Immunohistochemicallocalizationofguinea-pigleukotriene B
4
12-hydroxydehydrogenase/15-ketoprostaglandin 13-reductase
Toshiko Yamamoto
1
, Takehiko Yokomizo
1,2
, Akihide Nakao
3
, Takashi Izumi
1,2
and Takao Shimizu
1,2
1
The Department of Biochemistry and Molecular Biology,
2
CREST of Japan Science and Technology Corporation;
3
The Department of
Nephrology and Endocrinology, Faculty of Medicine, The University of Tokyo, Japan
We have cloned cDNA for leukotriene B
4
12-hydroxy-
dehydrogenase (LTB
4
12-HD)/15-ketoprostaglandin
13-reductase (PGR) from guinea-pig liver. LTB
4
12-HD
catalyzes the conversion of LTB
4
into 12-keto-LTB
4
in the
presence of NADP
1
, and plays an important role in
inactivating LTB
4
. The cDNA contained an ORF of 987 bp
that encodes a protein of 329 amino-acid residues with a
78% identity with porcine LTB
4
12-HD. The amino acids in
the putative NAD
1
/NADP
1
binding domain are well
conserved among the pig, guinea-pig, human, rat, and
rabbit enzymes. The guinea-pig LTB
4
12-HD (gpLTB
4
12-HD) was expressed as a glutathione S-transferase (GST)
fusion protein in Escherichia coli, which exhibited similar
enzyme activities to porcine LTB
4
12-HD. We examined the
15-ketoprostaglandin 13-reductase (PGR) activity of
recombinant gpLTB
4
12-HD, and confirmed that the K
cat
of the PGR activity is higher than that of LTB
4
12-HD
activity by 200-fold. Northern and Western blot analyses
revealed that gpLTB
4
12-HD/PGR is widely expressed in
guinea-pig tissues such as liver, kidney, small intestine,
spleen, and stomach. We carried out immunohistochemical
analyses of this enzyme in various guinea-pig tissues.
Epithelial cells of calyx and collecting tubules in kidney,
epithelial cells of airway, alveoli, epithelial cells in small
intestine and stomach, and hepatocytes were found to
express the enzyme. These findings will lead to the
identification of the unrevealed roles of PGs and LTs in these
tissues.
Keywords: leukotriene B
4
12-hydroxydehydrogenase;
leukotriene B
4
; cDNA cloning; 15-keto-prostaglandin
13-reductase; dual functioning enzyme.
Leukotriene B
4
(LTB
4
), a metabolite of arachidonic acid, is
a potent chemotactic factor stimulating polymorphonuclear
leukocytes, macrophages, and eosinophils through
G-protein-coupled receptors (leukotriene B
4
receptor;
BLT), and plays important roles in inflammatory responses
and host defense mechanisms [1,2]. LTB
4
also acts as a
regulator of transcription by binding to a peroxisome
proliferator-activated receptor alpha [3,4]. Arachidonic acid,
released from the cell membrane by cytosolic phospholipase
A
2
, is converted to 5-hydroperoxyeicosatetraenoic acid and
leukotriene A
4
(LTA
4
) by 5-lipoxygenase [5,6]. LTB
4
is
biosynthesized from LTA
4
by LTA
4
hydrolase expressed in
most tissues [7]. In human polymorphonuclear leukocytes,
LTB
4
is converted and inactivated to 20-hydroxy-LTB
4
and
further to 20-carboxy-LTB
4
[8– 11]. LTB
4
is reported to also
be produced in tissues other than leukocytes [12–17]. We
reported an alternative pathway for LTB
4
-inactivation in
various porcine tissues, and purified a cytosolic LTB
4
12-HD from porcine kidney [18]. The primary structure of
PGR, which catalyses the conversion of 15-keto-PG into
13,14-dihydro 15-keto-PG, was reported to be identical to
LTB
4
12-HD [19]. PGs mediate a wide range of
physiological processes, including ovulation, homeostasis,
platelet aggregation, control of water balance, and immune
response [20], and PGR is a critical enzyme that irreversibly
inactivates all types of PGs. Thus, we examined the PGR
activity with 15-keto-PGE
2
as a substrate using recombinant
gpLTB
4
12-HD. We also prepared a highly specific
polyclonal antibody against the enzyme using recombinant
gpLTB
4
12-HD/PGR. Using this antibody, we quantified the
enzyme protein in cytosolic fraction of various guinea-pig
tissues, and examined the precise localizationof this
enzyme by immunohistochemistry.
