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Chemico-Biological Interactions 178 (2009) 151–157

Contents lists available at ScienceDirect

Chemico-Biological Interactions

journa l homepage: www.e lsev ier .com/ locate /chembio int

haracterization of a rat NADPH-dependent aldo-keto reductase (AKR1B13)nduced by oxidative stress

atoshi Endoa,b,∗, Toshiyuki Matsunagaa, Hiroaki Mamiyaa, Akira Haraa, Yukio Kitadeb,azuo Tajimac, Ossama El-Kabbanid

Laboratory of Biochemistry, Gifu Pharmaceutical University, Gifu 502-8585, JapanUnited Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, Gifu 501-1193, JapanFaculty of Pharmaceutical Sciences, Hokuriku University, Kanazawa 920-1181, JapanMedicinal Chemistry and Drug Action, Monash Institute of Pharmaceutical Sciences, Victoria 3052, Australia

r t i c l e i n f o

rticle history:vailable online 19 September 2008

eywords:ldo-keto reductase superfamilyKR1B13ldose reductase

a b s t r a c t

A rat aldo-keto reductase (AKR1B13) was identified as a hepatoma-derived protein, exhibiting highsequence identity with mouse fibroblast growth factor (FGF)-induced reductase, AKR1B8. In this study,AKR1B13 was characterized in terms of its enzymatic properties, tissue distribution and regulation.Recombinant AKR1B13 exhibited NADPH-linked reductase activity towards various aldehydes and �-dicarbonyl compounds, which include reactive compounds such as methylglyoxal, glyoxal, acrolein,4-hydroxynonenal and 3-deoxyglucosone. The enzyme exhibited low NADP+-linked dehydrogenase activ-

-Deoxyglucosone-Dicarbonyl compoundxidative stress

ity towards aliphatic and aromatic alcohols, and was inhibited by aldose reductase inhibitors, flavonoids,benzbromarone and hexestrol. Immunochemical and reverse transcription-PCR analyses revealed that theenzyme is expressed in many rat tissues, endothelial cells and fibroblasts. Gene expression in YPEN-1 andNRK cells was up-regulated by treatments with submicromolar concentrations of hydrogen peroxide and1,4-naphthoquinone, but not with FGF-1, FGF-2, 5�-dihydrotestosterone and 17�-estradiol. These resultsindicate that AKR1B13 differs from AKR1B8 in tissue distribution and gene regulation, and suggest that it

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functions as a defense sys

. Introduction

The aldo-keto reductase (AKR) superfamily encompasses morehan 140 proteins with different physiological roles, most ofhich are NAD(P)(H)-dependent oxidoreductases that metabo-

ize carbohydrates, steroids, prostaglandins, and other endogenousldehydes and ketones, as well as xenobiotic compounds [1].mong members of this superfamily, aldose reductase (AR) belongs

o the AKR1B subfamily and catalyzes the first step in the polyolathway that has been a focus of interest because of its implication

n the pathogenesis of diabetic complications [2,3]. In addition toolyol synthesis, AR shows broad substrate specificity for carbonylompounds and is thought to play physiological roles in osmotic

Abbreviations: AKR, aldo-keto reductase; AR, aldose reductase; FGF, fibroblastrowth factor; HSD, hydroxysteroid dehydrogenase; TBE, 6-tert-butyl-2,3-epoxy-5-yclohexene-1,4-dione; RT, reverse transcription; U, unit; FBS, fetal bovine serum.∗ Corresponding author at: Laboratory of Biochemistry, Gifu Pharmaceutical Uni-

ersity, Gifu 502-8585, Japan. Tel.: +81 58 237 3931; fax: +81 58 237 5979.E-mail address: sendo@gifu-pu.ac.jp (S. Endo).

