HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 277, Issue 21, 18763-18768, May 24, 2002

This Article Abstract Full Text (PDF) All Versions of this Article: 277/21/18763    most recent M109447200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ogasawara, H. Articles by Izumi, T. Search for Related Content PubMed PubMed Citation Articles by Ogasawara, H. Articles by Izumi, T. Social Bookmarking                      What's this?

Characterization of Mouse Cysteinyl Leukotriene Receptors mCysLT₁ and mCysLT₂

DIFFERENTIAL PHARMACOLOGICAL PROPERTIES AND TISSUE DISTRIBUTION^*

Hideaki Ogasawara^§, Satoshi Ishii^§, Takehiko Yokomizo^§, Takashi Kakinuma^¶, Mayumi Komine^¶, Kunihiko Tamaki^¶, Takao Shimizu^§, and Takashi Izumi^§**

From the Departments of  Biochemistry and Molecular Biology and ^¶ Dermatology, Faculty of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, ^§ CREST of Japan Science and Technology Corporation, Bunkyo-ku, Tokyo 113-0033, and the ^** Department of Biochemistry, Gunma University School of Medicine, Maebashi, Gunma 371-8511, Japan

Received for publication, October 1, 2001, and in revised form, February 15, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

Cysteinyl leukotrienes (LTs) are important proinflammatory mediators. Their precise roles in mice need to be elucidated to interpret mouse models of inflammatory diseases. For this purpose, we cloned and characterized mouse receptors for cysteinyl LTs, mCysLT₁ and mCysLT₂. mCysLT₁ and mCysLT₂ were composed of 339 amino acids with 87.3% identity and 309 amino acids with 73.4% identity to human orthologues, respectively. A pharmacological difference was noted between mouse and human CysLT₂. Pranlukast, a specific inhibitor for human CysLT₁, antagonized mCysLT₂ responses as determined by Ca²⁺ elevation and receptor-induced promoter activation. The mRNA expressions of both mCysLTs were higher in C57BL/6 mice than in 129 mice. mCysLT₁ mRNA was expressed mainly in skin, lung, and small intestine. mCysLT₂ was seen more ubiquitously with high expressions in spleen, lung, and small intestine. By in situ hybridization we demonstrated for the first time that mCysLT₁ and mCysLT₂ were expressed in subcutaneous fibroblasts. The different pharmacological characteristics of CysLT₂ between human and mouse and the different distributions of CysLTs between mouse strains suggest that careful choice and interpretation are necessary for a study of CysLTs using animal models.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

Cysteinyl leukotrienes (LTs)¹ including LTC₄, LTD₄, and LTE₄ are inflammatory mediators previously known as SRS-A (slow reacting substances of anaphylaxis) (1-4). They are produced by LTC₄ synthase from the biologically inactive precursor LTA₄, a product of 5-lipoxygenation of arachidonic acid (5-7). LTC₄ synthase is expressed in inflammatory cells including mast cells, eosinophils, basophils, and monocytes/macrophages (7). The cysteinyl LTs are potent bronchoconstrictors and macrophage activators, and have been identified in urine and tissues in asthmatic patients (8-10). At least two cysteinyl LT receptors (CysLT₁ and CysLT₂) have been defined pharmacologically as G protein-coupled receptors. Most of the biological reactions of cysteinyl LTs including bronchospasm, plasma exudation, vasoconstriction, mucus secretion, and eosinophil recruitment are mediated through interaction with CysLT₁ (11). CysLT₁ antagonists, montelukast (Singulair^TM) (12, 13), zafirlukast (Accolate^TM) (14), and pranlukast (Onon^TM) (15) are currently used clinically for the treatment of bronchial asthma and allergic rhinitis. Human CysLT₁, human CysLT₂, and mouse CysLT₁ were recently cloned and characterized (16-21). Human CysLT₁ mRNA was detected in airway smooth muscle cells, tissue macrophages, monocytes, and eosinophils (16, 17). Human CysLT₂ mRNA was prominently expressed in lung macrophages, airway smooth muscle, cardiac Purkinje cells, adrenal medulla cells, peripheral blood leukocytes, placenta, spleen, and brain (18-20).

