Fig. 6. Domain structure and amino acid identity between human (M12674) and mouse (M38651) ER␣, chicken (X03805) and American alligator (AB115909) ER␣, and Japanese giant salamander and Xenopus (L20735) ER␣. The functional A/B to E/F domains are schematically represented with the numbers of amino acid residues indicated.

Estrogens are implicated in a wide array of reproductive activities in vertebrates, such as gonadal differentiation, maturation of the female reproductive tract, and reproductive behaviors (for representative reviews, see Iguchi et al., 2001; McLachlan, 2001; Moore et al., 2005). In vertebrates, estrogens appear to induce

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Fig. 7. ERE-dependent transcriptional activities of jgsER␣. (A) CHO-K1 cells are transiently transfected with the ERE-luciferase vector (1× ERE or 4× ERE) together with a jgsER␣ expression vector. (B) Cells were incubated with (E2(+)) or without (E2(−)) 100 nM of estradiol-17␤ (E2) for 48 h. Data are expressed as a ratio of steroid:vehicle (ethanol). Each column represents the mean of triplicate determinations, and vertical bars represent the means ± S.E.

both genomic and non-genomic cellular actions via nuclear and possibly G-coupled membrane receptors (see McLachlan, 2001; Bjornstrom and Sjoberg, 2005). In 1987, a full sequence for the nuclear estrogen receptor ER␣ (ESR1) was reported for an amphibian, X. laevis (Weiler et al., 1987). Since that time, only one additional sequence has been reported for amphibians, and that from an additional species of the same genus, X. tropicalis. We reported here the sequence for ER␣ in the Japanese giant salamander, the first such report for a salamander and this unique, endangered species. Although no data is available on the circulating concentrations of estrogens in the any species of giant salamander, we assume that like other amphibian species, estradiol-17␤ is an important reproductive steroid. Previous studies in frogs and salamanders have noted seasonal changes in plasma estradiol17␤ concentrations with changes in hepatic vitellogenesis, ovarian maturation of oocytes and oviductal proliferation and growth (for examples, see Kelley, 1982; Licht et al., 1983; Itoh and Ishii, 1990; Moore et al., 1992; Lynch and Wilczynski, 2005). Concentrations of circulating estradiol-17␤ in these amphibian species (range = high (pg/ml) – low (ng/ml) of plasma) are within the normal range of that reported for a wide array of other vertebrates, such as reptiles, birds and mammals (for examples, see Plotka et al., 1977; Cree et al., 1992; Cockrem and Seddon, 1994; Guillette et al., 1997). Our data suggest that the C domain (DNA-binding domain) of the ER␣ in the Japanese giant salamander is highly con-

Fig. 8. Transcriptional activities of jgsER␣. (A) Dose–response profile of jgsER␣ activation by E2. CHO-K1 cells were transiently transfected with the ERE-luciferase vector together with a jgsER expression vector. Cells were incubated with increasing concentrations of E2 (0.01–100 nM) or with no ligand for 48 h. Each point represents the mean of triplicate determinations, and vertical bars present the mean ± S.E. (B) Transcriptional activities of jgsER␣ for various steroids. CHO-K1 cells were transiently transfected with the ERE-luciferase vector together with a jgsER␣ expression vector. Cells were incubated with or without 100 nM various steroid for 48 h. E2, estradiol-17␤; T, testosterone; P, progesterone; E1, estrone; E3, estriol; EE2, 17␣-ethynylestradiol. Data are expressed as a ratio of steroid:vehicle. Each column represents the mean of triplicate determinations, and vertical bars represent the meas ± S.E. (C) Effects of jgsER␣ activation by nonylphenol (NP) and bisphenol-A (BPA). CHO-K1 cells were transiently transfected with the ERE-luciferase vector together with a jgsER expression vector. Cells were incubated with increasing concentrations of E2 (0.01–100 nM), NP (0.01–10 mM) or BPA (0.01–100 mM) for 48 h. Each point represents the mean of triplicate determinations, and vertical bars present the mean ± S.E.

served and very similar to that of other vertebrates. Likewise the ligand-binding domain, the E/F domain, was very similar to other estrogen receptors except, the N-terminal sequence of the Japanese giant salamander is shorter than that observed in other vertebrates. ER␣ sequences obtained from the majority mam-

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Fig. 9. Transactivation of jgsER␣ using GAL4 system. (A) CHO-K1 cells were transiently transfected with the pBIND or pBIND–jgsER␣ (pBIND–ER) together with pG5-luc vector. Cells were incubated without (E2(−)) or with (E2(+)) 100 nM of E2 for 48 h. (B) Dose–response profile of jgsER␣ activation by E2 or P. CHO-K1 cells were transiently transfected with the pG5-luc vector together with a jgsER expression vector (pBIND–jgsER␣). Cells were incubated with increasing concentrations of E2 (䊉) or P4 (
) (0.01 pM–100 nM) or with no ligand for 48 h. Each point represents the mean of triplicate determinations, and vertical bars present the mean ± S.E.

mals, birds and reptiles, to date, exhibit an N-terminal sequence of MTM(P/T)L(P/H), whereas the N-terminal sequence for the ER␣ of the Japanese giant salamander is MPLH, thus lacking the first MT sequence. Using the GenBank sequence database,

Fig. 10. Distribution of ER␣ in tissues of female Japanese giant salamander. Total RNA was prepared from kidney, spleen, lung, stomach, skin, small intestine, large intestine, ovary heart, pancreas and liver of female giant salamander. ␤-Actin was used as a positive control. Messenger RNA of ER␣ was detected by RT-PCR (see Section 2).