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009-2797/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.cbi.2008.09.010

gainst oxidative stress in rat tissues.© 2008 Elsevier Ireland Ltd. All rights reserved.

omeostasis, steroid and xenobiotic metabolism, and signal pro-essing and oxidative defense mechanisms [4–6].

cDNA isolation from human, mouse and rat tissues have pro-ided evidence that the AKR1B subfamily contains several enzymesith high sequence similarity (67–71%) to human and rodent ARs.

hese AR-like proteins include small intestine reductase (AKR1B10)n humans [7,8], mouse fibroblast growth factor (FGF)-induced pro-ein (AKR1B8) [9], mouse vas deferens protein (AKR1B7) [10], andat AR-like protein 1 (AKR1B13) [11]. AKR1B10 [8], AKR1B8 [9,12]nd AKR1B7 [13,14] were characterized to be NADPH-dependentnzymes, which catalyze the reduction of aliphatic and aromaticldehydes excluding aldoses. The three enzymes differ in their sub-trate specificity and kinetic constants for the aldehydes, despite ofheir high sequence identity (>80%). AKR1B10 efficiently reducesetinals [15] and some drug ketones [16], whereas AKR1B8 exhibitsigh catalytic efficiency for long-chain aliphatic aldehydes such as-hydroxynonenal derived from lipid peroxidation [12]. AKR1B7

educes isocaproaldehyde, a product of the side-chain cleavagef cholesterol in steroid hormone synthesis [13]. In addition, thehree enzymes differ in their tissue distribution and gene regula-ion. AKR1B10 is distributed in all human tissues [7,8], whereashe other two mouse enzymes show tissue-specific expression in

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52 S. Endo et al. / Chemico-Biologi

he testis, ovary, heart and adrenal gland (for AKR1B8) [9,17] andn the male reproductive organs and adrenal gland (for AKR1B7)17]. While AKR1B8 is induced by FGF-1 and FGF-2 [9], AKR1B7 isp-regulated by androgen and pituitary tropic hormones [10,14].lthough such inducers of AKR1B10 are unknown, recent clinicaltudies have pointed out that its expression is elevated in lesionsf lung and other cancers and the enzyme has been recognized as aiomarker of some cancers [8,18,19]. Rat AR-like protein, AKR1B13,as identified earlier as a tumor-associated protein with increased

evels in experimental hepatoma [11]. However, the biochemicalroperties of AKR1B13 have yet to be explored.

In this study, we have characterized AKR1B13 in terms of itsnzymatic properties, tissue distribution and gene regulation, andompared its substrate specificity and kinetic properties with thosef rat AR, AKR1C15 [20] and other hydroxysteroid dehydrogenasesHSDs) that reduce non-steroidal carbonyl compounds [21]. Theata indicated that AKR1B13 acts as a reductase for reactive car-onyl compounds derived from oxidative stress and glycation inat tissues including endothelial cells.

. Materials and methods

.1. Materials

Rat FGFs were obtained from Peprotech (Rocky Hill, NJ); resinsor column chromatography and an enhanced chemiluminescenceubstrate system were from Amersham Biosciences (Piscataway,J); and a pCold IV expression vector and Taq DNA polymeraseere from Takara (Kusatsu, Japan). Pfu DNA polymerase and

scherichia coli BL21 (DE3) pLysS were purchased from Strata-ene (La Jolla, CA) and Invitrogen (Carlsbad, CA), respectively. Ratrostatic endothelial cell line, YPEN-1, and renal epithelial cell

ine, NRK, were obtained from American Type Culture Collec-ion (Manassas, VA). Isocaproaldehyde [22], d-lactoaldehyde [23],-hydroxynonenal [24], 21-dehydrocortisol [25] and 6-tert-butyl-,3-epoxy-5-cyclohexene-1,4-dione (TBE) [26] were synthesized asescribed previously. AR inhibitors were kindly denoted by Dr. P.F.ador (University of Nebraska Medical Center) and Sanwa Kagakuenkyusho Co. (Mie, Japan). All other chemicals were of the highestrade that could be obtained commercially.

.2. cDNA isolation and site-directed mutagenesis

The cDNA for AKR1B13 (Accession no. NM 173136) was isolatedrom the total RNA preparation of rat liver by reverse transcriptionRT)-PCR. The RT and DNA techniques followed the standard proce-ures described by Sambrook et al. [27]. PCR was performed withfu DNA polymerase and a pair of sense and antisense primers,′-cccccatatggcaaccttcg-3′, and 5′-ccccggatcctcagtactct-3′, whichontain underlined NdeI and BamHI sites, respectively. The PCRroducts were purified, digested with the two restriction enzymes,nd ligated into the pCold IV vectors that had been digestedith the two restriction enzymes. The expression constructs were

ransfected into E. coli BL21 (DE3) pLysS cells. The insert of theloned cDNA was sequenced by using a Beckman CEQ2000XL DNAequencer, and its identity was verified with the cDNA.

