Immunohistochemical localization of guinea-pig leukotriene B412-hydroxydehydrogenase/15-ketoprostaglandin 13-reductase Toshiko Yamamoto1, Takehiko Yokomizo1,2, Akihide Nakao3, Takashi Iz
Trang 1Immunohistochemical localization of guinea-pig leukotriene B4
12-hydroxydehydrogenase/15-ketoprostaglandin 13-reductase
Toshiko Yamamoto1, Takehiko Yokomizo1,2, Akihide Nakao3, Takashi Izumi1,2and Takao Shimizu1,2
1 The Department of Biochemistry and Molecular Biology,2CREST of Japan Science and Technology Corporation;3The Department of Nephrology and Endocrinology, Faculty of Medicine, The University of Tokyo, Japan
We have cloned cDNA for leukotriene B4
12-hydroxy-dehydrogenase (LTB4 12-HD)/15-ketoprostaglandin
13-reductase (PGR) from guinea-pig liver LTB4 12-HD
catalyzes the conversion of LTB4into 12-keto-LTB4in the
presence of NADP1, and plays an important role in
inactivating LTB4 The cDNA contained an ORF of 987 bp
that encodes a protein of 329 amino-acid residues with a
78% identity with porcine LTB412-HD The amino acids in
the putative NAD1/NADP1 binding domain are well
conserved among the pig, guinea-pig, human, rat, and
rabbit enzymes The guinea-pig LTB4 12-HD (gpLTB4
12-HD) was expressed as a glutathione S-transferase (GST)
fusion protein in Escherichia coli, which exhibited similar
enzyme activities to porcine LTB412-HD We examined the
15-ketoprostaglandin 13-reductase (PGR) activity of
recombinant gpLTB4 12-HD, and confirmed that the Kcat
of the PGR activity is higher than that of LTB4 12-HD activity by 200-fold Northern and Western blot analyses revealed that gpLTB4 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 B4 12-hydroxydehydrogenase; leukotriene B4; cDNA cloning; 15-keto-prostaglandin 13-reductase; dual functioning enzyme
Leukotriene B4(LTB4), a metabolite of arachidonic acid, is
a potent chemotactic factor stimulating polymorphonuclear
leukocytes, macrophages, and eosinophils through
G-protein-coupled receptors (leukotriene B4 receptor;
BLT), and plays important roles in inflammatory responses
and host defense mechanisms [1,2] LTB4 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
A2, is converted to 5-hydroperoxyeicosatetraenoic acid and
leukotriene A4 (LTA4) by 5-lipoxygenase [5,6] LTB4 is
biosynthesized from LTA4by LTA4hydrolase expressed in
most tissues [7] In human polymorphonuclear leukocytes,
LTB4is converted and inactivated to 20-hydroxy-LTB4and further to 20-carboxy-LTB4[8 – 11] LTB4is reported to also
be produced in tissues other than leukocytes [12 – 17] We reported an alternative pathway for LTB4-inactivation in various porcine tissues, and purified a cytosolic LTB4 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 LTB4 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-PGE2as a substrate using recombinant gpLTB4 12-HD We also prepared a highly specific polyclonal antibody against the enzyme using recombinant gpLTB412-HD/PGR Using this antibody, we quantified the enzyme protein in cytosolic fraction of various guinea-pig tissues, and examined the precise localization of this enzyme by immunohistochemistry
M A T E R I A L S A N D M E T H O D S Reagents
LTB4is 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, leukotriene B4 receptor; GP, guinea-pig.