Ovalbumin sensitization and aerosol challenge in mice elicits release of LTB₄ and LTC₄ into bronchoalveolar lavage fluid, eosinophilia in the mucosa and the bronchoalveolar lavage fluid, and increased airway reactivity to methacholine (22). Although cysteinyl LTs are not established as bronchoconstrictors in mice, MK-571, a CysLT₁-selective antagonist, inhibits eosinophilia, bronchial hyperreactivity, and microvascular leakage of mice (23), suggesting a contribution of cysteinyl LTs in these processes. We cloned and characterized the mouse CysLT₁ (mCysLT₁) and CysLT₂ (mCysLT₂) to better study the roles of cysteinyl LTs in animal models of diseases.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

Antagonists-- Pranlukast was a generous gift from Ono Pharmaceutical Co. (Osaka, Japan). MK-571 and BAY u9773 were purchased from BIOMOL Research Laboratories (Plymouth Meeting, PA). Pranlukast and MK-571 were dissolved in 100% ethanol to make 10 mM stock solutions.

Cloning and Expression of mCysLT₁ and mCysLT₂-- A mouse genome library (129 inbred strain) in Fix II vector (Stratagene, La Jolla, CA) was screened with [-³²P]dCTP-labeled partial open reading frame (ORF) of human CysLT₁ (581 nucleotides), and a clone was isolated. The CysLT₁ ORF from C57BL/6 was obtained by PCR with a genome template using sense (5'-ATTCCTGGAGAACATGAATGG-3') and antisense (5'-CATTGTTCTGCACTGTAGATGAG-3') primers. A mouse expressed sequence tag clone with 88.4% identity in cDNA sequence to the human CysLT₁ was found during a routine BLAST search of the NCBI data base (GenBank^TM accession number AI506060), and it was purchased from Genome Systems (St. Louis, MO). These three clones were sequenced using an automated DNA sequencer 373A (Applied Biosystems, Foster City, CA) and found to be completely identical. The ORF of the expressed sequence tag clone was amplified by PCR with sense (5'-CGGGATCCCGAATGGAACTGAAAATCTGAC-3') and antisense (5'-GCTCTAGAGCTTATTCGTTACATATTTCTT-3') primers, subcloned into a pGEM-T Easy Vector (Promega, Madison, WI), digested with restriction enzymes (BamHI and XbaI), and subcloned again into an expression vector, pcDNA4HisMax (Invitrogen, Carlsbad, CA) to obtain pc4HM-mCysLT₁. For the cloning of a mouse CysLT₂ orthologue, the mouse genomic library in FixII vector was screened by plaque hybridization using [-³²P]dCTP-labeled full-length ORF of human CysLT₂ cDNA (19) as a probe. The fragments that hybridized to human CysLT₂ probe were sequenced as described above. The putative ORF was attached with an hemagglutinin tag at its N terminus and subcloned into an expression vector pcDNA3.1 (Invitrogen) between the KpnI site and the XbaI site to obtain pc3.1-mCysLT₂. The CysLT₂ ORF from C57BL/6 was obtained by PCR with a genome template using primers designed from the 129 genome sequence.

Cell Culture and Transfection-- HEK-293 cells and B103 cells were cultured in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% fetal calf serum (FCS; Sigma), 100 IU/ml penicillin, and 100 µg/ml streptomycin; PC12 cells in Dulbecco's modified Eagle's medium supplemented with 10% horse serum, 10% FCS, 100 IU/ml penicillin, and 100 µg/ml streptomycin; and CHO cells in Nutrient Mixture F-12 HAM (Sigma) supplemented with 10% FCS, 100 IU/ml penicillin, and 100 µg/ml streptomycin. Superfect (Qiagen, Valencia, CA) was used for the transfection of HEK-293 cells, B103 cells, and PC12 cells, and FuGENE 6 (Roche Molecular Biochemicals) was used for the transfection of CHO cells, according to the manufacturers' protocols. To obtain HEK-293 cells stably expressing mCysLT₁, the cells were transfected with pc4HM-mCysLT₁ and selected with 500 µg/ml Zeocin (Invitrogen). Two lines of the cells (named HEK 7-1 and HEK 7-3) were chosen by the increase of intracellular Ca²⁺ concentration ([Ca²⁺]_i) in response to LTD₄ (see below) and maintained in Dulbecco's modified Eagle's medium with 10% FCS, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 200 µg/ml Zeocin. The expression of mCysLT₁ was confirmed by Northern hybridization. HEK-293 cells transfected with the vector alone were also kept in a medium with Zeocin and used as a vector control. To obtain CHO cells stably expressing mCysLT₂, the cells were transfected with pc3.1-mCysLT₂ and selected with 1 mg/ml G418 (Invitrogen). Two lines of the cells (named CHO-7A1 and CHO-8B3) were chosen by Northern hybridization and maintained in F-12 with 10% FCS, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 300 µg/ml G418. CHO cells transfected with the vector alone were also kept in a medium with G418 and used as a vector control.