MATERIALS AND METHODS
Reagents
LTB
4
is a generous gift from Ono Pharmaceutical Company
(Osaka, Japan), and PGs were purchased from Cayman (Ann
Arbor, MI, USA). Nitrocellulose membrane was obtained
from Amersham (Cleveland, OH, USA). Silica gel 60 thin-
layer plates were purchased from MERCK (Rahway, NJ,
USA). Freund’s adjuvant was from DIFCO (Detroit, MI,
Correspondence to T. Yokomizo, The Department of Biochemistry and
Molecular Biology, Faculty of Medicine, The University of Tokyo,
Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan.
Fax: 1 81 3 3813 8732, Tel.: 1 81 3 5802 2925,
E-mail: yokomizo-tky@umin.ac.jp
Note: the nucleotide sequence reported in this paper has been submitted
to the GenBank/DDBJ/EMBL database with an accession number of
AB021219.
(Received 27 July 2001, revised 27 September 2001, accepted
27 September 2001)
Abbreviations:LTB
4
, leukotriene B
4
,5(S ),12(R)-dihydroxy-
6,14-cis-8,10-trans-eicosatetraenoic acid; LTB
4
12-HD, leukotriene B
4
12-hydroxydehydrogenase; LTA
4
, leukotriene A4; LXA
4
, lipoxin A
4
,
5(S ),6(R ),15(S)-trihydroxy-7,9,13-cis-11-trans-eicosatetraenoic acid;
GST, glutathione S-transferase; PGR, 15-ketoprostaglandin
13-reductase; 15-PGDH, 15-hydroxyprostaglandin dehydrogenase;
BLT, leukotrieneB4 receptor; GP, guinea-pig.
Eur. J. Biochem. 268, 6105–6113 (2001) q FEBS 2001
USA). NADH, NADPH, and NADP
1
were purchased from
Boehringer Mannheim (Mannheim, Germany).
CDNA cloning ofguinea-pig LTB
4
12-hydroxydehydrogenase
Based on the highly homologous sequences of pig, human,
and rabbit enzymes, a 19-mer sense (5
0
-TGATGGGGCA
GCAAGTGGC-3
0
) and 21-mer antisense (5
0
-GGGCATGT
TTTCAAATCCTTC-3
0
) oligonucleotide primers were
designed and synthesized. RT-PCR using these primers
was performed to obtain a partial cDNA fragment for
screening of the library.
Total RNA was prepared from the guinea-pig liver by a
cesium trifluoroacetate method [21]. Poly(A)
1
RNA was
purified using Oligotex-dT30 Super (Takara Shuzo, Kyoto,
Japan) according to the manufacturer’s protocol. An
oligo(dT)12–18-primed cDNA was synthesized from 1 mg
of poly(A)
1
RNA by a moloney murine leukemia virus
reverse transcriptase (Pharmacia, Sweden).
The PCRconditions were as follows: denaturation at
94 8C for 1 min, annealing at 55 8C for 2 min, and
elongation at 72 8C for 3 min. After 25 cycles of PCR, the
products were ethanol-precipitated and separated in a 1%
agarose gel, and a band of < 750 bp was recovered from the
gel using a Gel Purification kit (Qiagen, Crawley, West
Sussex, UK). The fragment was ligated into a T-vector
(Promega, Madison, WI, USA) by a T4 DNA ligase, and the
resulting constructs were used for the transformation of
E.coli strain JM109 (Competent high, TOYOBO, Japan).
The DNA was sequenced using an automated ABI 373 DNA
sequencer (PerkinElmer, Norwalk, CT, USA). The insert
was radiolabelled by random primer labelling, and used as a
probe to screen the guinea-pig liver cDNA library by plaque
hybridization.