Trang 2USA) NADH, NADPH, and NADP1were purchased from
Boehringer Mannheim (Mannheim, Germany)
CDNA cloning of guinea-pig LTB4
12-hydroxydehydrogenase
Based on the highly homologous sequences of pig, human,
and rabbit enzymes, a 19-mer sense (50-TGATGGGGCA
GCAAGTGGC-30) and 21-mer antisense (50-GGGCATGT
TTTCAAATCCTTC-30) 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 mg of
poly(A)1RNA, 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 2.7 106
plaque forming units:mg21 was thus obtained Clones
(4 105) were transferred to Hybond-N1nylon membranes
(Amersham, Little Chalfont, Bucks, UK) and screened by
hybridization with the [32P]dCTP-labelled probe The
hybridization was performed at 42 8C in a hybridization
buffer containing 10 Denhardt’s solution (0.2% Ficoll
400, 0.2% BSA, 0.2% polyvinylpyrrolidone), 0.5% SDS,
5 NaCl/Cit, 50 mg:mL21 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)1RNA 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
[32P]dCTP-labelled full-length gpLTB 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 NaCl/Cit,
5 Denhardt’s solution, 0.2% SDS, 100 mg:mL21salmon sperm DNA, 0.1% SDS, and 50% formamide The membrane was washed in 1 NaCl/Cit, 0.1% SDS at room temperature and then in 0.1 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 gpLTB4 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 mM isopropyl thio-b-D-galactoside (IPTG)
at 25 8C E coli was collected, suspended in NaCl/Pi containing 2 mM EDTA, 1 mM dithiothreitol, 0.5 mM
phenylmethanesulfonyl fluoride, and 0.7 mg:mL21 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/Pi, the protein was eluted with 50 mM Tris/HCl, pH 8.0, containing 10 mM GSH The purity of the protein was determined by SDS/PAGE after staining with Coomassie Brilliant Blue
Assay of gpLTB412-HD and 15-ketoprostaglandin 13-reductase (PGR) activity
The gpLTB412-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 0.1M sodium phosphate,
pH 7.4, 1 mM 2-mercaptoethanol, 1 mM NADH, and various concentrations (0 – 60 mM) of 15-keto-PGE2 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 mL of 2 M NaOH was added to terminate the reaction The amount of 15-keto-PGE2 remaining was measured by reading the maximal absorption
at 500 nm (red chromophores) The Km and Vmax values were determined by Lineweaver – Burk plots
Preparation of affinity-purified anti-(gpLTB412-HD/PGR) Ig
The purified GST – LTB412-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 (4.6 150 mm Tosoh, Tokyo, Japan) The buffer used is solution A (20 mMTris/HCl pH 7.5) and solution B (20 mM
Tris/HCl, 2 M NaCl) One millilitre of thrombin-treated GST – LTB4 12-HD (about 2 mg) was injected onto the FPLC, and the absorbed protein (LTB412-HD) was eluted with 20 mM Tris/HCl pH 7.5 in a increasing gradient of
Trang 3NaCl up to 1.4M(70% of solution B) for 35 min The flow
rate was 1 mL:min21
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 LTB4 12-HD (2 mg) to epoxy-activated Sepharose 6B (0.5 g) The adsorbed antibody was eluted with 2 mL of 0.1Mglycine/HCl buffer (pH 2.5), and immediately neutralized with 100 mL of 1MTris (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 NAD1/
NADP1binding pocket [22].
Trang 4and stored at 4 8C with 1% (w/v) BSA and 0.02% (w/v)
NaN3 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 of guinea-pig were excised and
homogen-ized in 4 vol (v/w) of 50 mM potassium phosphate buffer
(pH 7.5) containing 2 mM EDTA, 1 mM dithiothreitol,
1 mM phenylmethanesulfonyl fluoride, and 0.7 mg:mL21
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:mL21) for
2 h at room temperature or overnight at 4 8C The membranes were washed in NaCl/Tris/Tween [20 mM Tris/HCl pH 7.5,
150 mMNaCl, 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 of guinea-pig were excised, cut into small blocks and fixed in 10% (v/v) formalin in NaCl/Pifor 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.
Trang 5day 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:mL21 affinity-purified
anti-(LTB412-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/Pi
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-pLTB412-HD/
PGR) Ig
R E S U L T S
CDNA cloning of the gpLTB412-HD/PGR
Using the 750-bp cDNA fragment obtained by PCR as a
probe, cDNAs for gpLTB4 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 gpLTB4
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
NAD1/NADP1binding domain [22] are well conserved in
guinea-pig, pig [22], human [22], rabbit [24], and rat [25]
enzymes
Expression of gpLTB4 12-HD/PGR cDNA and
charac-terization of the recombinant enzyme gpLTB412-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 Vmax and Km
values of the purified recombinant protein against LTB4
were 1.7 ^ 0.2 mU:mg21 and 93 ^ 9.2 mM, respectively
(means ^ SD, n ¼ 3) The Vmax and Km values
against 15-keto-PGE2 were 345 ^ 26 mU:mg21 and
35 ^ 8.5 mM, respectively (means ^ SD, n ¼ 3) The
Vmaxof PGR activity was higher than LTB412-HD activity
by 200-fold
Northern and Western blot analyses
Figure 3A shows the tissue distribution of gpLTB412-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 gpLTB4 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 gpLTB4 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 localization of gpLTB412-HD/PGR gpLTB412-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 [32P]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 12-HD/PGR antibody.
