Substrate specificity and inhibitor sensitivity of rabbit 20α-hydroxysteroid dehydrogenase

Article abstract of DOI:10.1248/bpb.b13-00342

In this study, we examined the substrate specificity and inhibitor sensitivity of rabbit 20α-hydroxysteroid dehydrogenase (AKR1C5), which plays a role in the termination of pregnancy by progesterone inactivation. AKR1C5 moderately reduced the 3-keto group of only 5α-dihydrosteroids with 17β- or 20α/β-hydroxy group among 3-ketosteroids. In contrast, the enzyme reversibly and efficiently catalyzed the reduction of various 17- and 20-ketosteroids, including estrogen precursors (dehydroepiandrosterone, estrone and 5α-androstan-3β- ol-17-one) and tocolytic 5β-pregnane-3,20- dione. In addition to the progesterone inactivation, the formation of estrogens and metabolism of the tocolytic steroid by AKR1C5 may be related to its role in rabbit parturition. AKR1C5 also reduced various non-steroidal carbonyl compounds, including isatin, an antagonist of the C-type natriuretic peptide receptor, and 4-oxo-2-nonenal, suggesting its roles in controlling the bioactive isatin and detoxification of cytotoxic aldehydes. AKR1C5 was potently and competitively inhibited by flavonoids such as kaempferol and quercetin, suggesting that its activity is affected by ingested flavonoids.

Full text of DOI:10.1248/bpb.b13-00342

1514
Note
Biol. Pharm. Bull. 36(9) 1514–1518 (2013)
Vol. 36, No. 9
Substrate Specificity and Inhibitor Sensitivity of Rabbit
20α-Hydroxysteroid Dehydrogenase
,a
Satoshi Endo,* Yuki Arai,a Akira Hara,b Yukio Kitade,b Yasuo Bunai,c Ossama El-Kabbani,d and
Toshiyuki Matsunagaa
a Laboratory of Biochemistry, Gifu Pharmaceutical University; Gifu 502–1196, Japan: b Department of Biomolecular
c
Science, Faculty of Engineering, Gifu University: Department of Legal Medicine, Graduate School of Medicine, Gifu
d
University; Gifu 501–1193, Japan: and Department of Medicinal Chemistry, Victorian College of Pharmacy, Monash
University; Parkville, Victoria 3052, Australia.
Received May 6, 2013; accepted June 18, 2013
In this study, we examined the substrate specificity and inhibitor sensitivity of rabbit 20α-hydroxysteroid
dehydrogenase (AKR1C5), which plays a role in the termination of pregnancy by progesterone inactivation.
AKR1C5 moderately reduced the 3-keto group of only 5α-dihydrosteroids with 17β- or 20α/β-hydroxy group
among 3-ketosteroids. In contrast, the enzyme reversibly and efficiently catalyzed the reduction of various 17-
and 20-ketosteroids, including estrogen precursors (dehydroepiandrosterone, estrone and 5α-androstan-3β-
ol-17-one) and tocolytic 5β-pregnane-3,20-dione. In addition to the progesterone inactivation, the formation
of estrogens and metabolism of the tocolytic steroid by AKR1C5 may be related to its role in rabbit partu-
rition. AKR1C5 also reduced various non-steroidal carbonyl compounds, including isatin, an antagonist of
the C-type natriuretic peptide receptor, and 4-oxo-2-nonenal, suggesting its roles in controlling the bioactive
isatin and detoxification of cytotoxic aldehydes. AKR1C5 was potently and competitively inhibited by flavo-
noids such as kaempferol and quercetin, suggesting that its activity is affected by ingested flavonoids.
Key words aldo-keto reductase 1C5; 20α-hydroxysteroid dehydrogenase; 4-oxo-2-nonenal; 17β-hydroxysteroid
dehydrogenase; dehydroepiandrosterone; isatin
20α-Hydroxysteroid dehydrogenase (20α-HSD, EC 1.1.1.49) 20α-HSD (AKR1C5) that shares 80% sequence identity with
catalyzes the conversion of an active progestin, progesterone, AKR1C1 was molecularly cloned from the ovary,6) but its
into 20α-hydroxyprogesterone, and plays a major role in the substrate specificity for steroids and PGs differs from that of
termination of pregnancy.1) The cDNAs for 20α-HSDs have the human enzyme. AKR1C5 displays comparable activity
been cloned from human2) and other mammalian tissues.3–6) for the 3α-, 17β- and 20α-reduction of 5α-dihydrotestosterone,
The mammalian enzymes are composed of 323-amino acids, 4-androstene-3,17-dione and progesterone, respectively,18) and
and belong to the aldo-keto reductase (AKR) superfam- is identical to ovarian PGE2 9-ketoreductase.19) However, it
ily (http://www.med.upenn.edu/akr/).1) Among them, human remains unknown whether AKR1C5 exhibits such a broad
20α-HSD (AKR1C1) is the most characterized enzyme. specificity for other steroids and nonsteroidal compounds
AKR1C1 reduces various 20-ketosteroids, and exhibits moder- reported for the human and rodent 20α-HSDs. In this study,
ate 3α- and 3β-HSD activities for 5β-dihydro-3-ketosteroids we examined the substrate specificity of AKR1C5 for various
and 5α-dihydro-3-ketosteroids, respectively, although its steroids and nonsteroidal compounds, in order to elucidate its
17β-HSD activity is negligibly low.7–9) It also shows broad role other than the progesterone inactivation. Additionally, we
substrate specificity for endogenous and xenobiotic non- compared the inhibitor sensitivity of AKR1C5 with that of
steroidal carbonyl compounds, which include lipid peroxida- AKR1C1.