Library construction and screening
Poly(A)
1
RNA was isolated from the guinea-pig liver as
described above. cDNA was synthesized from 5 mgof
poly(A)
1
RNA, using a SuperScript II Choice System (Life
Technologies, Gaithersburg, ND, USA). The cDNA was
inserted into the Eco RI site of Lambda ZAPII vector
(Stratagene, La Jolla, CA, USA). A library of 2.7 Â 10
6
plaque forming units
:
mg
21
was thus obtained. Clones
(4 Â 10
5
) were transferred to Hybond-N
1
nylon membranes
(Amersham, Little Chalfont, Bucks, UK) and screened by
hybridization with the [
32
P]dCTP-labelled probe. The
hybridization was performed at 42 8C in a hybridization
buffer containing 10 Â Denhardt’s solution (0.2% Ficoll
400, 0.2% BSA, 0.2% polyvinylpyrrolidone), 0.5% SDS,
5 Â NaCl/Cit, 50 mg
:
mL
21
salmon sperm DNA. After
tertiary screening, five clones were isolated, and the
plasmids were recovered by excision in vivo (clone nos
2–6) and sequenced.
Northern blot analysis
Five mg of poly(A)
1
RNA isolated from guinea-pig tissues
was separated in a 1% (w/v) denaturing agarose gel, and
transferred on to a Gene Screen Plus membrane (NEN,
Boston, MA, USA). The membrane was hybridized with a
[
32
P]dCTP-labelled full-length gpLTB
4
12-HD/PGR or a
glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
cDNA (Clontech, Palo Alto, CA, USA) for overnight at
42 8C in a hybridization buffer containing 4 Â NaCl/Cit,
5 Â Denhardt’s solution, 0.2% SDS, 100 mg
:
mL
21
salmon
sperm DNA, 0.1% SDS, and 50% formamide. The
membrane was washed in 1 Â NaCl/Cit, 0.1% SDS at
room temperature and then in 0.1 Â NaCl/Cit, 0.1% SDS at
60 8C. The membrane was subjected to autoradiography and
analyzed with a Bas-2000 system analyzer (Fuji Film,
Japan).
Expression and purification of recombinant gpLTB
4
12-HD/PGR
The cDNA insert was digested from clone no.2 by Eco RI
and subcloned into an Eco RI site of pGEX-1 Lambda T
vector (Pharmacia). An E. coli strain JM109 was trans-
formed by heat shock, and the recombinant protein was
induced by 0.2 m
M isopropyl thio-b-D-galactoside (IPTG)
at 25 8C. E. coli was collected, suspended in NaCl/P
i
containing 2 mM EDTA, 1 mM dithiothreitol, 0.5 mM
phenylmethanesulfonyl fluoride, and 0.7 mg
:
mL
21
pep-
statin A, and disrupted with a sonicator (Otake, Tokyo,
Japan). The sonicate was centrifuged for 5 min at 8000 g,
and 30 mL (for 1 L of E. coli culture) of GSH– Sepharose
was added to the supernatant. After washing with NaCl/P
i
,
the protein was eluted with 50 m
M Tris/HCl, pH 8.0,
containing 10 m
M GSH. The purity of the protein was
determined by SDS/PAGE after staining with Coomassie
Brilliant Blue.
Assay of gpLTB
4
12-HD and 15-ketoprostaglandin
13-reductase (PGR) activity
The gpLTB
4
12-HD activity was assayed according to the
procedure described by Yokomizo et al. [22]. The PGR
activity was assayed by the chromophor method described
by Hansen [23]. The assay conditions were as follow: the
reaction mixtures contained 0.1
M sodium phosphate,
pH 7.4, 1 m
M 2-mercaptoethanol, 1 mM NADH, and
various concentrations (0–60 m
M) of 15-keto-PGE
2
. The
reaction was started by adding 3.5 mg of enzyme in a final
volume of 50 mL. The mixture was incubated at 37 8C for 0
and 5 min, and 12.5 mLof2
M NaOH was added to
terminate the reaction. The amount of 15-keto-PGE
2
remaining was measured by reading the maximal absorption
at 500 nm (red chromophores). The K
m
and V
max
values
were determined by Lineweaver–Burk plots.
