Mol. Hum. Reprod. Advance Access originally published online on April 26, 2004
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Molecular Human Reproduction, Vol. 10, No. 7, pp. 527-533, 2004
Molecular Human Reproduction vol. 10 no. 7 © European Society of Human Reproduction and Embryology 2004; all rights reserved
Characterization of htAKR, a novel gene product in the aldo-keto reductase family specifically expressed in human testis
1Department of Pharmacology, Kyoto Prefectural University of Medicine, Kamigyo-ku, Kyoto 602-8566, Japan 2Department of Urology, Kyoto Prefectural University of Medicine, Kamigyo-ku, Kyoto 602-8566, Japan 3Department of Anatomy, Kyoto Prefectural University of Medicine, Kamigyo-ku, Kyoto 602-8566, Japan *The two authors contributed equally to this work.
4 To whom correspondence should be addressed.; Email: nchihiro{at}koto.kpu-m.ac.jp
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Abstract |
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In human testis, expression of a novel member of the aldo-keto reductase family was identified. Based on its testis-specific expression, we termed this protein human testis aldo-keto reductase (htAKR). In addition to four major isoforms, the existence of multiple alternatively spliced products of htAKR was detected using RT-PCR followed by nested PCR. htAKR was a homologue of mouse liver keto-reductase, AKR1E1, with close similarity in their genomic organizations. htAKR4, the longest isoform, was expressed as a non-fused native form. It exhibited a limited activity toward 9,10-phenanthrenequinone, while no activity toward the steroids or prostaglandins was demonstrated. Using the laser capture microdissection technique and RT-PCR, expression of htAKR was detected in testicular germ cells as well as in interstitial cells. The levels of htAKR mRNA in the tissues obtained from seminoma were much lower than those in normal testes. A significant decline in the htAKR expression was observed when NEC8, a cell line originated from a human testicular germ cell tumour, was exposed to phorbol 12-myristate 13-acetate or 5
-dihydrotestosterone. These results indicate that the expression of htAKR, down-regulated in the testicular tumour, is possibly controlled by mitogenic and hormonal signals. Key words: aldo-keto reductase/alternative splicing/mouse vas deferens protein/NEC8/seminoma
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Introduction |
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Aldo-keto reductases (AKR) represent monomeric oxidoreductases with a (ß/
)8 barrel structure catalysing NAD(P)H-dependent conversion of a wide variety of aldehydes to the corresponding alcohols (Bohren et al., 1989
; Wilson et al., 1992; Jez et al., 1997). AKR compose a large superfamily consisting of >100 proteins and a systematic nomenclature has been adopted (Hyndman et al., 2003). The substrates for AKR include steroids and prostaglandins as well as xenobiotic aldehydes, sugars and bile acids, indicating that AKR participate in various pathophysiological events (Jez et al., 1997). For instance, aldose reductase (AKR1B1) has been implicated in the pathogenesis of diabetic complications and numerous aldose reductase inhibitors were developed as possible therapeutic agents (Yabe-Nishimura, 1998).
The AKR family includes the mouse vas deferens protein (MVDP), classified as AKR1B7, which is predominantly expressed in mouse vas deferens (Pailhoux et al., 1990). MVDP exhibits oxidoreductase activity toward certain aldehydes (Lefrancois-Martinez et al., 1999), and its expression is regulated by androgens, suggesting a possible role in germ cell proliferation and maturation (Pailhoux et al., 1990). Recently, Baron et al. (2003) reported increased level of MVDP mRNA in mouse adult Leydig cells exposed to hCG.
In an attempt to isolate the human counterpart of MVDP, we identified a novel AKR with a high sequence identity with MVDP (Nishinaka et al., 2003). This new member of the AKR family, htAKR (human testis aldo-keto reductase), formerly designated as HTSP (human testis specific protein), was predominantly expressed in human testis but not in other organs such as vas deferens, adrenal glands or ovary. We here report unusual properties of this novel testis-specific member of AKR family.