we found that the Zebra finch (Taeniopygia guttata) exhibits a similar MTLH amino acid sequence of the N-terminal region of ER␣ (L79911) (Jacobs et al., 1996). Presently, we have no data to indicate whether this modification is unique to a few species or more common, nor can we currently deduce a possible role for this reduction. We also examined the relative similarity of the sequences of ER␣ between related species, such that we compared the amino acid sequences of human and mouse (mammalian) ER␣, chicken and alligator (archosaur) ER␣, and salamander and Xenopus (amphibian) ER␣. Chicken and alligator belong historically to different classes (bird versus reptile, respectively), but are currently considered the Archosauria; their amino acid sequences for ER␣ show high similarity (over all homology is 89.6%) which indicates strong selection for a stable sequence as these groups last had a common ancestor, based on molecular clock data, approximately 240 million years before the present (mybp) (Kumar and Hedges, 1998). Strong conservation is also seen when human and mouse sequences for ER␣ are compared; these groups last shared an ancestor approximately 40 mybp (Kumar and Hedges, 1998). In contrast to mammalian and archosaurian phylogeny, great debate still exists on the origins of the modern amphibians, the Lissamphibia. Amphibians are thought to be a monophyletic group, with three extant orders (Anura, Caudata and Gymnophiona). The origins and interrelationships among these groups are currently under intense study (for reviews, see Feller and Hedges, 1998; Meyer and Zardoya, 2003). We observed that the similarity in ER␣ sequence for the two amphibians groups available, the Japanese Giant salamander and Xenopus is 77.7%. This value is lower than that of the other comparisons discussed above, but is remarkable as salamanders and frogs have been estimated to have separated evolutionarily 357 mypb (405–317 mybp) (San Mauro et al., 2005). Further study on the molecular evolution of the steroid receptors in Anura, Caudata and Gymnophiona could reveal evolutionary relationships among amphibians, as it appears that strong selection for conserved amino acid sequences of these receptors may allow us to use these data to develop phylogenies as we have previously proposed for crocodilians (see Katsu et al., 2004, 2006). To date, the only amphibian ERs cloned (full length and partial) are from two Xenopus species, X. laevis (Weiler et al., 1987), X. tropicalis (Wu et al., 2003; Takase et al., unpublished data) and the Japanese giant salamander (this study). ER cloning from other amphibians will promote an understanding of the molecular evolution of the ER in amphibians, and as well as amniote vertebrates given this groups ancestral position in the evolution of amniotes. We have already amplified DNA fragments of ERs from several amphibian species that will be published in the future. In this study, we examine the transactivation of jgsER␣ using reporter gene assay systems. Conventional assay using EREresponsive-element containing reporter construct is suitable for analysis of ER-induced transactivation by estrogens. However, the maximal activity is not high (about five-fold in this study). We also applied the GAL4-system for transactivation of ER by estrogens. This system is modified from the mammalian

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two-hybrid assay system. The reporter gene containing GAL4binding site and GAL4–DNA binding domain fused estrogen receptor (GAL4–ER) construct are introduced into culture cells. The expressed GAL4–ER can bind to GAL4-binding site of reporter gene, however, reporter gene activity is not activated before exposure to an estrogenic ligand. Estrogens interact with the GAL4–ER construct and induce a conformational change leading to reporter gene activation. In fact, we found E2 can activate the reporter gene (Fig. 9). This activation reached 50fold, within the same dose range for activation in conventional transactivation assays. So, this system could provide information on both the binding of ER and steroids as well as the transactivation by of a receptor by steroids. Further we found that the A/B domain of the ER is not necessary for this assay system (Katsu et al., unpublished data). This work was performed, in part, to develop in vitro assay techniques to study the basic endocrinology of an endangered species as well as to determine if this species is uniquely susceptible to the endocrine disruptive effects of various environmental contaminants capable of acting as ER agonists or antagonists. For example, recent studies have demonstrated that ER␣ from the Japanese medaka (O. latipes) is more responsive to a number of environmental estrogens, such as nonylphenol, when compared to the response of mammalian ER␣ (Kang et al., 2003; Ministry of the Environment, 2004). Although the Japanese giant salamander lives primarily in rural aquatic habitats, contamination of those environments with a wide array of endocrine disrupting chemicals (EDCs) is present. Surveys for the presence of various estrogenic EDCs has been performed in Japan and chemicals such as nonylphenol are commonly found (Tsuda et al., 2000), although not at the relatively high levels required to activate the jgsER␣. However, we remain concerned that various environmental chemicals with endocrine activity could alter the reproductive potential of captive or wild stock as well as potentially affect the sex determination and growth of larvae as has been reported for a number of fish, amphibian and reptile populations (see Guillette and Gunderson, 2001; Guillette and Iguchi, 2003; Milnes et al., 2006). Thus, future studies are needed to examine the regulation of ER␣ in detail and focus on ontogenic and sexually dimorphic responses in amphibians. In addition to the examination of the interaction of ER␣ with endogenous steroids, work must examine the possible role of environmental contaminants affecting the developmental and reproductive biology of salamanders. Given the continuous use of DDT in tropical regions of Africa, Asia and South America with malaria threats, it is worth further study to examine possible interactions between this pesticide and its metabolites and developmental effect in amphibians as many common persistent environmental contaminants have been shown to interact with vertebrate estrogen receptors (for review, see Rooney and Guillette, 2000). Thus, studies should examine the molecular interactions between steroid receptors from a specific species and native ligands as well as common contaminants in that species’ environment. In summary, we cloned, sequenced and characterized the ER␣ from the Japanese giant salamander, the first such sequence from any species of the Caudata. This is the first isolation of functional

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