Preparation of affinity-purified anti-(gpLTB
4
12-HD/PGR)
Ig
The purified GST–LTB
4
12-HD was digested by thrombin
protease (10 U per 300 mg of fusion protein) at 37 8C for
18 h, followed by purification using an FPLC system
(Pharmacia, Uppsala, Sweden) with a Blue 5PW column
(4.6 Â 150 mm Tosoh, Tokyo, Japan). The buffer used is
solution A (20 m
M Tris/HCl pH 7.5) and solution B (20 mM
Tris/HCl, 2 M NaCl). One millilitre of thrombin-treated
GST– LTB
4
12-HD (about 2 mg) was injected onto the
FPLC, and the absorbed protein (LTB
4
12-HD) was eluted
with 20 m
M Tris/HCl pH 7.5 in a increasing gradient of
6106 T. Yamamoto et al. (Eur. J. Biochem. 268) q FEBS 2001
NaCl up to 1.4 M (70% of solution B) for 35 min. The flow
rate was 1 mL
:
min
21
.
For initial immunization, the purified protein (300 mg)
was emulsified with Freund’s complete adjuvant and
administered to New Zealand White rabbits by multiple
subcutaneous injections. The protein (100 mg) with
incomplete adjuvant was used for booster injections. The
titer of antisera was determined by enzyme-linked
immunosorbent assay (ELISA). The antisera (2 mL) were
applied to an affinity column, which was prepared by
coupling the recombinant LTB
4
12-HD (2 mg) to epoxy-
activated Sepharose 6B (0.5 g). The adsorbed antibody was
eluted with 2 mL of 0.1
M glycine/HCl buffer (pH 2.5), and
immediately neutralized with 100 mLof1
M Tris (pH 7.5)
Fig. 1. Structure of LTB
4
12-HD. (A)
Nucleotide and deduced amino-acid sequences of
gpLTB
4
12-HD/PGR. The nucleotide sequence of
the isolated clone contains a 987-bp of open
reading frame encoding 329 amino acids. The stop
codon and primers used for RT-PCR are indicated
by an asterisk and underlines, respectively. (B)
Comparison of amino-acid sequences of
guinea-pig, human [22], pig [22], rabbit [24] and
rat [25] LTB
4
12-HD. The amino acids conserved
in five species are indicated by asterisks. The
boxed amino acids are required for the enzyme
activity possibly by forming a putative NAD
1
/
NADP
1
binding pocket [22].
q FEBS 2001 Guinea-pigleukotriene B
4
12-hydroxydehydrogenase (Eur. J. Biochem. 268) 6107
and stored at 4 8C with 1% (w/v) BSA and 0.02% (w/v)
NaN
3
. For negative control antibody, the IgG fraction was
prepared with Hi-Trap protein G column (Pharmacia) from
the preimmune serum.
Western blot analysis
Various tissues ofguinea-pig were excised and homogen-
ized in 4 vol. (v/w) of 50 m
M potassium phosphate buffer
(pH 7.5) containing 2 m
M EDTA, 1 mM dithiothreitol,
1m
M phenylmethanesulfonyl fluoride, and 0.7 mg
:
mL
21
pepstatin A with a physcotron homogenizer (Microtec,
Chiba, Japan). The homogenate was centrifuged at 1000 g
for 15 min, and resulting supernatant was further centri-
fuged at 100 000 g for 60 min. The final supernatant was
recovered as a cytosolic fraction. Cytosolic fractions (10 mg
protein) were subjected to 10% SDS/PAGE and were
transferred to a nitrocellulose membrane (Hybond ECL,
Amersham, Cleveland, OH, USA). Recombinant protein
(25 ng) was used as a positive control. The membranes were
blocked with Block Ace (Yukijirushi, Sapporo, Japan) and then
incubated with the affinity-purified antibody (3.8 ng
:
mL
21
)for
2 h at room temperature or overnight at 4 8C. The membranes
were washed in NaCl/Tris/Tween [20 m
M Tris/HCl pH 7.5,
150 m
M NaCl, 0.1% (v/v) Tween-20] and incubated with anti-
(rabbit IgG) Ig conjugated with horseradish peroxidase
(Zymed, San Francisco, CA, USA), diluted 1 : 15 000 in
NaCl/Tris/Tween. The immunoreactive bands were visualized
using an ECL detection kit (Amersham).
Immunohistochemical staining
The tissues ofguinea-pig were excised, cut into small blocks
and fixed in 10% (v/v) formalin in NaCl/P
i
for more than one
Fig. 2. Characterization of recombinant proteins. (A) E. coli homogenates (lane 1 and 5, guinea-pig: lane 3 and 7, pig) and 2 mg of purified
recombinant protein (lane 2 and 6, guinea-pig: lane 4 and 8, pig) were separated in a 10% SDS/PAGE. Lane 1–4 were stained with Coomassie
brilliant blue and lane 5–8 were transferred to a nitrocellulose membrene and immunostained using anti-GST Ig. (B,C) Kinetic profiles of the
recombinant gpLTB
4
12-HD/PGR for LTB
4
(B) and for 15-keto-PGE
2
(C). These Lineweaver–Burk plots (n ¼ 3, means ^ SD) are representatives
of three independent experiments with similar results.