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Materials
Normal human testis samples were obtained from fertile middle-aged patients castrated for treatment of prostate cancer. Cancer tissue samples were obtained from three patients with stage I seminoma and one patient with para-aortic lymph node metastasized stage IIA seminoma. All tissue samples were obtained with informed consent. The research protocol of this study was approved by the Ethics Committee at Kyoto Prefectural University of Medicine. Multiple tissue northern (MTN) blots were purchased from Clontech (USA). [
-32P]dCTP was from ICN Biomedicals, Inc. (USA). The bovine Factor Xa was purchased from Haematologic Technologies, Inc. (USA). Other reagents were of the highest grade available.
Northern blotting
Northern blotting was performed as described previously (Nishinaka and Yabe-Nishimura, 2001). cDNA probes were labelled with [-32P]dCTP by random priming, and purified with G-25 spin columns. The blotted membrane was hybridized with a 1 x 106 cpm/ml radiolabelled probe at 42°C in a buffer containing 5 x standard saline phosphate EDTA (SSPE), 1% sodium dodecyl sulphate (SDS), 5 x Denhardt's solution, and 50% formamide. The blots were first rinsed with 2 x standard saline citrate (SSC) containing 0.5% SDS, and then washed twice with 0.1 x SSC containing 0.5% SDS for 30 min at 55°C. The radioactive signals were analysed using a BAS 2000 Bioimaging Analyzer (Fuji Film, Japan).
RT-PCR
RT-PCR was performed with the one-step RT-PCR system (Qiagen K.K. Tokyo, Japan). The primers used in this study were as follows: primer F(R): 5'-TCCTTGGTGGAAACAGCATG-3'; primer A: 5'-CACACAGGCTGTCTTGGAGA-3'; primer C: 5'-TTCAGACACCAATAATTCAATCTG-3'; primer D2: 5'-TGGACGAGAGCAACATGGTT-3'; primer A2: 5'-ATCTGGGCTGAGCAGAAACA-3'. Real-time PCR was performed using GeneAmp 5700 gene detection system (Applied Biosystems Japan, Japan) with SYBR green RT-PCR System (Qiagen). The primers used for the detection of htAKR were 5'-ATGGGAGATATCCCAGCCGTG-3' and 5'-CCAAATAGTTCAGCTTCAAGG-3'. With these primers, the first 290 nucleotides downstream of the initiation codon that are common to all htAKR isoforms were amplified. The level of htAKR expression was normalized by that to ß-actin with primers 5'-TGAACCCCAAGGGCCAACCGC-3' (forward) and 5'-TTGTGCTGGGTGCCAGGGCA-3' (reverse), or the amount of total RNA. The primers used for amplification of RBMY1, 5'-ATGCACTTCAGAGATACGG-3' and 5'-CCTCTCTCCACAAAACCAACA-3' were synthesized according to Friel et al. (2002).
Laser capture microdissection
To isolate testicular germ cells and interstitial cells from tissue samples, laser capture microdissection was performed with Arcturus LM200 (Olympus, Japan) according to the manufacturer's instructions. Total RNA was extracted from the dissected cells and subjected to RT-PCR.
Cell culture
NEC8, a cell line originated from human testicular germ cell tumour, was obtained from Health Science Research Resources Bank (Japan). The cells were grown in Roswell Park Memorial Institute medium supplemented with 10% fetal bovine serum at 37°C under an atmosphere of 95% air and 5% CO2.
Expression and purification of human testis-specific protein
The largest isoform, htAKR4, was expressed as a fusion protein with maltose binding protein (MBP). htAKR4 cDNA, amplified with primers SalI-P (5'-ACGCGTCGACATGGGAGATATCCCAGCCGTGGGCCTC-3') and SalI-C (5'-ACGCGTCGACACCAATAATTCAATCTGT-3'), was digested with SalI, and ligated into a pMAL-c2x vector (New England Biolabs, Inc., USA) to generate pMAL-htAKR4. The nucleotide sequence of htAKR4 was confirmed with an ABI Prism 310 sequencer. Transformants of the E. coli strain DH5 harbouring pMAL-htAKR4 were grown in Luria Broth containing 50 µg/ml ampicillin. The expression of MBP-fused htAKR4 was induced by the addition of 0.1 mmol/l isopropyl-ß-D-1-thiogalactopyronoside. Cells were collected, resuspended in buffer A, consisting of 20 mmol/l Tris-HCl (pH 8.0), 200 mmol/l NaCl, 1 mmol/l EDTA, 1 mmol/l phenylmethylsulphonyl fluoride, and 1 mmol/l dithiothreitol, and lysed by incubation with 0.75 mg/ml lysozyme. Following ultrasonic disruption and centrifugation, the supernatant fraction was mixed with amylose resin (New England Biolabs). After washing with buffer A, the MBP-fused htAKR4 was eluted with 10 mmol/l maltose. The purified fusion protein was dialysed against 20 mmol/l Tris-HCl (pH 7.5) buffer containing 150 mmol/l NaCl, and digested with Factor Xa. htAKR4 removed from MBP was purified by Q-Sepharose anion exchange chromatography (Amersham-Pharmacia Biotech). The purity of the htAKR4 protein was verified by SDS-polyacrylamide gel electrophoresis.