Trang 6shown) To confirm the localization of 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)
D I S C U S S I O N LTB4is 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] LTB4is produced mainly in leukocytes, but also in various tissues [12 – 17] LTB4is inactivated by omega-oxidation to yield 20-hydroxy-LTB4and 20-carboxy-LTB4in leukocytes [8 – 11] The cytochrome P450, a responsive enzyme for omega-oxidation of LTB4, was cloned from human leukocytes (CYP4F3) [11] and liver (CYP4F2) [27] Several inactivating pathways of LTB4other than omega-oxidation have been reported [18,28 – 30]
In the present study, we have cloned cDNA for gpLTB4 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 Immunohistochemical localization 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 of guinea-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.
Trang 7identical to rabbit LTB412-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 LTB412-HD [25] These
results suggest that LTB4 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, LTB4 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 LTB412-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; NAD1/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 (NAD1-dependent) and type II
(NADP1-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 LTB4 12-HD [19] We
examined the PGR activity using a recombinant gpLTB4
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
LTB4 in the presence of NADP1 In agreement with the
previous work, this enzyme has a much higher PGR activity
(345 mU:mg21) than LTB412-HD activity (1.7 mU:mg21)
Therefore, the enzyme apparently can function as PGR
in vivo Additionally, the enzyme also functions as a
reductase on 15-oxo-LXA4 [41] PGs and LTB4 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 localization of LTB4
12-HD (PGR) Thus, we prepared a highly specific
polyclonal antibody against gpLTB4 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 (LTB4 12-HD) activities [45], and the majority of PGs are inactivated through the pulmonary circulation PGE2is an important cyclooxygen-ase product of airway epithelium [46], and cultured airway smooth muscle cells are capable of generating large amounts
of PGE2 [47] PGE2 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 LTA4hydrolase [7] Wenzel et al reported that the airway of asthmatic patients contains high amounts
of LTB4and peptide leukotrienes [49] Specific stainings for PGE2and 15-PGDH were observed in parietal and epithelial cells of rat stomach [50] These results are consistent with the localization of PGR observed in this paper PGE2acts as
a constrictor of longitudinal muscle from stomach to colon PGE2 also inhibits gastric acid secretion stimulated by feeding, histamine, or gastrin Mucus secretion in the stomach and small intestine are enhanced by PGE2 These effects help to maintain the integrity of the gastric mucosa
In kidney, PGE2 is an important regulator of water and mineral balance LTB4 12-HD (PGR) and 15-PGDH are expressed in papillary region (Fig 5) and in proximal renal tubule [51], respectively These results suggest that PGE2is inactivated in the proximal tubule by 15-PGDH and metabolized irreversibly by PGR in the papillary tubules
A recently cloned low-affinity LTB4 receptor, BLT2, is abundantly expressed in human liver [52] LTA4hydrolase is also expressed in liver [7] Thus, highly concentrated LTB4 produced in inflammatory lesion binds to BLT2, and may mediate some unknown functions in liver CYP4F2, another LTB4inactivating enzyme, is also expressed in liver [27] The expression of two inactivating enzymes suggests that liver in the major site of LTB4degradation LTB4 12-HD (PGR) is an important enzyme that regulates the tissue contents of LTB4 and PGE2 that contribute physiological and pathological processes
In conclusion, we have cloned, characterized, and immunohistochemically localized gpLTB4 12-HD/PGR This enzyme is a dual functioning enzyme that acts on both LTB4 and 15-keto-PGs Considering the reported reductase activity on 15-oxo-LXA4 [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 LTB4 12-HD/15-oxo-LXA4reductase during inflammatory status Information on tissue distributions and localizations of this enzyme will be useful to reveal the biological and pathological roles of PG, LTB4and LXs in these tissues
A C K N O W L E D G E M E N T S
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.
Trang 8R E F E R E N C E S
1 Shimizu, T & Wolfe, L.S (1990) Arachidonic acid cascade and
signal transduction J Neurochem 55, 1 – 15.
2 Yokomizo, T., Izumi, T., Chang, K., Takuwa, Y & Shimizu, T.
(1997) A G-protein-coupled receptor for leukotriene B 4 that
mediates chemotaxis Nature 387, 620 – 624.
3 Devchand, P.R., Keller, H., Peters, J.M., Vazquez, M., Gonzalez,
F.J & Wahli, W (1996) The PPARa-leukotriene B 4 pathway to
inflammation control Nature 384, 39 – 43.