tion products (4-hydroxy-2-nonenal10) and 4-oxo-2-nonenal11)),
prostaglandin (PG) D2,9) p-quinones and drug ketones.3,12,13) In
MATERIALS AND METHODS
the reverse reaction, AKR1C1 exhibits dihydrodiol dehydro-
genase activity, which oxidizes trans-dihydrodiol metabolites
Materials Nicotinamide adenine dinucleotide (NAD+),
of aromatic hydrocarbons.14) Due to possible involvement of nicotinamide adenine dinucleotide phosphate (NADP+) and
AKR1C1 in inactivation of active steroids and cancer develop- their reduced forms (NADH and NADPH) were obtained
ment, its various inhibitors have been reported.11,15)
from Oriental Yeast Co. (Tokyo, Japan); steroids were from
In other mammals, 20α-HSDs of monkeys (AKR1C25),3,9) Steraloids (Newport, RI, U.S.A.); 4-oxo-2-nonenal, 4-hydroxy-
rats (AKR1C8)12,16,17) and mice (AKR1C18)9) show dual 3α- 2-nonenal and PGD2 were from Cayman Chemical (Ann
and 3β-HSD activities and broad substrate specificity for Arbor, MI, U.S.A.). trans-Benzene dihydrodiol,20) 6-tert-
20-ketosteroids and xenobiotic compounds. AKR1C25 shares butyl-2,3-epoxy-5-cyclohexen-1,4-dione (TBE),21) isocapro-
94% amino acid sequence identity with AKR1C1 exhibiting aldehyde,22) 4-oxo-2-nonenal23) and 4-oxo-2-hexanal24) were
almost identical properties including PGD2 reductase activ- synthesized as described previously. 3-Deoxyglucosone and
ity and inhibitor sensitivity, whereas AKR1C8 and AKR1C18 befunolol were gifts from Nippon Zoki Pharmaceutical Co.
share lower amino acid sequence identity with AKR1C1 (Osaka, Japan) and Kaken Pharmaceutical Co. (Tokyo, Japan),
(72 and 69%, respectively), do not reduce PGD2 and show respectively.
an inhibitor sensitivity different from AKR1C1. In rabbits,
Preparation of Recombinant Enzymes A cDNA for
AKR1C5 was isolated from a total RNA sample of ovaries of
a Japanese white rabbit by reverse transcription (RT)-poly-
The authors declare no conflict of interest.
*To whom correspondence should be addressed. e-mail: sendo@gifu-pu.ac.jp
© 2013 The Pharmaceutical Society of Japan

September 2013
1515
merase chain reaction (PCR). The preparation of total RNA, as their 17β-hydroxy metabolites. Similarly, AKR1C5 reacted
RT, and DNA techniques followed the standard procedures towards various 20-ketosteroids as excellent substrates. The
described by Sambrook et al.25) The PCR was performed reduced products of 5α/β-pregnane-3,20-diones and progester-
using Pfu DNA polymerase (Stratagene) and a pair of sense one were identified as their 20α-hydroxy metabolites, indicat-
and antisense primers, 5′-ttttcatatgatggatcccaaatttcagcg-3′ ing that the enzyme preferentially reduces the 20-keto group
and 5′-ttttgtcgacttaatattcatcagaaaatgggt-3,′ which contain of the 3,20-diketosteroids. Thus, the binding of the 17- and
underlined NdeI and SalI sites, respectively. The PCR prod- 20-ketosteroids to the enzyme may not be significantly affect-
ucts were purified, digested with the two restriction enzymes ed with their differences in the fusion types between A- and
(Invitrogen), and ligated into the pCold I vectors (TaKaRa) B-rings, the unsaturated bond on the rings and the configura-
that had been digested with the two restriction enzymes. The tion of 3-hydroxy group on A-ring.