Ca²⁺ Response Assay-- The cells stably expressing mouse CysLTs were loaded with 3 µM Fura2-AM (Dojindo, Kumamoto, Japan) in a modified HEPES-Tyrode's bovine serum albumin buffer (25 mM Hepes-NaOH, pH 7.4, 140 mM NaCl, 2.7 mM KCl, 1.0 mM CaCl₂, 12 mM NaHCO₃, 5.6 mM D-glucose, 0.37 mM NaH₂PO₄, 0.49 mM MgCl₂, and 0.1% (w/v) bovine serum albumin (Wako, Osaka, Japan)) containing 0.01% Cremophor EL (Sigma) at 37 °C for 1 h. The cells were detached from dishes with PBS containing 2 mM EDTA, collected, centrifuged at 240 × g for 5 min in a 15-ml tube, and resuspended in the modified HEPES-Tyrode's bovine serum albumin buffer (2 × 10⁶ cells/ml). The change in the fluorescence ratio (340 nm/380 nm) in response to LTs was monitored using a Ca²⁺ analyzer, CAF-100 (Jasco, Tokyo, Japan). [Ca²⁺]_i was estimated as described previously (24). A receptor antagonist was applied 5 min before stimulation with LTC₄ or LTD₄.

Reporter Gene Assay-- We have recently established a zif268-driven promoter assay induced by receptor activation (25), and the original method was modified. Briefly, 1 × 10⁴ B103 cells were seeded in collagen-coated 96-well microplates (Asahi Techno Glass, Tokyo, Japan) and transfected with 25 ng of pc4HM-mCysLT₁ or vector alone in combination with 150 ng of zif268-firefly luciferase/pGL2, which was a generous gift of Dr. T. Naito at Japan Tobacco Inc. (Tokyo, Japan). They were incubated for 48 h and treated with LTs in a serum-free medium. Receptor antagonists were applied 15 min before the stimulation. The cells were lysed after 4 h of incubation at 37 °C. Luciferase activity was determined by measuring luminescent signals using a luciferase reporter gene assay system, PICAGENE Dual Seapansy (Toyo Ink, Tokyo, Japan) and a Top Count luminescence counter (Packard, Meriden, CT). For the assay of CysLT₂, 2 × 10⁵ PC12 cells were transfected with 250 ng of pc3.1-mCysLT₂ or vector alone, 300 ng of zif268-firefly luciferase/pGL2, and 250 ng of thymidine kinase-Renilla luciferase/pRL (Promega) and seeded in collagen-coated 24-well plates. After serum starvation for 1.5 h, they were stimulated with ligands for 6 h. Firefly and Renilla luciferase activities were measured using PICAGENE Dual Seapansy and a Mini Lumat LB9506 luminometer (Berthold, Bad Wildbad, Germany). Firefly luciferase values were standardized to Renilla values.

Northern Blotting-- Total RNA was extracted from 129+Ter/Sv Jcl (Clea Japan, Tokyo, Japan) and C57BL/6J Jcl (Clea Japan) mouse tissues including brain, heart, lung, liver, spleen, kidney, small intestine, skeletal muscle, and skin, using Isogen (Wako, Osaka, Japan). Poly(A)⁺ RNA was isolated from 200 µg of the total RNA using a µMACS mRNA isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). The RNA samples were denatured, electrophoresed on 0.7% formaldehyde-agarose gels, and transferred onto nylon membranes Hybond-N+ (Amersham Biosciences) as described (26). The membranes were hybridized with [-³²P]dCTP-labeled ORF of mCysLT₁, mCysLT₂, or human glyceraldehyde-3-phosphate dehydrogenase (G3PDH) at 65 °C for 2 h in a Rapid Hyb hybridization solution (Amersham Biosciences). The membranes were washed at 65 °C in 0.2× SSC, 0.1% SDS for 1 h and subjected to autoradiography for 5 days (mCysLT₁ and mCysLT₂) or overnight (G3PDH).