4 Devchand, P.R., Hihi, A.K., Perroud, M., Schleuning, W.D.,
Spiegelman, B.M & Wahli, W (1999) Chemical probes that
differentially modulate peroxisome proliferator-activated receptor
a and BLTR, nuclear and cell surface receptors for leukotriene B 4
J Biol Chem 274, 23341 – 23348.
5 Uozumi, N., Kume, K., Nagase, T., Nakatani, N., Ishii, S., Tashiro,
F., Komagata, Y., Maki, K., Ikuta, K., Ouchi, Y., Miyazaki, J &
Shimizu, T (1997) Role of cytosolic phospholipase A2 in allergic
response and parturition Nature 390, 618 – 622.
6 Ueda, N., Kaneko, S., Yoshimoto, T & Yamamoto, S (1986)
Purification of arachidonate 5-lipoxygenase from porcine
leuko-cytes and its reactivity with hydroperoxyeicosatetraenoic acids.
J Biol Chem 261, 7982 – 7988.
7 Ohishi, N., Minami, M., Kobayashi, J., Seyama, Y., Hata, J.,
Yotsumoto, H., Takaku, F & Shimizu, T (1990) Immunoligical
quantitation and immunohistochemical localization of leukotriene
A 4 hydrolase in guinea pig tissues J Biol Chem 265, 7520 – 7525.
8 Powell, W.S (1984) Property of leukotriene 20-hydroxylase from
polymorphonuclear leukocytes J Biol Chem 259, 3082 – 3089.
9 Shak, S & Goldstein, I.M (1984) Omega-oxydation is the major
pathway for catabolism of leukotriene B 4 in human
polymorpho-nuclear leukocytes J Biol Chem 259, 10181 – 10187.
10 Soberman, R., Harper, T.W., Murphy, R.C & Austen, K.F (1985)
Identification and functional characterization of leukotriene b 4
20-hydroxylase of human polymorphonuclear leukocytes Proc.
Natl Acad Sci USA 82, 2292 – 2295.
11 Kikuta, Y., Kusunose, E., Endo, K., Yamamoto, S., Sogawa, K.,
Fujii, K.Y & Kusunose, M (1993) A novel form of cytochrome
P-450 family 4 in human polymorphonuclear leukocytes cDNA
cloning and expression of leukotriene B 4 omega-hydroxylase.
J Biol Chem 268, 9376 – 9380.
12 Rahman, M.A., Nakazawa, M., Emancipator, S.N & Dunn, M.J.
(1988) Increased leukotriene B 4 synthesis in immune injured rat
glomeruli J Clin Invest 81, 1945 – 1952.
13 Sola, J., Godessart, N., Vila, L., de Puig, L., Fogh, K., Ziboh, V.A.,
Kristensen, P., Schmedes, A & Kragballe, K (1993) Leukotriene
B 4 formation during human neutrophil keratinocyte interactions:
evidence for transformation of leukotriene A 4 by putative
keratinocyte leukotriene A 4 hydrolase J Invest Dermatol 100,
293 – 298.
14 Iversen, L., Fogh, K., Ziboh, V.A., Kristensen, P., Schmedes, A &
Kragballe, K (1993) Leukotriene B 4 formation during human
neutrophil keratinocyte interactions: evidence for transformation of
leukotriene A 4 by putative keratinocyte leukotriene A 4 hydrolase.
J Invest Dermatol 100, 293 – 298.
15 Cattell, V., Cook, H.T., Smith, J., Salmon, J.A & Moncada, S.
(1987) Leukotriene B 4 production in normal rat glomeruli.
Nephrol Dial Transplant 2, 154 – 157.
16 Spurney, R.F., Ruiz, P., Pisetsky, D.S & Coffman, T.M (1991)
Enhanced renal leukotriene production in murine lupus: role of
lipoxygenase metabolites Kidney Int 39, 95 – 102.
17 Nakao, A., Watanabe, T., Ohishi, N., Toda, A., Asano, K.,
Taniguchi, S., Nosaka, K., Noiri, E., Suzuki, T., Sakai, T.,
Kurokawa, K., Shimizu, T & Kimura, S (1999) Ubiquitous
localization of leukotriene A 4 hydrolase in the rat nephron Kidney
Int 55, 100 – 108.