.3. Expression and purification of recombinant enzymes

The E. coli cells were cultured in a LB medium (1 l) con-

aining ampicillin (50 �g/ml) at 37 ◦C until the absorbance at00 nm reached 0.5. Isopropyl-�-d-thiogalactopyranoside (1 mM)as added and the culture was continued for 24 h at 15 ◦C. The cell

xtract was prepared as described previously [28]. The recombi-ant enzymes were purified at 4 ◦C by subsequent three column

Tsoyw

eractions 178 (2009) 151–157

hromatography steps in 10 mM Tris–HCl buffer, pH 8.0, contain-ng 5 mM 2-mercaptoethanol, 0.5 mM EDTA and 20% (v/v) glycerolo stabilize the enzyme. The E. coli cell extract was dialyzed againsthe Tris buffer and applied to a Q-Sepharose column (2 cm × 40 cm).he enzyme was eluted with a linear gradient of 0–0.1 M NaCl.he enzyme fractions were concentrated by ultrafiltration, andel-filtrated through a Sephadex G-100 column (2.5 cm × 90 cm).he enzyme fractions were applied to a Red-Sepharose column1.5 cm × 5 cm), and the enzyme was eluted with the buffer con-aining 0.5 mM NADP+ after the column was washed with the bufferithout NADP+.

Rat AKRs (1B4, 1C8, 1C9, 1C15, 1C16, 1C17, 1C24 and 17�-HSDype 5) were expressed from their cDNAs and purified to homo-eneity [20,21,29–31]. Purity was confirmed by SDS-PAGE, androtein concentration was determined by a bicinchoninic acid pro-ein assay reagent kit (Pierce, Rockford, IL) using bovine serumlbumin as the standard.

.4. Assay of enzyme activity

The reductase activity of AKRs was determined by measuringhe rate of change in NADPH absorbance at 340 nm. The standardeaction mixture consisted of 0.1 M potassium phosphate, pH 7.4,.1 mM NADPH, substrate and enzyme in a total volume of 2.0 ml.n the purification of AKR1B13, 0.1 M potassium phosphate, pH.0, and 0.1 mM pyridine-3-aldehyde were used as the buffer andubstrate, respectively. The dehydrogenase activity was assayed byeasuring the change in NADPH fluorescence (at 455 nm with an

xcitation wavelength of 340 nm) in the phosphate buffer, pH 7.4,ontaining 0.25 mM NADP+ and an appropriate amount of alcoholubstrate. The reductase activity for all-trans-retinal was measuredy the method of Parés and Julià [32], except that 0.1 M potas-ium phosphate, pH 7.4, containing 0.01% Tween 80 was useds the buffer. The IC50 (inhibitor concentrations required for 50%nhibition) values for inhibitors were determined with 0.1 mMyridine-3-aldehyde as the substrate. The substrates and inhibitors,hich are hardly soluble in water, were dissolved in methanol or

0% methanol, and added into the reaction mixture, in which thenal concentration of methanol was less than 1.2%. The concentra-ion of methanol did not affect the activity of the enzymes. One unitU) of enzyme activity was defined as the amount of enzyme thatatalyzes the oxidation or formation of 1 �mol NADPH per minutet 25 ◦C. Kinetic constants for substrates and coenzymes, and IC50alues were calculated by using the appropriate programs of thenzFitter (Biosoft, Cambridge, UK), and are expressed as the meansf two determinations.