Enzyme assay
AKR activity was measured as described previously (Nishinaka et al., 1992). For the determination of the reductase activity, the reaction mixture consisted of 100 mmol/l sodium phosphate (pH 7.0), 66 µmol/l NADPH, and various substrates in a final volume of 0.1 ml. For dehydrogenase activity, the reaction mixture consisted of 100 mmol/l sodium phosphate (pH 6.0), 66 µmol/l NADP+, and substrates. The assay was performed at 25°C by monitoring the absorbance at 340 nm with a Shimadzu UV-160 spectrophotometer (Kyoto, Japan). The assay mixture for prostaglandin F synthase activity consisted of 0.4 µmol/l [1-14C]PGH2, 0.1 mol/l potassium phosphate buffer (pH 6.5), 5 mmol/l glucose-6-phosphate, 0.5 mmol/l NADP+, and 1 IU of glucose-6-phosphate dehydrogenase. The reaction was conducted for 2 min at 24°C. Prostaglandins were extracted in diethylether:methanol:1 mol/l citric acid (30:4:0.1). The products were separated by thin layer chromatography and analysed by a BAS 2000 Bioimaging Analyzer.
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Results |
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Comparison of the genomic structure of htAKR with mouse AKR1E1
The deduced amino acid sequence of the longest isoform htAKR4 was compared with other AKR family members (Figure 1A
). While htAKR4 demonstrated only 56.5% sequence identity with MVDP and 56.0% with human aldose reductase, it showed the greatest (69.0%) identity with mouse liver keto-reductase, AKR1E1 (Bohren et al., 1997). The high throughput genomic (HTG) sequence database search further confirmed the greatest similarity between the htAKR gene and the AKR1E1 gene. They commonly have shorter exon 1 and exon 7 compared with other AKR including human aldose reductase (AKR1B1). The C-terminal region of AKR1E1 was similar to those of htAKR2 and htAKR3, the shorter isoforms of htAKR (Nishinaka et al., 2003). The apparent difference was the insertion of 19 amino acids in htAKR4 (amino acids 109–127; boxed in Figure 1A). This insertion in htAKR seemed to occur due to a single base substitution at the putative splice acceptor site of exon 4 (bold in Figure 1B).
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htAKR is exclusively expressed in human testis
Figure 2
shows the expression profile of htAKR in human tissues. Except for the testis, no hybridization signal was detected in the spleen, thymus, prostate, uterus, small intestine, colon, or leukocytes, even though poly-A+ RNA was blotted on the membrane. No signal was detected on the membrane blotted with poly-A+ RNA isolated from the heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas (data not shown). These results indicated that htAKR is predominantly expressed in human testis.
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Expression of multiple alternatively spliced isoforms of htAKR in human testis
To confirm the expression of htAKR isoforms in human testes, RT-PCR, followed by nested PCR, was performed (Figure 3
). The first round RT-PCR was carried out with primer F(R) and primer A. For the detection of htAKR1 and 4, the second round nested PCR was performed with primer D2 and primer C. For the detection of htAKR2 and 3, the second round was performed with primer D2 and primer A2. As demonstrated in Figure 3, the bands corresponding to the four isoforms were detected, indicating that these isoforms are actually expressed in human testes. In addition to these isoforms, several products of different sizes were simultaneously amplified. These results suggest that there are multiple isoforms of htAKR expressed in the testis.