6108 T. Yamamoto et al. (Eur. J. Biochem. 268) q FEBS 2001
day at room temperature. Tissue blocks were dehydrated and
replaced for paraffin. The paraffin-embedded tissues were
sliced into 3-mm of sections by a microtome, mounted on
3-aminopropyl-triethoxy-silan-coated glass slides, and
dewaxed in xylene. Xylene was removed in graded
concentrations of ethanol and replaced with water. Samples
were treated with 0.1% trypsin for 25 min at 37 8C. Slides
were incubated with 3.8 mg
:
mL
21
affinity-purified anti-
(LTB
4
12-HD) Ig for 1 h at room temperature, followed by
incubation at room temperature for 15 min with
biotinylated secondary antibody (Dako, Carpinteria, CA,
USA) and incubated at room temperature for 15 min
with alkaline phosphatase-conjugated streptavidin label
(Dako). Slides were washed three times with NaCl/P
i
after each incubation. Color was developed using
naphthol phosphate and Fast Red (Dako) dissolved in 0.1
M Tris/HCl, pH 8.2. For negative control, affinity-purified
preimmune IgG was used instead of anti-(g-pLTB
4
12-HD/
PGR) Ig.
RESULTS
CDNA cloning of the gpLTB
4
12-HD/PGR
Using the 750-bp cDNA fragment obtained by PCR as a
probe, cDNAs for gpLTB
4
12-HD were isolated from a
guinea-pig liver cDNA library by plaque hybridization.
Among five positive clones (clone nos 2–6), three clones
(2,3 and 6) were revealed to encode full length gpLTB
4
12-HD/PGR. The nucleotide sequence of this insert (clone
2) and its deduced amino-acid sequence are shown in
Fig. 1A. The ORF consists of 987 bp and encodes a
protein of 329 amino-acid residues. The calculated
molecular mass is 35 729. The identity between guinea-
pig and porcine enzymes is 78% at the amino-acid level
as shown in Fig. 1B. The amino acids in the putative
NAD
1
/NADP
1
binding domain [22] are well conserved in
guinea-pig, pig [22], human [22], rabbit [24], and rat [25]
enzymes.
Expression of gpLTB
4
12-HD/PGR cDNA and charac-
terization of the recombinant enzyme gpLTB
4
12-HD/PGR
was expressed in E. coli as a GST–fusion protein, and
purified by affinity chromatography as described in the
Experimental procedures. The enzyme was purified to
homogeneity as shown in Fig. 2A. The V
max
and K
m
values of the purified recombinant protein against LTB
4
were 1.7 ^ 0.2 mU
:
mg
21
and 93 ^ 9.2 mM, respectively
(means ^ SD, n ¼ 3). The V
max
and K
m
values
against 15-keto-PGE
2
were 345 ^ 26 mU
:
mg
21
and
35 ^ 8.5 m
M, respectively (means ^ SD, n ¼ 3). The
V
max
of PGR activity was higher than LTB
4
12-HD activity
by 200-fold.
Northern and Western blot analyses
Figure 3A shows the tissue distribution of gpLTB
4
12-HD/
PGR mRNA in guinea-pig tissues. The mRNA is expressed
most abundantly in small intestine, followed by liver,
kidney, and colon. Two bands of 1.6 and 2.8 kb were
detected in all these tissues. These two bands may represent
alternative spliced variants of gpLTB
4
12-HD/PGR or
mRNAs driven by different promoters. In various guinea-pig
tissues, a single protein of 36 kDa was observed in Western
blotting (Fig. 3B). There were no extra bands observed
suggesting that this antibody is specific for gpLTB
4
12-HD/PGR. The enzyme protein was expressed most
abundantly in liver, stomach, spleen, and small intestine,
followed by kidney, and colon. The affinity-purified
preimmune IgG did not show any signals on Western
blotting (data not shown).