.5. Tissue distribution analysis

Tissues were excised from 12 to 14 week-old male and femaleistar rats, and their total RNAs and cytosolic fractions were

repared as described [27,28]. The total RNA samples were sub-ected to RT-PCR using Taq DNA polymerase. PCR amplificationonsisted of an initial denaturation step at 94 ◦C for 5 min, and 30ycles of denaturation (94 ◦C/30 s), annealing (30 s) and extension72 ◦C/2 min), and a final incubation step at 72 ◦C for 5 min. Therimers and annealing temperatures for the amplification of theDNAs for AKRs (1A3, 1B4, 1B13 and 1C15) are listed in Table 1. TheDNA for rat �-actin was also amplified as an internal control usinghe specific primers that were obtained from Toyobo (Osaka, Japan).

he PCR products were separated by agarose gel electrophore-is, and revealed with ethidium bromide. The cytosolic fractionsf the tissue homogenates were subjected to Western blot anal-sis using antibodies against the purified recombinant AKR1B13,hich were raised in rabbits [33]. The immunoreactive proteins

S. Endo et al. / Chemico-Biological Interactions 178 (2009) 151–157 153

Table 1Primers used in the RT-PCR analysis.

Amplified cDNA for Sequence of sense primer Annealing (◦C)

AKR1A3 5′-aacatatgacggcctccagt-3′ 57AAA

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Table 2Kinetic constants for aldehydes.

Substrate Km (�M) kcat (min−1) kcat/Km (min−1 �M−1)

Aromatic aldehydes4-Nitrobenzaldehyde 3.5 34 9.8Pyridine-3-aldehyde 12 31 2.6Pyridine-4-aldehyde 20 38 1.92-Phenyl-2-propenal 52 26 0.50Benzaldehyde 63 25 0.41

Alkanals1-Nonanal 5.0 34 6.81-Decanal 5.5 21 3.81-Octanal 14 42 3.01-Butanal 15 40 2.61-Hexanal 23 45 1.91-Propanal 300 7.8 0.03

Alkenals2-Decenal 10 8.7 0.902-Nonenal 17 9.3 0.552-Hexenal 75 40 0.544-Hydroxynonenal 30 11 0.372,4-Decadienal 48 12 0.242,4-Nonadienal 64 12 0.18Acrolein 120 16 0.13

Other aldehydes

iarasewd(wtCvaefficiently than dl-glyceraldehyde. The enzyme also reduced a 2-carbon aldehyde, glycolaldehyde, although the Km value was high.

AKR1B13 highly reduced �-dicarbonyl compounds (Table 3). Atendency toward higher catalytic efficiency with increasing chainlength was observed in a series of aliphatic �-diketones, similar

Table 3Substrate specificity for �-dicarbonyl compounds.

Substrate Km (�M) kcat (min−1) kcat/Km (min−1 �M−1)

�-Diketones2,3-Heptanedione 6.5 36 5.616-Ketoestrone 5.7 11 1.92,3-Hexanedione 14 15 1.1Phenyl-1,2-propanedione 56 23 0.41Isatin 69 17 0.252,3-Pentanedione 200 23 0.121S-Camphorquinone 200 20 0.013,4-Hexanedione 310 14 0.042,3-Butanedione 680 20 0.03

Other �-dicarbonyls

KR1B4 5′-ggcaccaagatgcccaccc-3′ 65KR1B13 5′-atggcaaccttcgtgg-3′ 57KR1C15 5′-atggatctcaaacatagtcg-3′ 57

ere visualized using the enhanced chemiluminescence substrateystem.

.6. Cell culture experiments

Rat coronary aortic endothelial cells and fibroblasts were iso-ated and cultured according to the method of Domenighetti etl. [34] with minor modification [35]. YPEN-1 and NRK cells wereultured in Dulbecco’s modified Eagle medium [supplementedith sodium bicarbonate (1.5 mg/ml), 0.1 mM non-essential amino

cids, 1.0 mM sodium pyruvate, heparin (30 �g/ml), 5% fetal bovineerum (FBS), penicillin (100 U/ml) and streptomycin (100 �g/ml)]nd Eagle’s minimum essential medium (supplemented with.1 mM non-essential amino acids, 5% FBS, penicillin and strepto-ycin), respectively, at 37 ◦C in a humidified incubator containing

% CO2. After the cells were grown to 70–80% confluent in growthedium, the medium was replaced with another containing 2% FBS,

xcept that the medium containing 0.5% FBS was used in the treat-ents of FGF-1 and FGF-2. The cells were then treated with agents.

he cell collection and preparation of the total RNA samples wereerformed as described [35]. The expression levels of the cDNAs forKRs (1B4, 1B13 and 1C15) were analyzed by RT-PCR as describedbove. The densities of the cDNAs for the AKRs and �-actin wereeasured using a Gel Doc 2000 and an attached program, Quantityne (Bio-Rad). All cell incubations were performed in triplicate.