sequence of the coding region of the cDNA was confirmed to
In the reverse reaction, AKR1C5 oxidized various 17β- and
encode the 323-amino acid sequences of AKR1C57) fused to 20α-hydroxysteroids (Table 2), and the oxidized products of
the N-terminal 6-His tag by DNA sequencing. The expression 5α/β-androstane-3α,17β-diols were identified as their 17-keto
construct was transfected into Escherichia coli BL21 (DE3) metabolites. The results suggest that the oxidoreduction of
pLysS, and the recombinant AKR1C5 was expressed and puri- the 17β- and 20α-hydroxy groups on the steroid substrates by
fied to homogeneity from the cell extracts using the nickel- the enzyme is reversible. In contrast, no significant activities
charged Sepharose 6FF resin (GE Healthcare) as described were observed with 5α/β-androstan-3α/β-ol-17-ones, 5α/β-
previously.24) Recombinant AKR1C1 was expressed from the pregnan-3α/β-ol-20-ones, 5α/β-pregnane-3α,21-diol-20-ones,
cDNA and purified to homogeneity as described previously.26) 4-pregnen-3α-ol-20-one and lithocholic acid as substrates,
Assay of Enzyme Activity The dehydrogenase and re- indicating that the reduction of the 5α-dihydro-3-ketosteroids
ductase activities of the recombinant proteins were assayed at is apparently irreversible.
25°C by measuring the rate of change in NADPH fluorescence
The stereospecific reduction of only 5α-dihydro-3-
and its absorbance, respectively, in 0.1M potassium phosphate ketosteroids by AKR1C5 is clearly different from those by
buffer, pH 7.4, containing enzyme, substrate and coenzyme AKR1C1 and rodent 20α-HSDs (AKR1C8 and AKR1C18)
(0.25 mM NADP+ and 0.1mM NADPH, respectively).24) The that act as reversible 3α- and 3β-HSDs towards 5α- and
reductase activity for all-trans-retinal was measured by the 5β-dihydrosteroids, respectively.8,9,12) In addition, the high
method of Parés and Julià,27) except that 0.1M potassium phos- 17β-HSD activity is a unique property of AKR1C5, because
phate, pH 7.4, containing 0.01% Tween 80 was added as the the 17-keto- and 17β-hydroxy-steroids listed in Tables 1 and 2
buffer.
were not significantly reduced or oxidized by AKR1C1 (data
The apparent Km and kcat values for coenzymes and sub- not shown), AKR1C189) and AKR1C8.12,16) A crystallographic
strates were determined by fitting the initial velocities to the study of AKR1C5-NADP+-testosterone ternary complex has
Michaelis–Menten equation. The inhibitor constant, Ki, was shown that the substrate specificity of the enzyme is medi-
estimated from the Dixon plot of the velocities obtained in
the substrate β-ionol range (20–200µM) with three concentra-
tions of the inhibitor. The kinetic constants and IC50 values are
expressed as the means of at least three determinations. The
standard deviations of the determinations were less than 15%,
unless otherwise noted.
Product Identification The reaction was conducted at
37°C for 30min in a 2.0-mL reaction mixture, containing
0.2mM NADPH, substrate (0.05–0.1mM), enzyme (0.1mg),
and 0.1M potassium phosphate, pH 7.4. The products were
extracted into 4mL ethyl acetate, and identified by TLC and
liquid chromatography/mass spectrometry.9,24) The reduced
products of ONE were also analyzed by TLC.23)
Table 1. Kinetic Constants for Ketosteroids
Km
kcat
kcat/Km−1
(min−1μM
)
Substrate
(μM) (min−1
)
a)
3-Ketosteroids
5α-Pregnan-20α-ol-3-one
5α-Dihydrotestosterone
17-Ketosteroids
5.9
3.4
4.6
1.5
0.78
0.44
4-Androstene-3,17-dione
5β-Androstan-3α-ol-17-one
DHEA
1.0
1.7
6.0
4.1
4.2
2.8
1.1
2.9
6.7
6.0
2.6
4.0
9.3
5.1
4.8
2.7
1.0
2.6
5.9
5.0
2.6
2.4
1.6
5α-Androstan-3α-ol-17-one
DHEA-3-sulfate
1.2
1.1
5β-Androstan-3β-ol-17-one
Estrone
0.96
0.91
0.90
0.88
0.83
RESULTS AND DISCUSSION
7α-Hydroxy-DHEA
Specificity for Steroids The 3-, 17- and 20-ketosteroids
reduced by the purified recombinant AKR1C5 in the pres-
ence of NADPH are listed in Table 1. Among 3-ketoste-
roids, 5α-dihydrotestosterone (5α-androstan-17β-ol-3-one) and
5α-pregnan-20α-ol-3-one were reduced into their 3α-hydroxy
metabolites, but their 5β-isomers and ∆4-unsaturated-3-ke-