Immunohistochemical localizationof gpLTB
4
12-HD/PGR
gpLTB
4
12-HD/PGR immunoreactivities were observed in
epithelial cells and muscular coat both of stomach (Fig. 4A)
and small intestine (Fig. 4C,D). In kidney, the intense
signals were observed in the epithelial cells of calyx and
collecting tubules (Fig. 4E,F). In lung, cartilages were
stained strongly, and bronchial smooth muscle, epithelial
cells, and alveoli were weakly stained (Fig. 4G). The signals
were observed strongly in hepatocytes around the vein
(Fig. 4H). Most of the splenocytes were weakly stained
(data not shown). The purified preimmune IgG did not show
any signals in all the tissues examined (Fig. 4B, data not
Fig. 3. Tissue distribution of gpLTB
4
12-HD/PGR. (A) Northern
blot analysis of gpLTB
4
12-HD/PGR in guinea-pig tissues. Poly(A)
1
RNAs (5 mg) were applied as follows: lane 1, lung; lane 2, leukocytes;
lane 3, colon; lane 4, small intestine; lane 5, kidney; lane 6, liver. The
membrane was hybridized with [
32
P]dCTP-labelled full-length guinea-
pig LTB
4
12-HD (upper panel) and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) (lower panel). The length of RNA markers
are shown in the left. (B) Western blot analysis of gpLTB
4
12-HD/PGR
in guinea-pig tissues. Cytosolic fractions prepared from various tissues
(10 mg protein) and recombinant gpLTB
4
12-HD/PGR (25 ng, control)
were loaded and separated in 10% SDS/PAGE and transferred to a
nitrocellulose membrane. lane 1, control; lane 2, liver; lane 3, kidney;
lane 4, small intestine; lane 5, colon; lane 6, lung; lane 7, brain; lane 8,
ovary; lane 9, heart; lane 10, spleen; lane 11, stomach. The membrane
was blotted with a purified gpLTB
4
12-HD/PGR antibody.
q FEBS 2001 Guinea-pigleukotriene B
4
12-hydroxydehydrogenase (Eur. J. Biochem. 268) 6109
shown). To confirm the localizationof the enzyme in kidney,
we performed an immunoblot analysis using the preparation
from papilla, medulla, and cortex of kidney. Papilla showed
intense signals, and very faint signal was observed in cortex
on Western blotting. No signal was detected in medulla
(Fig. 5).
DISCUSSION
LTB
4
is a lipid mediator of the inflammatory responses and
activates leukocytes to migrate from vessels, to generate
superoxide anions, and to release lysosomal enzymes [1,26].
LTB
4
is produced mainly in leukocytes, but also in various
tissues [12–17]. LTB
4
is inactivated by omega-oxidation to
yield 20-hydroxy-LTB
4
and 20-carboxy-LTB
4
in leukocytes
[8– 11]. The cytochrome P450, a responsive enzyme for
omega-oxidation of LTB
4
, was cloned from human
leukocytes (CYP4F3) [11] and liver (CYP4F2) [27]. Several
inactivating pathways of LTB
4
other than omega-oxidation
have been reported [18,28–30].
In the present study, we have cloned cDNA for gpLTB
4
12-HD/PGR by screening a guinea-pig liver cDNA library
using a cDNA fragment isolated by PCR using primers of
highly homologous sequences of pig, human, and rabbit
enzymes. The guinea-pig cDNA contained an ORF of
987 bp and encoded a protein of 329 amino acids. The
guinea-pig enzyme shares a 78% identity with the porcine
enzyme and a 80% identity with the human enzyme at the
amino-acid level. AdRab-F protein that is presumably
Fig. 4. Immunohistochemicallocalization of
gpLTB
4
12-HD/PGR in guinea-pig tissues.
The guinea-pig tissues were fixed with 10%
formalin and embedded in paraffin. For
detection, anti-(gpLTB
4
12-HD/PGR) Ig,
alkaline phosphatase-conjugated streptavidin-
biotinylated secondary Ig, and 2,2-azino-
bis(3-ethylbenzthiazoline-sulfonic acid) were
used. LTB
4
12-HD is stained in red. (A)
Stomach, (B) stomach (preimmune IgG), (C)
small intestine, (D) small intestine (ciliary end),
(E) kidney (papillary region), (F) kidney
(collecting tubules), (G) lung, (H) liver.