. Results and discussion

.1. Purification of recombinant AKR1B13

SDS-PAGE analysis of the E. coli cell extracts displayed thathe cells transfected with the expression plasmids harboring theKR1B13 cDNA overexpressed a 36-kDa protein, which was notresent in the control cells transfected with the vector alone. Thisell extract exhibited NADPH-linked reductase activity towards.1 mM pyridine-3-aldehyde (0.27 U/mg) at pH 6.0. The recombi-ant AKR1B13 was purified by assaying the pyridine-3-aldehydeeductase activity and detecting the 36-kDa protein on SDS-PAGEnalysis. The overall yield of the activity resulting from the AKR1B13urification was low (2%) because of the presence of an E. coli-erived 30-kDa pyridine-3-aldehyde reductase in the cell extract.he purified AKR1B13 with specific activity of 1.4 U/mg showed aingle band on SDS-PAGE (data not shown). The enzyme was elutedt a low molecular weight of approximately 35 kDa on the Sephadex-100 column chromatography step, suggesting its monomericature.

.2. Properties of AKR1C13

The purified AKR1B13 showed strict coenzyme specificity forADPH, and no NADH-linked reductase activity was observed. The

m and Vmax values for NADPH determined with 1 mM pyridine-3-ldehyde as the substrate at pH 7.4 were 2.6 �M and 0.85 U/mg,espectively. The NADPH-linked pyridine-3-aldehyde reductasectivity was gradually increased by decreasing pH from 8.0 to.0. Therefore, the substrate specificity of AKR1B13 was exam-

d-Lactoaldehyde 73 17 0.23dl-Glyceraldehyde 2,000 35 0.02Glycolaldehyde 1,500 12 0.008

ned at a physiological pH of 7.4. The enzyme reduced aromaticnd aliphatic aldehydes (Table 2). Aromatic aldehydes were highlyeduced by the enzyme, but 2-carboxybenzaldehyde did not serves the substrate. Long-chain aliphatic aldehydes were excellentubstrates, with Km values in low �M range. Within a series of lin-ar alkanals, the catalytic efficiency (kcat/Km) tended to increaseith increasing chain lengths. It should be noted that aldehy-es with branched side chains, such as 0.1 mM isocaproaldehyde4-methylpentanal), 50 �M farnesal and 20 �M all-trans-retinal,ere not reduced by the enzyme. Introduction of double bonds

ended to result in small increase in Km and decrease in kcat in6-, C9- and C10-alkenals. In the 3-carbon aldehydes, acrolein con-ersely showed lower Km and higher kcat values than 1-propanal,nd d-lactoaldehyde (2-hydroxy-1-propanal) was reduced more

Phenylglyoxal 16 25 1.621-Dehydrocortisol 9.5 12 1.33-Deoxyglucosone 70 12 0.17Methylglyoxal 260 26 0.10Glyoxal 1,280 12 0.009

154 S. Endo et al. / Chemico-Biological Int

Table 4Kinetic constants for alcohols oxidized byAKR1B13.