18 Yokomizo, T., Izumi, T., Takahashi, T., Kasama, T., Kobayashi, Y.,
Sato, F., Taketani, Y & Shimizu, T (1993) Enzymatic inactivation
of leukotriene B 4 by a novel enzyme found in the porcine kidney Purification and properties of leukotriene B 4 12-hydroxydehydro-genase J Biol Chem 268, 18128– 18135.
19 Ensor, C.M., Zhang, H & Tai, H.H (1998) Purification, cDNA cloning and expression of 15-oxoprostaglandin 13-reductase from pig lung Biochem J 330, 103 – 108.
20 Campbell, W.B & Halushka, P.V (1996) Lipid-derived autacoids eicosanoids and platelet-activating factor Pharmacol Basis Therapeutics 9, 601 – 616.
21 Zarlenga, D.S & Gamble, H.R (1987) Simultaneous isolation of preparative amounts of RNA and DNA from Trichinella spiralis by cesium trifluoroacetate isopycnic centrifugation Anal Biochem.
162, 569 – 574.
22 Yokomizo, T., Ogawa, Y., Uozumi, N., Kume, K., Izumi, T & Shimizu, T (1996) cDNA cloning, expression, and mutagenesis study of leukotriene B 4 12-hydroxydehydrogenase J Biol Chem.
271, 2844 – 2850.
23 Hansen, H.S (1979) Purification and characterization of a15-keto-prostaglandin delta 13-reductase from bovine lung Biochim Biophys Acta 574, 136 – 145.
24 Boll, W., Schmid, C.T., Semenza, G & Mantei, N (1993) Messenger RNAs expressed in intestine of adult but not baby rabbits Isolation of cognate cDNAs and characterization of a novel brush border protein with esterase and phospholipase activity.
J Biol Chem 268, 12901 – 12911.
25 Primiano, T., Li, Y., Kensler, T.W., Trush, M.A & Sutter, T.R (1998) Identification of dithiolethione-inducible gene-1 as a leukotriene B 4 12-hydroxydehydrogenase: implications for chemoprevention Carcinogenesis 19, 999 – 1005.
26 Yokomizo, T., Izumi, T & Shimizu, T (2001) Leukotriene B 4 : metabolism and signal transduction Archi Biochem Biophys 385,
231 – 241.
27 Kikuta, Y., Kusunose, E., Kondo, T., Yamamoto, S., Kinoshita, H.
& Kusunose, M (1994) Cloning and expression of a novel form of leukotriene B 4 omega-hydroxylase from human liver FEBS Lett.
348, 70 – 74.
28 Harper, T.W., Garrity, M.J & Murphy, R.C (1986) Metabolism of leukotriene B 4 in isolated rat hepatocytes Identification of a novel 18-carboxy-19, 20-dinor leukotriene B 4 metabolite J Biol Chem.
261, 5414 – 5418.
29 Kaever, V., Martin, M., Fauler, J., Marx, K.H & Resch, K (1987) A novel metabolic pathway for leukotriene B 4 in different cell types: primary reduction of a double bond Biochim Biophys Acta 922,
337 – 344.
30 Wainwright, S.L & Powell, W.S (1991) Mechanism for the formation of dihydro metabolites of 12-hydroxyeicosanoids Conversion of leukotriene B 4 and 12-hydroxy-5,8,10,14-eicosate-traenoic acid to 12-oxo intermediates J Biol Chem 266, 20899– 20906.
31 Primiano, T., Gastel, J.A., Kensler, T.W & Sutter, T.R (1996) Isolation of cDNAs representing dithiolethione-responsive genes Carcinogenesis 17, 2297 – 2303.
32 Lee, S.C & Levine, L (1975) Prostaglandin metabolism II Identification of two 15-hydroxyprostaglandin dehydrogenase types J Biol Chem 250, 548 – 552.
33 Ensor, C.M & Tai, H.H (1995) 15-Hydroxyprostaglandin dehydrogenase J Lipid Med Cell Signal 12, 313 – 319.
34 Kaplan, L., Lee, S.C & Levine, L (1975) Partial purification and some properties of human erythrocyte prostaglandin 9-ketoreduc-tase and 15-hydroxyprostaglandin dehydrogenase Arch Biochem Biophys 167, 287 – 293.
35 Westbrook, C., Lin, Y.M & Jarabak, J (1977) NADP-linked 15-hydroxyprostaglandin dehydrogenase from human placenta: partial purification and characterization of the enzyme and identification of an inhibitor in placental tissue Biochem Biophys Res Commun 76, 943 – 949.