Fig. 5. Immunoblot analysis of gpLTB
4
12-HD/PGR in papilla,
medulla and cortex ofguinea-pig kidney. Cytosolic fraction (20 mg
protein) and recombinant LTB
4
12-HD (25 ng) were loaded and
separated in 10% SDS/PAGE and transferred to a nitrocellulose
membrane and immunostained using the prepared anti-(gpLTB
4
12-HD/PGR) Ig. lane 1, control; lane 2, papilla; lane 3, medulla; lane
4, cortex.
6110 T. Yamamoto et al. (Eur. J. Biochem. 268) q FEBS 2001
identical to rabbit LTB
4
12-HD expressed in the intestine of
adult, but not baby rabbits, was cloned by Boll et al. [24].
Primiano et al. isolated several cDNA clones representing
dithiolethione-responsive genes from rat liver [31], and one
of the isolated cDNAs proved to be LTB
4
12-HD [25]. These
results suggest that LTB
4
12-HD mRNA is up-regulated
during development and with various stimuli. The amino-
acid alignment of this enzyme from five species is shown in
Fig. 1B. In guinea-pig, LTB
4
12-HD mRNA is highly
expressed in small intestine (Fig. 3A). In contrast, the
enzyme is richest in kidney and liver in human [22]. In
guinea-pig, two bands of 1.6 and 2.8 kb were detected by
using either full length cDNA (Fig. 3A) or ORF (data not
shown) as a probe. Northern and Western blot analyses
(Fig. 3A,B) revealed that LTB
4
12-HD is widely distributed
in various tissues of guinea-pig. The difference in tissue
distributions observed in Northern and Western blots may be
due to the differences of mRNA stability, or efficiency of
protein translation in these tissues.
PGs as well as LTs are lipid mediators derived from
arachidonic acid. PGs are mainly inactivated by two
enzymes sequentially; NAD
1
/NADP1-dependent
15-PGDH and PGR. 15-PGDH oxidizes the 15-hydroxyl
group of PGs to 15-keto group. 15-PGDH consists of two
types of type I (NAD
1
-dependent) and type II
(NADP
1
-dependent) enzymes [32]. The type I enzyme
was cloned or purified from various species [33]. The type II
enzyme was purified from various species [32,34– 37].
15-Keto-PGs are reduced to the 13,14-dihydro 15-keto-PGs
by PGR. The reaction catalyzed by 15-PGDH is reversible,
but that of PGR is irreversible in vivo [23,35]. Accordingly,
PGR is an important enzyme for the complete inactivation of
PGs. PGR was purified from human [38], bovine [23], and
chicken [39]. As reported by Kitamura et al. PGR exhibits
different cofactor requirements, and is different in size [40].
Ensor et al. reported that the primary structure of porcine
lung PGR is identical to porcine LTB
4
12-HD [19]. We
examined the PGR activity using a recombinant gpLTB
4
12-HD/PGR, and found this enzyme is a dual functioning
enzyme that has a catalytic activity for the reduction of the
13,14-double bond of 15-keto-PGs in the presence of NADH
or NADPH, and the oxidation of the 12-hydroxy group of
LTB
4
in the presence of NADP
1
. In agreement with the
previous work, this enzyme has a much higher PGR activity
(345 mU
:
mg
21
) than LTB
4
12-HD activity (1.7 mU
:
mg
21
).
Therefore, the enzyme apparently can function as PGR
in vivo. Additionally, the enzyme also functions as a
reductase on 15-oxo-LXA
4
[41]. PGs and LTB
4
are
different in the biological activities and the sites of
action. Examples of a single enzyme with dual enzyme
activities are seen in other enzymes of eicosanoid
metabolism, such as 5-lipoxygenase [42,43], and 12-lipox-
ygenase [44].
There are no reports on the precise localizationof LTB
4
12-HD (PGR). Thus, we prepared a highly specific
polyclonal antibody against gpLTB
4
12-HD/PGR using a
recombinant guinea-pig enzyme. The epithelial cells and
muscular coat in stomach (Fig. 4A) and small intestine
(Fig. 4C,D), the epithelial cells of calyx and collecting
tubules in kidney (Fig. 4E,F), the airway smooth muscle,
epithelial cells, alveoli, and cartilages in lung (Fig. 4G), and
hepatocytes around the vein (Fig. 4H) were found to express
this enzyme. Spleen (data not shown) were diffusely stained.