tosteroids were not the substrates. In contrast, the enzyme
efficiently reduced 17-keto groups of 5α/β-androstanes,
dehydroepiandrosterone (DHEA, 5-androsten-3β-ol-17-one)
and its 7α-hydroxy and 3-sulfate derivatives, as well as
4-androstene-3,17-dione and estrone. The reduced prod-
ucts of 4-androstene-3,17-dione, 5β-androstan-3α-ol-17-one,
5α-androstan-3β-ol-17-one, DHEA and estrone were identified
Estrone-3-sulfate
5α-Androstan-3β-ol-17-one
20-Ketosteroids
Progesterone
0.6
0.5
1.0
1.0
1.0
1.3
2.6
6.2
1.6
1.1
1.9
1.8
1.7
1.9
1.0
1.1
2.7
2.2
1.9
1.8
1.7
1.5
0.38
0.18
5α-Pregnane-3,20-dione
4-Pregnen-3α-ol-20-one
5β-Pregnan-3α-ol-20-one
5β-Pregnane-3,20-dione
5α-Pregnan-3α-ol-20-one
5β-Pregnane-3α,21-diol-20-one
5α-Pregnane-3α,21-diol-20-one
a) 5β-Androstan-17β-ol-3-one, 5β-pregnan-20α-ol-3-one, testosterone and 20α-
hydroxyprogesterone were not reduced.

1516
Vol. 36, No. 9
ated by three structural elements; the relaxation of loop B is similar to those of AKR1C13,12,13) and AKR1C18,9) but the
(residues 223–230), the nature of the residue at position 54 and kinetic constants for the substrates are different among the
the residues in C-terminal tail.18) In particular, the relaxation three enzymes. In fact, AKR1C1 oxidized β-ionol at lower Km
movement of loop B induced upon the binding of the substrate (1.2 µM) and kcat values (3.2min−1) than the values of AKR1C5.
is proposed to be mostly related to the 17β-HSD activity of
Physiological Roles Although AKR1C5 predominantly
AKR1C5.
functions in the metabolism of progesterone in the ovarian
Specificity for Non-steroidal Compounds AKR1C5 and luteal tissues,6,19) its broad substrate specificity for com-
reduced various endogenous and xenobiotic carbonyl com- pounds other than progesterone and PGE2 suggests follow-
pounds (Table 3). Among the endogenous substrates, isatin ing novel roles. 1) The enzyme efficiently reduced estrone,
was the most excellent substrate, showing the highest kcat/Km 4-androstene-3,17-dione and DHEA, which are precursors
value. Most of the endogenous aldehydes were reduced at of 17β-estradiol synthesis. In addition to the inactivation of
low Km values, which were comparable to those for the progesterone, the estrogen synthesis by AKR1C5 may be
steroidal substrates. These substrates include cytotoxic al- related to its major role in the termination of pregnancy.
dehydes (4-oxo-2-nonenal, 4-hydroxy-2-nonenal and 4-oxo- AKR1C5 is suggested to be implicated in luteolysis by pro-
2-hexenal)23,28,29) and all-trans-retinal, and the reduced product ducing PGF2α from PGE2,19) but the reported Km and kcat/Km
of 4-oxo-2-nonenal was identified as its alcohol metabolite, values for PGE2 are much higher and lower, respectively, than
4-oxo-2-nonenol. The enzyme also showed high activities for those for the above 17-ketosteroids. Recently, 17β-estradiol
xenobiotic pyridine-3-aldehyde, p-quinones (2-tert-butyl-1,4- has been reported to control time of luteolysis in rats and
benzoquinone, TBE and menadione) and ketones (4-nitro- heifers.31,32) Thus, it is possible that the reductive 17β-HSD
acetophenone, befunolol, α-tetralone and ketotifen). While activity of AKR1C5 is involved in regressing post-partum
AKR1C5 is known to reduce PGE2,19) no reductase activity rabbit corpora lutea. 2) 5-Androstene-3β,17β-diol, the reduced
was detected with 0.1mM PGD2 as the substrate. In addition to metabolite of DHEA by AKR1C5, binds to estrogen receptor
the inability to reduce PGD2, the high kcat/Km values for isatin (ER) β,33) which is expressed in rabbit ovary and uterus.34)
and the p-quinones of AKR1C5 is distinct from the low values Additionally, AKR1C5 reduced 5α-androstan-3β-ol-17-one
of AKR1C1.13,30) In contrast, AKR1C5 is similar to AKR1C18 into 5α-androstane-3β,17β-diol, which is another ligand of
in the high activity for isatin,9) but the two enzymes clearly ERβ.35) Since rabbit aldose reductase-like protein (AKR1B19)
differ in the reactivity to befunolol, which is not reduced by that converts 5α-androstane-3,17-dione into 5α-androstan-3β-
AKR1C18.