Trang 936 Watanabe, K., Shimizu, T., Iguchi, S., Wakatsuka, H., Hayashi, M.
& Hayaishi, O (1980) An NADP-linked prostaglandin D
dehydrogenase in swine brain J Biol Chem 255, 1779 – 1782.
37 Watanabe, T., Shimizu, T., Narumiya, S & Hayaishi, O (1982)
NADP-linked 15-hydroxyprostaglandin dehydrogenase for
prosta-glandin D in human blood platelets Arch Biochem Biophys 216,
372 – 379.
38 Westbrook, C & Jarabak, J (1978) 15-Ketoprostaglandin delta13
reductase from human placenta: purification, kinetics, and inhibitor
binding Arch Biochem Biophys 185, 429 – 442.
39 Lee, S.C & Levine, L (1974) Purification and properties of
chicken heart prostaglandin delta13-reductase Biochem Biophys.
Res Commun 61, 14 – 21.
40 Kitamura, S., Katsura, H & Tatsumi, K (1996) Multiplicity of rat
liver 15-ketoprostaglandin delta 13-reductases Prostaglandins 52,
35 – 49.
41 Clish, C.B., Levy, B.D., Chiang, N., Tai, H.H & Serhan, C.N.
(2000) Oxidoreductases in lipoxin A 4 metabolic inactivation:
15-oxoprostaglandin 13-reductase/leukotriene B 4
12-hydroxyde-hydrogenase is a multifunctional eicosanoid oxidoreductase J Biol.
Chem 275, 25372– 25380.
42 Shimizu, T.R., A ˆ dmark, O & Samuelssom, B (1984) Enzyme with
dual lipoxygenase activities cayalyzes leukotriene A 4 synthesis
from arachidonic acid Proc Natl Acad Sci USA 81, 689 – 693.
43 Minami, M., Ohishi, N., Mutoh, H., Izumi, T., Bito, H., Wada, H.,
Seyama, Y., Toh, H & Shimizu, T (1990) Leukotriene A 4
hydrolase is a zinc-containing aminopeptidase Biochem Biophys.
Res Commun 173, 620 – 626.
44 Romano, M., Chen, X.S., Takahashi, Y., Yamamoto, S., Funk, C.D.
& Serhan, C.N (1993) Lipoxin synthase activity of human platelet
12-lipoxygenase Biochem J 296, 127 – 133.
45 Anggard, E., Larsson, C & Samuelsson, B (1971) The distribution
of 15-hydroxy prostaglandin dehydrogenase and prostaglandin-delta 13-reductase in tissues of the swine Acta Physiol Scand 81,
396 – 404.
46 Churchill, L., Chilton, F., Resau, J.H., Bascom, R., Hubbard, W.C.
& Proud, D (1989) Cyclooxygenase metabolism of endogenous arachidonic acid by cultured human tracheal epithelial cells Am Rev Respir Dis 140, 449 – 459.
47 Delamere, F., Holland, E., Patel, S., Bennett, J., Pavord, I & Knox, A (1994) Production of PGE 2 by bovine cultured airway smooth muscle cells and its inhibition by cyclo-oxygenase inhibitors Br J Pharmacol 111, 983 – 988.
48 Pavord, I.D & Tattersfield, A.E (1995) Bronchoprotective role for endogenous prostaglandin E 2 Lancet 345, 436 – 438.
49 Wenzel, S.E., Trudeau, J.B., Kaminsky, D.A., Cohn, J., Martin, R.J.
& Westcott, J.Y (1995) Effect of 5-lipoxygenase inhibition on bronchoconstriction and airway inflammation in nocturnal asthma.
Am J Respir Crit Care Med 152, 897 – 905.
50 Kobayashi, K., Higuchi, K., Arakawa, T., Matsumoto, T & Nagura,
H (1992) Effect of sofalcone on localization of 15-hydroxypros-taglandin dehydrogenase, an enzyme that metabolizes prostaglan-din E 2 , in rat gastric mucosa: an immunohistochemical study.
J Clin Gastroenterol 14, S39 – S42.
51 Wright, J.T.J.R & Corder, C.N (1979) NAD 1 -15-hydroxyprosta-glandin dehydrogenase distribution in rat kidney J Histochem Cytochem 27, 657 – 664.
52 Yokomizo, T., Kato, K., Terawaki, K., Izumi, T & Shimizu, T (2000) A second leukotriene B 4 receptor, BLT2: a new therapeutic target in inflammation and immunological disorders J Exp Med.
192, 421 – 432.