Báo cáo Y học: Immunohistochemical localization of guinea-pig leukotriene B4 12-hydroxydehydrogenase/15-ketoprostaglandin 13-reductase pdf

Immunohistochemical localization of guinea-pig leukotriene 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 localization of 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, leukotriene B4 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 of guinea-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-pig leukotriene 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 of guinea-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 of guinea-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 localization of 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-pig leukotriene B 4 12-hydroxydehydrogenase (Eur. J. Biochem. 268) 6109 shown). 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). 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. 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. 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 localization of 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 localization of 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. 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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