Substrate Km (�M) kcat (min−1) kcat/Km (min−1 �M−1)

1-Decanol 82 1.4 0.021-Nonanol 740 3.7 0.0051-Hexanol 970 4.1 0.0041-Butanol 63,000 8.2 0.0001Benzyl alcohol 2,100 2.3 0.0012-Phenyl-1-propanol 780 0.7 0.0009S-1-Indanola (0.4)S-1-Teralola (0.2)Pyridine-3-methanola (0.1)

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a The kinetic constants for the substrates were not determined due to the lowctivities. The values in the parentheses were calculated from the specific activitiesor 1.0 mM substrates.

o the series of linear alkanals. By contrast, the enzyme exhib-ted different reactivity towards �-diketones with aromatic orlicyclic rings. The enzyme reduced other �-dicarbonyls with anldehyde and ketone or aldehyde groups, in which phenylglyoxalnd 21-dehydrocortisol showed high catalytic efficiency comparedith methylglyoxal and glyoxal. While monosaccharides such as-glucuronate, d-glucose and d-xylose were not reduced by thenzyme, 3-deoxyglucosone was moderately reduced. The enzymeeduced a p-quinone, TBE (Km = 67 �M, kcat = 37 min−1), but didot exhibit reductase activity towards other p-quinones, aromaticnd aliphatic monoketones, 3- and 20-ketosteroids, and �- and �-iketones, such as 2,5-hexanedione and 2,4-pentanedione.

In the reverse reaction using NADP+ as the coenzyme, AKR1B13xidized several aromatic and aliphatic alcohols (Table 4). Thecat/Km values for the alcohol substrates were much lower thanhose for the corresponding carbonyl products, when the kineticonstants were compared in each oxidoreduction pair, such asliphatic alcohols vs alkenals, and benzyl alcohol vs benzalde-yde. The enzyme did not oxidize R-1-indanol, 2-cyclohexen-1-ol,-phenyl-1-propanol, xylitol and sorbitol.

The pyridine-3-aldehyde reductase activity of AKR1B13 wasnhibited by AR inhibitors, such as zopolestat, minalrestat, tol-estat, fidarestat and sorbinil, whose IC50 values were 0.03, 0.6,.8, 8.5 and 30 �M, respectively. The enzyme was also inhib-

ted by flavonoids that are inhibitors of AR [36]. The IC50 valuesor quercetin, myricetin, daizein and quercetin were 4.8, 6.8, 10nd 17 �M, respectively. However, the enzyme activity was notffected by 0.1 mM Wy14643, a peroxisome proliferators activated

eceptor � agonist, which inhibits both AR [37] and AKR1B1038]. AKR1B13 was inhibited by inhibitors of the AKR1C subfamilynzymes, benzbromarone, hexestrol and zearalenone (IC50 val-es were 0.8, 3.2, and 25 �M, respectively) [39]. No significant

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able 5omparison of kinetic constants for reactive carbonyl compounds among rat AKRs.

KR Methylglyoxal 3-Deoxyglucosone Gly

Km kcat/Km Km kcat/Km Km

B13a 0.26 100 0.070 170 1B4a 0.10 2,100 0.079 440 0A3b 2.4 400 1.7 190C15c 0.015 1,700 0.020 40 1C9a 3.2 10 5.5 30C8a 26 10 n.a.7�HSD5a 0.77 20 n.a. 35A1d 7.2 6 nd 45

a The Km (mM) and kcat/Km (min−1 mM−1) values of AKRs (1B13, 1B4, 1C9, 1C8 and 17�Hufficiently high to determine kinetic constants. nd, not determined.

b The kinetic constants determined at pH 7.0 are taken from [47].c The kinetic constants determined at pH 7.4 are taken from [20].d The kinetic constants determined at pH 6.6 are taken from [41].

eractions 178 (2009) 151–157

nhibition (less than 25%) was observed with aldehyde reduc-ase inhibitors (0.5 mM valproate, diphenic acid and barbital) [40],�-HSD inhibitors (0.1 mM indomethacin and 10 �M medrox-progesterone acetate) [31,39] and an AKR1C15 inhibitor (0.1 mM′,3′′,5′,5′′-tetrabromophenolphthalein) [20].