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Characterization of the recombinant htAKR expressed in E. coli
To characterize the enzymatic property of htAKR, the non-fused native form of htAKR4 was prepared. htAKR4 was first expressed as a fusion protein with a maltose binding protein, digested with Factor Xa, and purified by Q-Sepharose anion exchange chromatography (Figure 4
). Determination of the oxidoreductase activity of htAKR4 revealed that 9,10-phenanthrenequinone was the only substrate reduced by this isoform. The values of KM and kcat with NADPH toward 9,10-phenanthrenequinone were 44.3 µmol/l and 4.0 x 10–3 s–1 respectively. The KM values toward NADPH and NADH were 166 µmol/l and 2.3 mmol/l respectively (Table I). The reductase activity of htAKR4 was undetectable toward typical substrates for aldehyde reductase (EC 1.1.1.2, AKR1A1) or aldose reductase (AKR1B1), including DL-glyceraldehyde, 4-nitroacetophenone and menadione. The recombinant protein did not show any oxidoreductase activity toward the keto or hydroxyl groups at the C-3, C-17, and C-20 positions of such steroids as androsterone, 4-androstene-3,17-dione, 5-dihydrotestosterone and estrone. Finally, htAKR4 did not show any prostaglandin F synthase activity, reducing PGD2 and PGE2 to PGF2, or converting PGH2 to PGF2 (data not shown).
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Localization of htAKR mRNA in the testis
To define the localization of htAKR in the testicular tissue, we isolated the germ cells and interstitial cells by the laser capture microdissection technique. A typical dissection process is shown in Figure 5A–E
. The total RNA was extracted from each sample, and subjected to RT-PCR. The separation between germ cells and interstitial cells was confirmed by the germ cell-specific expression of RBMY1 (RNA binding motif protein linked Y chromosome 1), a possible azoospermia factor (Friel et al., 2002) (data not shown). As shown in Figure 5F, the expression of htAKR was observed in germ cells as well as in interstitial cells, indicating that htAKR is uniformly expressed in the testicular tissue.
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htAKR is down-regulated in the testicular tumour
The expression of htAKR was investigated in the cells obtained from the testicular tumours. The total RNA was extracted from tissue samples obtained from four patients with seminoma, and subjected to real-time PCR. As shown in Figure 6
, an apparent decrease in htAKR expression was observed in all the tested tissues. The expression levels in these samples were <10% of the normal testicular tissues. We further studied the expression of htAKR in NEC8 (Figure 7), a cell line originated from a human testicular germ cell tumour. The level of htAKR expression was <10% of the normal tissues. When NEC8 cells were treated with phorbol 12-myristate 13-acetate (PMA), a significant reduction in the expression of htAKR was demonstrated at 8 and 24 h. Similarly, the htAKR expression was significantly reduced in the cells treated with 5-dihydrotestosterone for 8 h. These results indicate that the expression of htAKR, down-regulated in the testicular tumour, is possibly controlled by mitogenic and hormonal signals.
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Discussion |
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In search of the homologue of htAKR in the database, we found the closest genomic organization between the htAKR gene and the murine AKR1E1 gene. The database indicated that the htAKR gene is localized at human chromosome 10p15.1, while the AKR1E1 is mapped to mouse chromosome 13. Although these two genes are homologous at the genomic level, there was an apparent divergence in the amino acid sequence of htAKR. htAKR contained 19 additional amino acids at amino acids 109–127. This region is unique to htAKR, and has never been observed in any other AKR identified to date. This insertion appears to be created by a single nucleotide substitution at the corresponding splice acceptor sequence in exon 4 of htAKR, making a different splicing site to generate an extra amino acid sequence. These observations suggest that htAKR is a member of the AKR1E group.
The tissue distribution of htAKR was further analysed with MTN membranes. Among 19 different tissues so far tested, htAKR expression was only seen in the testis. According to an Expressed Sequence Tag (EST) database search, htAKR clone was identified in the brain and the retina of normal human subjects other than testis. The expression of htAKR in the brain, however, should be very low since our northern blot analysis failed to detect htAKR expression in the brain.