ol-17-one is expressed in many rabbit tissues including the
In the reverse reaction, AKR1C5 oxidized xenobiotic ovary,24) AKR1C5 might be involved in the synthesis of this
alicyclic and aliphatic alcohols, including trans-benzene ERβ ligand in concert with AKR1B19. The production of the
dihydrodiol that is a representative substrate of dihydrodiol
dehydrogenase12,14) (Table 2). Among the xenobiotic substrates,
Table 3. Kinetic Constants for Non-steroidal Carbonyl Compounds
β-ionol was most efficiently oxidized. The substrate specificity
Km
(μM)
kcat
kcat/Km−1
(min−1μM
)
Substrate
(min−1
)
Table 2. Kinetic Constants for Hydroxysteroids and Other Alcohols
Endogenous ketones
Isatin
0.6
8.2
9.5
7.2
5.8
11
14
Km
(μM)
kcat
kcat/Km−1
(min−1μM
)
Diacetyl
19
88
0.50
0.082
0.080
0.008
Substrate
(min−1
)
Methylglyoxal
Acetoin
17β-Hydroxysteroids
5β-Androstane-3α,17β-diol
5α-Androstane-3α,17β-diol
5-Androstene-3β,17β-diol
5β-Androstan-17β-ol-3-one
5α-Androstane-3β,17β-diol
5β-Androstane-3β,17β-diol
Testosterone
162
1400
1.4
15
16
11
3-Deoxyglucosone
Endogenous aldehydes
4-Oxo-2-nonenal
1-Nonanal
4.2
3.1
6.2
2.2
2.5
3.8
6.0
9.2
1.9
2.0
3.4
0.67
2.8
1.9
1.8
6.7
3.7
1.5
0.9
7.6
5.5
7.4
1.9
2.1
0.86
0.80
0.26
0.52
0.16
1-Hexanal
2.6
0.34
0.22
0.20
0.15
0.11
0.098
0.057
0.021
trans-2-Nonenal
trans-2-Hexenal
Acrolein
1.2
13
1.3
18
1.5
17β-Estradiol
14
2.1
5α-Dihydrotestosterone
20α-Hydroxysteroids
5β-Pregnane-3α,20α-diol
5α-Pregnane-3α,20α-diol
5β-Pregnan-20α-ol-3-one
20α-Hydroxyprogesterone
5α-Pregnan-20α-ol-3-one
Non-steroidal alcohols
β-Ionol
4-Oxo-2-hexenal
4-Hydroxy-2-nonenal
all-trans-Retinal
Isocaproaldehyde
Xenobiotics
5.9
5.3
8.9
0.69
0.52
0.51
1.4
7.8
0.8
5.7
2.2
17
2.2
1.8
1.6
1.3
0.86
1.5
9.2
2.9
9.5
68
TBQa)
1.0
1.2
2.0
11
11
11
4-Nitroacetophenone
TBE
6.7
10
5.6
5.0
18
112
131
152
260
180
14
0.61
Befunolol
34
7.0
7.9
7.5
3.2
1.2
0.79
0.13
0.12
0.060
0.011
(S)-1-Tetralol
13
12
0.12
Pyridine-3-aldehyde
Menadione
61
18
(S)-1-Indanol
0.092
0.027
0.025
0.011
4-Chromanol
4.1
α-Tetralone
53
trans-Benzene dihydrodiol
Geraniol
6.5
2.0
Ketotifen
111
a) 2-tert-Butyl-1,4-benzoquinone.

September 2013
1517
ER ligands by AKR1C5 may also stimulate roles of ERβ in ferol, naringenin galandin and myricetin), suggesting that at
the ovary and uterus.36,37) 3) The efficient reduction of po- least the presence of two hydroxy groups at positions 3′ and
tent tocolytic 5β-pregnane-3,20-dione38,39) by AKR1C5 may 4′ on the structure of galandin is a structural rationale for
be related to the role of the enzyme in parturition. 4) Isatin, the potent inhibition of AKR1C5 by flavonoids. The inhibi-
the best substrate of AKR1C5, has recently been reported to tion patterns of AKR1C5 by phenolphthalein, benzbromarone,
antagonize rat brain receptor for C-type natriuretic peptide quercetin and kaempferol were competitive with respect to
(CNP),40) which is also secreted from mouse growing follicles β-ionol, showing Ki values of 4.7 0.5, 41 5, 13 2 and 30
and stimulates preantral and antral follicle growth.41) AKR1C5 6nM, respectively. This indicates that the inhibitors bind to the
may be involved in ovarian follicle development by metabo- substrate-binding site of the enzyme. Therefore, the difference
lizing the CNP receptor antagonist, isatin. 5) The enzyme in the inhibitor sensitivity between AKR1C5 and AKR1C1
also reduced other α-dicarbonyls (diacetyl and methylglyoxal) is reflected by the difference between the substrate-binding
and various aliphatic aldehydes at low Km values, which are pockets of the two enzymes.