.3. Comparison of reactivity to carbonyl compounds among ratKRs

AKR1B13 shows 82% sequence identity with human AKR1B10nd mouse AKR1B7, but is clearly different from the human andouse enzymes [13,15] in its inability to reduce all-trans-retinal

nd isocaproaldehyde. The three AR-like enzymes also differ fromach other in the susceptibility to AR inhibitors: in contrast to highnhibition of AKR1B13, AR inhibitors moderately inhibit AKR1B1038] and have no effect on AKR1B7 [14]. The substrate specificitynd inhibitor sensitivity of AKR1B13 are similar to that of anotherouse AR-like protein AKR1B8 [9,12], which shares 95% sequence

dentity with AKR1B13. The �-diketones found in the present studyo be good substrates of AKR1B13 may be reduced by AKR1B8.owever, the kinetic constants for several aromatic and aliphaticldehydes are different between the two enzymes. It would benteresting to elucidate the structural factors affecting the differ-nce in the kinetic constants between the rat and mouse enzymesn a future study.

The substrate specificity of AKR1B13 overlaps with thosef AR, aldehyde reductase, aflatoxin B1-aldehyde reductase andSDs in the AKR1C subfamily, but clearly differs in the follow-

ng terms. Unlike AR [15] and AKR1C15 [20], AKR1B13 did notatalyze the reduction of all-trans-retinal, isocaproaldehyde andldoses. Additionally, AKR1B13 did not reduce d-glucuronic acid,-carboxybenzaldehyde and ketosteroids, which are representa-ive substrates of aldehyde reductase [40], aflatoxin B1-aldehydeeductases [41,42] and the HSDs [21,30,31,39], respectively. Thus,KR1B13 seems to show somewhat narrow substrate specificityompared with these other AKRs. However, it reduced reactivendogenous carbonyl compounds, which include the lipid-derivedldehydes, such as 4-hydroxynonenal [43] and acrolein [44], and-dicarbonyl compounds, such as 3-deoxyglucosone, glyoxal andethylglyoxal, that are generated in the course of glycation and

ugar metabolism [45,46]. The kinetic constants for the reactivendogenous carbonyl compounds were compared among eight

urified recombinant AR (AKR1B4), 3�-HSD (AKR1C9), 20�-HSDAKR1C8) and rat 17�-HSD type 5 (17�HSD5). Judging from them and kcat/Km values, AR most efficiently reduces methylgly-xal, glyoxal and acrolein. AKR1C15 is also an excellent reductase

oxal Acrolein 4-Hydroxynonenal

kcat/Km Km kcat/Km Km kcat/Km

.28 9 0.12 130 0.030 370

.56 50 0.041 490 0.033 420nd nd – nd

.6 10 0.14 210 0.002 2,900LA 1.0 8 LALA LA LA0.2 0.87 9 n.a.2 LA LA

SD5) were determined at pH 7.4. n.a., no activity was detected. LA, low activity not

cal Interactions 178 (2009) 151–157 155

fetcw[dt

3

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c

Fig. 1. Tissue distribution of AKR1B13 and its mRNA in rat tissues in comparisonwith those of other AKRs (1B4, 1A3 and 1C15). Panels a and c–f: RT-PCR analysis forexpression of mRNAs for AKR1B13 (a), AKR1B4 (c), AKR1A3 (d), AKR1C15 (e), and�-actin (f), whose cDNA sizes amplified are 951, 445, 978, 975 and 764 base pairs,rmtK

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FAmT*

S. Endo et al. / Chemico-Biologi

or methylglyoxal and 4-hydroxynonenal. AKR1B13 is the thirdffective reductase for these carbonyl compounds. In particular,he Km and kcat/Km values for 3-deoxyglucosone of AKR1B13 areomparable to those of AR and aldehyde reductase, respectively,hich were reported to be major 3-deoxyglucosone reductases

22,40,47]. Thus, AKR1B13 appears to function as a defense system,etoxifying these reactive carbonyl compounds occurring in ratissues.

.4. Tissue distribution and regulation of gene expression

The tissue distribution of the AKR1B13 mRNA was initiallyssessed by RT-PCR using the primer specific to the cDNA forKR1B13. As illustrated in Fig. 1a, the mRNA was detected in all

emale rat tissues besides the brain. A similar expression pattern ofhe transcript in male rat tissues was observed, and there was noex difference in expression levels of the mRNA (data not shown).he 36-kDa immunoreactive protein was observed in several ratissues on Western blotting (Fig. 1b) using the anti-AKR1B13 poly-lonal antibodies, which did not cross-react with rat AKRs (1B4,C8, 1C9, 1C15, 1C16, 1C17, 1C24 and 17�HSD5). The expression ofKR1B13 might be tissue-specifically regulated as some discrep-ncy between the levels of its mRNA and protein in several tissuesas observed. The results suggest that AKR1B13 is expressed in

lmost all rat tissues, but its contents in rat tissues other thanhe lung, stomach and adrenal gland are low. The tissue distri-ution is essentially identical to the previous two-dimensionallectrophoresis analysis of AKR1B13 as a tumor-associated proteinn some rat tissues [11], but differs from that of the mouse homolog,