The enzymatic activity of htAKR4 was nearly 100 times less efficient than that of murine AKR1E1. Recombinant htAKR4 protein demonstrated a limited activity to reduce 9,10-phenanthrenequinone, one of the xenobiotic compounds. While some AKR catalyse the oxidoreduction of steroid hormones (Nishinaka et al., 1991; Petrash et al., 1997; Penning et al., 2000), htAKR4 did not show any detectable reductase or dehydrogenase activity toward such steroids as androgens, estrogens and progestins. While some AKR are involved in the metabolic conversion of prostaglandins (Watanabe, 2002), htAKR4 did not catalyse the conversion of PGH2 to PGF2, or the reduction of PGD2 or PGE2. The reason why htAKR shows such a limited activity may be due to the amino acid substitutions at the putative active site of the AKR family involved in cofactor binding. In fact, the KM value for NADPH (166 µmol/l) was much higher than that of human aldose reductase (4 µmol/l) (Nishimura et al., 1991). Furthermore, three-dimensional structure modelling by the Swiss-Model program illustrated that the extra 19 amino acids at amino acids 109–127 form another loop structure (Borhani et al., 1992). This unique structure of htAKR may therefore hinder the binding of the substrate and cofactor.
The physiological function of htAKR remains unclear. To elucidate the role of htAKR in the testis, we investigated the localization of htAKR mRNA by the laser capture microdissection and RT-PCR. The expression of htAKR was detected in isolated germ cells as well as in interstitial cells, indicating a uniform expression of htAKR in the testicular tissue. htAKR thus appeared not to be involved in the cell type-specific events in human testis. Since increased expression of some AKR was reported during hepatocellular carcinogenesis, the expression of htAKR may be associated with tumorigenesis (Zeindl-Eberhart et al., 1994; Scuric et al., 1998). When the expression of htAKR was investigated in tissue samples obtained from testicular tumours, the level of the htAKR transcript was much lower in seminoma cells, compared with normal tissue. We also observed the reduced htAKR expression in PMA- or 5-dihydrotestosterone-treated human testicular tumour cell line. These observations suggest that the expression of htAKR is down-regulated by mitogenic and hormonal stimuli.
htAKR may also participate in the regulation of spermatogenesis. As shown in Figure 3
, htAKR is expressed as multiple alternatively spliced isoforms. A unique feature of spermatogenic cells is that a number of mRNA differ in size and structure from transcripts of the same gene in somatic cells by usage of spermatogenic cell-specific transcription start sites, alternative splicing and upstream polyadenylation sites (Kleene, 2001). It is speculated that such unique spermatogenic cell-specific transcripts have structural or regulatory functions in meiosis and the differentiation of sperm (Kleene, 2001). For instance, cAMP-response element modulator (CREM) is present as multiple alternatively spliced isoforms in mammalian testes and known to play an important role in spermatogenesis (Don and Stelzer, 2002). Another feature of spermatogenic cells is that the levels of proteins encoded by these mRNA are often very low or undetectable (Kleene, 2001). In the course of this study, we produced polyclonal antibodies against the recombinant htAKR4 to identify the expression of the htAKR4 protein in the testis. While these antibodies reacted with the recombinant htAKR, the protein in the tissue was undetectable by western blot analyses. The fact that htAKR existed as multiple alternatively spliced isoforms with its unusual patterns of transcription and translation appears to follow the features reported in spermatogenic cells. In this context, htAKR is a unique member in the AKR family, since no other member in the AKR family has been known to exhibit similar expression pattern as htAKR.
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Acknowledgements |
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The authors thank Drs Kikuko Watanabe and Noriko Koda (University of East Asia, Yamaguchi, Japan) for assaying the prostaglandin F synthase activity, and Drs Masahito Oyamada and Tetsuro Takamatsu (Department of Pathology, Kyoto Prefectural University of Medicine) for the assistance in use of the laser capture microdissection equipment. The authors also acknowledge the assistance of Dr Atsuo Adachi at the start of this study.
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Submitted on February 2, 2004; resubmitted on March 18, 2004; accepted on March 25, 2004.
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