comparable or superior to those of rabbit aldose reductase and
AKR1B19.24) It is possible that AKR1C5 acts as a detoxifica-
REFERENCES
tion enzyme for reactive α-dicarbonyl compounds42) and cyto-
toxic aldehydes23,28,29) in the ovary, where 4-hydroxy-2-none-
1) Penning TM. Molecular endocrinology of hydroxysteroid dehydro-
genases. Endocr. Rev., 18, 281–305 (1997).
2) Stolz A, Hammond L, Lou H, Takikawa H, Ronk M, Shively JE.
nal and reactive oxygen species are increased by age-related
oxidative stress.43)
cDNA cloning and expression of the human hepatic bile acid bind-
ing protein. A member of the monomeric reductase gene family. J.
Biol. Chem., 268, 10448–10457 (1993).
Inhibitor Sensitivity The NADP+-linked β-ionol de-
hydrogenase activity of AKR1C5 was inhibited by known
AKR1C1 inhibitors (Table 4). Among them, phenolphthalein
inhibited AKR1C5 more potently than AKR1C1, whereas
3) Higaki Y, Kamiya T, Usami N, Shintani S, Shitaishi H, Ishikura S,
Yamaomoto I, Hara A. Molecular characterization of two monkey
3,′3,″5′,5″-tetrabromophenolphthalein (TBPP) and benzbroma-
dihydrodiol dehydrogenases. Drug Metab. Pharmacokinet., 17,
348–356 (2002).
rone, potent competitive inhibitors of AKR1C1,15) showed less
inhibition of AKR1C5. The low sensitivity towards TBPP and
benzbromarone is similar to that of AKR1C18,9) but AKR1C5
was relatively resistant to inhibition by sulfobromophthalein,
a potent AKR1C18 inhibitor showing an IC50 value of 20nM.9)
Compared to AKR1C1, AKR1C5 was potently inhibited by
flavonoids, suggesting that its activity is affected by ingested
flavonoids. Quercetin with hydroxy groups at positions 3′ and
4′ inhibited AKR1C5 more potently than its analogs (kaemp-
4) Miura R, Shiota K, Noda K, Yagi S, Ogawa T, Takahashi M. Mo-
lecular cloning of cDNA for rat ovarian 20α-hydroxysteroid dehy-
drogenase (HSD1). Biochem. J., 299, 561–567 (1994).
5) Ishida M, Chang KT, Hirabayashi K, Nishihara M, Takahashi M.
Cloning of mouse 20α-hydroxysteroid dehydrogenase cDNA and its
mRNA localization during pregnancy. J. Reprod. Dev., 45, 321–329
(1999).
6) Lacy WR, Washenick KJ, Cook RG, Dunbar BS. Molecular clon-
ing and expression of an abundant rabbit ovarian protein with
20α-hydroxysteroid dehydrogenase activity. Mol. Endocrinol., 7,
58–66 (1993).
Table 4. Inhibitor Sensitivity of AKR1C5 and AKR1C1
7) Penning TM, Burczynski ME, Jez JM, Hung CF, Lin HK, Ma H,
Moode M, Palackal N, Ratnam K. Human 3α-hydroxysteroid dehy-
drogenase isoforms (AKR1C1–AKR1C4) of the aldo-keto reductase
superfamily: functional plasticity and tissue distribution reveal roles
in the inactivation and formation of male and female sex hormones.
Biochem. J., 351, 67–77 (2000).
8) Steckelbroeck S, Jin Y, Gopishetty S, Oyesanmi B, Penning TM.
Human cytosolic 3α-hydroxysteroide dehydrogenases of the aldo-
keto reductase superfamily display significant 3β-hydroxysteroid de-
hydrogenase activity: implications for steroid hormone metabolism
and action. J. Biol. Chem., 279, 10784–10795 (2004).
9) Ishikura S, Nakajima S, Kaneko T, Shintani S, Usami N, Yamamoto
I, Carbone V, El-Kabbani O, Hara A. Comparison of stereoselec-
tive reduction of 3- and 20-oxosteroids among mouse and primate
20α-hydroxysteroid dehydrogenases. Enzymology and molecular
biology of carbonyl metabolism 12. (Weiner H, Plapp B, Lindahl
R, Maser E eds.), Purdue University Press, West Lafayette, pp.
341–351 (2005).