KR1B8 [17]. The expression patterns of AKR1B13 in rat tissues arelso slightly different from those of AR (AKR1B4), aldehyde reduc-ase (AKR1A3) and AKR1C15 (Fig. 1c–e).

RT-PCR analysis for the AKR1B13 mRNA in primary-culturedells of rat coronary aorta showed that it was expressed in both

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ig. 2. Expression of AKR1B13 in rat vascular endothelial cells and its induction by 1,4-napKR1B4 and AKR1C15 in the primary-cultured endothelial cells (EC), fibroblasts (F) andRNAs for AKR1B13 (�), AKR1C15 (�) and AKR1B4 ( ) in YPEN-1 (B) and NRK (C) cel

he expression of the mRNA transcripts was analyzed by RT-PCR, and its level is expresseSignificant difference from the untreated control, p < 0.05.

espectively. Panel b: Western blot analysis using the anti-AKR1B13 antibodies. Theolecular weights of the immunoreactive bands were 36-kDa, which was identical

o recombinant AKR1B13. Tissues: Br, brain; Lu, lung; He, heart; St, stomach; Li, liver;i, kidney; Ad, adrenal gland; Si, small intestine; Co, colon; and Ov, ovary.

ndothelial cells and fibroblasts (Fig. 2A). In the rat endothelialell line YPEN-1, the mRNA, together with those for AKR1B4 andKR1C15, was also expressed. Because human AR, mouse AKR1B8nd AKR1B7 have been reported to be up-regulated by estrogen48], FGFs [9] and androgen [10], respectively, the effects of theseteroids and factors on the expression of the AKR1B13 transcript

n YPEN-1 cell and the primary-cultured fibroblasts were exam-ned. 17�-Estradiol, 5�-dihydrotestosterone, FGF-1 and FGF-2 didot have significant effects on the expression level of the AKR1B13RNA in the two cells (data not shown). This, together with the

ifferences in tissue distribution patterns, implies that AKR1B13

hthoquinone and H2O2. (A): RT-PCR analysis for expression of mRNAs for AKR1B13,YPEN-1 cells. (B and C): Effects of 1,4-naphthoquinone and H2O2 on expression ofls. The cells were treated for 4 h with the indicated concentrations of the agents.d as the fold ± S.E. (n = 3) of the intensity of the cDNA band in the untreated cells.

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56 S. Endo et al. / Chemico-Biologi

iffers from human AR and the two mouse AR-like enzymes in itsene regulation.

Substrate specificity suggests that AKR1B13 acts as a detox-fication enzyme of reactive carbonyl compounds derived fromxidative stress. p-Quinones are known to generate reactive oxy-en species by redox cycles [49]. We examined the effects of,4-naphthoquinone and hydrogen peroxide on the expression ofhe mRNAs for AKR1B13, AKR1B4 and AKR1C15 in YPEN-1 cells.he treatments with the two compounds increased the expres-ion of the AKR1B13 mRNA in a dose-dependent manner (Fig. 2B).n contrast, the expression of the AKR1B4 mRNA was slightly ele-ated only by 1,4-naphthoquinone, and that of the AKR1C15 mRNAended to be increased only by the addition of hydrogen peroxide.ydrogen peroxide and 1,4-naphthoquinone similarly enhanced

he expression of AKR1B13 mRNA in the rat kidney cell line, NRKFig. 2C). The results support the physiological function of AKR1B13s a defense system against oxidative stress.

cknowledgments

This work was supported by a Grant-in-Aid for Young Scientistsrom the Japan Society for the Promotion of Science.

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