These signals were considered to represent the specific
immunoreactivity, because they were not observed by
staining with preimmune IgG (Fig. 4B). Lung is particularly
rich in 15-PGDH and PGR (LTB
4
12-HD) activities [45],
and the majority of PGs are inactivated through the
pulmonary circulation. PGE
2
is an important cyclooxygen-
ase product of airway epithelium [46], and cultured airway
smooth muscle cells are capable of generating large amounts
of PGE
2
[47]. PGE
2
acts as relaxant of airway smooth
muscle, and has protective roles in the airway against
inflammation [48]. Airway smooth muscle and epithelial
cells also express LTA
4
hydrolase [7]. Wenzel et al. reported
that the airway of asthmatic patients contains high amounts
of LTB
4
and peptide leukotrienes [49]. Specific stainings for
PGE
2
and 15-PGDH were observed in parietal and epithelial
cells of rat stomach [50]. These results are consistent with
the localizationof PGR observed in this paper. PGE
2
acts as
a constrictor of longitudinal muscle from stomach to colon.
PGE
2
also inhibits gastric acid secretion stimulated by
feeding, histamine, or gastrin. Mucus secretion in the
stomach and small intestine are enhanced by PGE
2
. These
effects help to maintain the integrity of the gastric mucosa.
In kidney, PGE
2
is an important regulator of water and
mineral balance. LTB
4
12-HD (PGR) and 15-PGDH are
expressed in papillary region (Fig. 5) and in proximal renal
tubule [51], respectively. These results suggest that PGE
2
is
inactivated in the proximal tubule by 15-PGDH and
metabolized irreversibly by PGR in the papillary tubules.
A recently cloned low-affinity LTB
4
receptor, BLT2, is
abundantly expressed in human liver [52]. LTA
4
hydrolase is
also expressed in liver [7]. Thus, highly concentrated LTB
4
produced in inflammatory lesion binds to BLT2, and may
mediate some unknown functions in liver. CYP4F2, another
LTB
4
inactivating enzyme, is also expressed in liver [27].
The expression of two inactivating enzymes suggests that
liver in the major site of LTB
4
degradation. LTB
4
12-HD
(PGR) is an important enzyme that regulates the tissue
contents of LTB
4
and PGE
2
that contribute physiological
and pathological processes.
In conclusion, we have cloned, characterized, and
immunohistochemically localized gpLTB
4
12-HD/PGR.
This enzyme is a dual functioning enzyme that acts on
both LTB
4
and 15-keto-PGs. Considering the reported
reductase activity on 15-oxo-LXA
4
[41], the enzyme is
unique in that it can function within three distinct eicosanoid
pathways, which are functionally and physiologically
separated. It is plausible that this enzyme acts as a PGR
under normal conditions, and as an LTB
4
12-HD/15-oxo-
LXA
4
reductase during inflammatory status. Information on
tissue distributions and localizations of this enzyme will be
useful to reveal the biological and pathological roles of PG,
LTB
4
and LXs in these tissues.
ACKNOWLEDGEMENTS
We are grateful to Ono Pharmaceutical Co., Ltd (Osaka, Japan) for
supplying LTB
4
. We also thank Drs M. Minami, K. Kume, I. Ishii, S.
Ishii, and I. Waga for discussion. This work was supported in part by,
Grants-in-Aid from the Ministry of Education, Science, Sports, and
Culture of Japan, and grants from the Yamanouchi Foundation for
Metabolic Disorders, the Uehara Memorial Foundation, and the Cell
Science Research Foundation.
q FEBS 2001 Guinea-pigleukotriene B
4
12-hydroxydehydrogenase (Eur. J. Biochem. 268) 6111
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q FEBS 2001 Guinea-pigleukotriene B
4
12-hydroxydehydrogenase (Eur. J. Biochem. 268) 6113
. Immunohistochemical localization of guinea-pig leukotriene B
4
12-hydroxydehydrogenase/15-ketoprostaglandin 13-reductase
Toshiko Yamamoto
1
,. Technology Corporation;
3
The Department of
Nephrology and Endocrinology, Faculty of Medicine, The University of Tokyo, Japan
We have cloned cDNA for leukotriene