IC50 (μM)a)
Inhibitor
AKR1C5
AKR1C1
AKR1C1 inhibitors
Phenolphthalein
Benzbromarone
TBPP
0.022
0.12
3.0
4.6b)
0.048b)
0.033b)
8.3b)
Hexestrol
6.4
Mefenamic acid
Sulfobromophthalein
Flufenamic acid
Flavonoidsc)
Quercetin
10
2.2
11
>20
26
5.3b)
0.059
0.11
0.37
0.41
0.64
1.4
7.3
0.31
2.6
5.5
Kaempferol
Naringenin
Galangin
Myricetin
10) Burczynski ME, Sridhar GR, Palackal NT, Penning TM. The
reactive oxygen species- and Michael acceptor-inducible human
aldo-keto reductase AKR1C1 reduces the α,β-unsaturated aldehyde
4-hydroxy-2-nonenal to 1,4-dihydroxy-2-nonene. J. Biol. Chem.,
276, 2890–2897 (2001).
a) The values of AKR1C5 and AKR1C1 were determined in the oxidation of
0.18mM β-ionol and 0.1mM (S)-1-teralol, respectively. b) The values are taken from
ref. 15. c) Structures of the flavonoids are shown below. Naringenin is a kaempferol
derivative without 3-hydroxy group.
11) El-Kabbani O, Dhagat U, Hara A. Inhibitors of human
20α-hydroxysteroid dehydrogenase (AKR1C1). J. Steroid Biochem.
Mol. Biol., 125, 105–111 (2011).
12) Matsunaga T, Shintani S, Hara A. Multiplicity of mammalian re-
ductases for xenobiotic carbonyl compounds. Drug Metab. Pharma-
cokinet., 21, 1–18 (2006).

1518
Vol. 36, No. 9
13) Matsunaga T, Endo S, Takemura M, Soda M, Yamamura K, Tajima
K, Miura T, Terada T, El-Kabbani O, Hara A. Reduction of cyto-
toxic p-quinone metabolites of tert-butylhydroquinone by human
aldo-keto reductase (AKR) 1B10. Drug Metab. Pharmacokinet., 27,
553–558 (2012).
14) Matsuura K, Deyashiki Y, Sato K, Ishida N, Miwa G, Hara A.
Identification of amino acid residues responsible for differences in
substrate specificity and inhibitor sensitivity between two human
liver dihydrodiol dehydrogenase isoenzymes by site-directed muta-
genesis. Biochem. J., 323, 61–64 (1997).
and modulators: probing with chimaeric enzymes. Biochem. J., 336,
429–436 (1998).
27) Parés X, Julià P. Isoenzymes of alcohol dehydrogenase in retinoid
metabolism. Methods Enzymol., 189, 436–441 (1990).
28) Esterbauer H, Schaur RJ, Zollner H. Chemistry and biochemistry
of 4-hydroxynonenal, malonaldehyde and related aldehydes. Free
Radic. Biol. Med., 11, 81–128 (1991).
29) Long EK, Picklo MJ Sr. trans-4-Hydroxy-2-hexenal, a product of
n-3 fatty acid peroxidation: make some room HNE.... Free Radic.
Biol. Med., 49, 1–8 (2010).
15) Higaki Y, Usami N, Shintani S, Ishikura S, El-Kabbani O, Hara A.
Selective and potent inhibitors of human 20α-hydroxysteroid dehy-
drogenase (AKR1C1) that metabolizes neurosteroids derived from
progesterone. Chem. Biol. Interact., 143–144, 503–513 (2003).
16) Ma H, Penning TM. Characterization of homogeneous recombinant
rat ovarian 20α-hydroxysteroid dehydrogenase: fluorescent proper-
ties and inhibition profile. Biochem. J., 341, 853–859 (1999).
17) Mao J, Duan RW, Zhong L, Gibori G, Azhar S. Expression, puri-
fication and characterization of the rat luteal 20α-hydroxysteroid
dehydrogenase. Endocrinology, 138, 182–190 (1997).
30) O’connor T, Ireland LS, Harrison DJ, Hayes JD. Major differences
exist in the function and tissue-specific expression of human afla-
toxin B1 aldehyde reductase and the principal human aldo-keto re-
ductase AKR1 family members. Biochem. J., 343, 487–504 (1999).
31) Goyeneche AA, Telleria CM. Exogenous estradiol enhances apopto-
sis in regressing post-partum rat corpora lutea possibly mediated by
prolactin. Reprod. Biol. Endocrinol., 3, 40 (2005).
32) Araujo RR, Ginther OJ, Ferreira JC, Palhão MM, Beg MA, Wilt-
bank MC. Role of follicular estradiol-17β in timing of luteolysis in
heifers. Biol. Reprod., 81, 426–437 (2009).
18) Couture JF, Legrand P, Cantin L, Labrie F, Luu-The V, Breton R.
Loop relaxation, a mechanism that explains the reduced specificity
of rabbit 20α-hydroxysteroid dehydrogenase, a member of the aldo-
keto reductase superfamily. J. Mol. Biol., 339, 89–102 (2004).
19) Wintergalen N, Thole HH, Galla HJ, Schlegel W. Prostaglandin
E2 9-reductase from corpus luteum of pseudopregnant rabbit
is a member of the aldo-keto reductase superfamily featuring
20α-hydroxysteroid dehydrogenase activity. Eur. J. Biochem., 234,
264–270 (1995).
33) Saijo K, Collier JG, Li AC, Katzenellenbogen JA, Glass CK. An
ADIOL-ERβ-CtBP transrepression pathway negatively regulates
microglia-mediated inflammation. Cell, 145, 584–595 (2011).
34) Monje P, Boland R. Subcellular distribution of native estrogen
receptor α and β isoforms in rabbit uterus and ovary. J. Cell. Bio-
chem., 82, 467–479 (2001).
35) Weihua Z, Lathe R, Warner M, Gustafsson J.-Ǻ. An endocrine
pathway in the prostate, ERβ, AR, 5α-androstane-3β,17β-diol, and
CYP7B1, regulates prostate growth. Proc. Natl. Acad. Sci. U.S.A.,
99, 13589–13594 (2002).
20) Platt K, Oesch F. An improved synthesis of trans-5,6-dihydroxy-1,3-
cyclohexadiene (trans-1,2-dihydroxy-1,2-dihydrobenzene). Synthe-
sis, 7, 449–450 (1977).
36) Harris HA. Estrogen receptor-β: recent lessons from in vivo studies.
Mol. Endocrinol., 21, 1–13 (2007).
21) Tajima K, Hashizaki M, Yamamoto K, Mizutani T. Metabolism of
3-tert-butyl-4-hydroxyanisole by horseradish peroxidase and hydro-
gen peroxide. Drug Metab. Dispos., 20, 816–820 (1992).
22) Matsuura K, Deyashiki Y, Bunai Y, Ohya I, Hara A. Aldose reduc-
tase is a major reductase for isocaproaldehyde, a product of side-
chain cleavage of cholesterol, in human and animal adrenal glands.
Arch. Biochem. Biophys., 328, 265–271 (1996).
37) Pastore MB, Jobe SO, Ramadoss J, Magness RR. Estrogen
receptor-α and estrogen receptor-β in the uterine vascular endotheli-
um during pregnancy: functional implications for regulating uterine
blood flow. Semin. Reprod. Med., 30, 46–61 (2012).
38) Thornton S, Terzidou V, Clark A, Blanks A. Progesterone metabo-
lite and spontaneous myometrial contractions in vitro. Lancet, 353,
1327–1329 (1999).
23) Doorn JA, Srivastava SK, Petersen DR. Aldose reductase catalyzes
reduction of the lipid peroxidation product 4-oxonon-2-enal. Chem.
Res. Toxicol., 16, 1418–1423 (2003).
24) Endo S, Matsunaga T, Kumada S, Fujimoto A, Ohno S, El-Kabbani
O, Hu D, Toyooka N, Mano J, Tajima K, Hara A. Characterization
39) Sheehan PM. A possible role for progesterone metabolites in human
parturition. Aust. N. Z. J. Obstet. Gynaecol., 46, 159–163 (2006).
40) Medvedev A, Crumeyrolle-Arias M, Cardona A, Sandler M, Glover
V. Natriuretic peptide interaction with [3H]isatin binding sites in rat
brain. Brain Res., 1042, 119–124 (2005).
of rabbit aldose reductase-like protein with 3β-hydroxysteroid dehy- 41) Sato Y, Cheng Y, Kawamura K, Takae S, Hsueh AJ. C-type natri-
drogenase activity. Arch. Biochem. Biophys., 527, 23–30 (2012).
25) Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Labora-
tory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring
Harbor (1989).
uretic peptide stimulates ovarian follicle development. Mol. Endo-
crinol., 26, 1158–1166 (2012).
42) Thornalley PJ. Dicarbonyl intermediates in the maillard reaction.
Ann. N. Y. Acad. Sci., 1043, 111–117 (2005).
26) Matsuura K, Hara A, Deyashiki Y, Iwasa H, Kume T, Ishikura S,
Shiraishi H, Katagiri Y. Roles of the C-terminal domains of human
dihydrodiol dehydrogenase isoforms in the binding of substrates
43) Lim J, Luderer U. Oxidative damage increases and antioxidant gene
expression decreases with aging in the mouse ovary. Biol. Reprod.,
84, 775–782 (2011).