Quantitative Real Time Reverse Transcriptase-PCR-- Total RNA was prepared as described above from 129 and C57BL/6 mouse adrenal gland, peritoneal macrophages, and spleen. For elicitation of peritoneal macrophages, the animals were injected with 2 ml of 4% thioglycollate broth 4 days prior to sacrifice and peritoneal lavage using ice-cold PBS with 2 mM EDTA. cDNA was synthesized from 1 µg of total RNA using Superscript II (Invitrogen) and 50 ng of random hexamers according to the manufacturer's protocol, and 2 µl of the cDNA was diluted in 38 µl of 10 mM Tris-HCl, 1 mM EDTA (pH 8.0) for PCR. PCR was carried out using a LightCycler System (Roche Molecular Biochemicals), and the products were detected by measuring the binding of the fluorescence dye SYBR Green I to double-stranded DNA. The PCR reactions were set up in microcapillary tubes in a volume of 20 µl. The reaction components were 1 µl of diluted cDNA, 1 × FastStart DNA Master SYBR Green I (Roche Molecular Biochemicals), a final concentration of 3 mM MgCl₂, and 1 µM upstream and downstream primers. pc4HM-mCysLT₁, pc3.1-mCysLT₂, and an expressed sequence tag clone containing mG3PDH cDNA (GenBank^TM accession number BF537941) purchased from IncyteGenomics (Palo Alto, CA) were used as standards. Primers were chosen so that they would yield PCR products identical in DNA sequence from 129 and C57BL/6 inbred strains. The following primers were used: mCys1-760+, 5'-CAACGAACTATCCACCTTCACC-3'; mCys1-923, 5'-AGCCTTCTCCTAAAGTTTCCAC-3'; mCys2-662+, 5'-GTCCACGTGCTGCTCATAGG-3'; mCys2-843, 5'-ATTGGCTGCAGCCATGGTC-3'; mG3PDH-879+, 5'-AGGTTGTCTCCTGCGACTTC-3'; and mG3PDH-1089, 5'-CTTGCTCAGTGTCCTTGCTG-3'. These primer pairs result in PCR products of 164 (mCysLT₁), 182 (mCysLT₂), and 211 bp (G3PDH). The standards and the samples were simultaneously amplified using the same reaction master mixture. The reactions were incubated at 95 °C for 10 min to activate the polymerase, followed by 50 cycles of amplification. Each cycle of PCR included 3 s of denaturation at 95 °C, 3 s of primer annealing at 67 °C for G3PDH, 65 °C for mCysLT₁, and 68 °C for mCysLT₂, and 10 s of extension at 72 °C. The temperature ramp was 20 °C/s. At the end of the extension steps, the fluorescence of each sample was measured to allow quantification of the cDNAs. After cycling, melting curves of the PCR products were acquired by stepwise increase of the temperature from 5 °C above the annealing temperature to 95 °C. Using LightCycler analysis software, the SYBR Green I signal of each sample was plotted versus the number of cycles, and the crossing points were obtained. These crossing points correlate inversely with the log of the initial template concentration. The levels of mRNA were estimated by subtracting the initial levels of target DNA in PCR reactions without reverse transcription, which represents genomic contamination. Then the mRNA levels were normalized to the level of G3PDH mRNA.

In Situ Hybridization-- Paraffin sections of the skin samples from 129 and C57BL/6 mice fixed in 10% formalin were investigated as described previously (27, 28) by using a slightly modified nonradioactive in situ hybridization technique with digoxigenin-labeled RNA probes. Briefly, paraffin-embedded tissues were cut to 4-µm-thin sections, mounted onto silane-coated slides, deparaffinized, and treated with proteinase K (5 µg/ml in PBS) for 10 min at 24 °C and glycine (2 mg/ml in PBS) for 15 min at 24 °C. Then the sections were acetylated with acetic anhydride (1 ml in 400 ml of 0.1 M triethanolamine, pH 8.0) for 15 min at 24 °C. After washing with PBS, the samples were soaked in 2× SSC with 50% formamide, subjected to hybridization. Fragments of cDNAs for mCysLTs (mCysLT₁ ORF at 687-887 and mCysLT₂ ORF at 18-222) were amplified by PCR using upstream primers with a recognition sequence for HindIII and downstream primers with a recognition sequence for EcoRI, and subcloned into pSPT18 by directional cloning. The plasmids were linearized using HindIII to prepare the antisense probes and EcoRI for the sense probes. The probes were labeled with digoxigenin-11-UTP using a DIG RNA labeling kit (Roche Molecular Biochemicals). The labeled RNA probes (1 µg/ml) in a mixture containing 50% formamide, 10% dextran sulfate, 2× SSC, 1 mg/ml tRNA, 1 mg/ml salmon sperm DNA, and 0.1% bovine serum albumin were placed on the slides and coverslipped. Hybridization was performed in a humidified chamber for 16 h at 42 °C for the mCysLT₁ probe and 50 °C for the mCysLT₂ probe. The slides were washed in 2× SSC with 50% formamide for 20 min three times at 42 °C. Nonhybridized probes were digested in 20 µg/ml RNase A, 500 mM NaCl, 1 mM EDTA, and 10 mM Tris-HCl (pH 8.0) for 30 min at 37 °C. They were then rinsed for 20 min in 0.1× SSC three times at 42 °C. The digoxigenin-labeled probes were visualized using a DIG nucleic acid detection kit (Roche Molecular Biochemicals) according to the manufacturer's protocol. The slides were counterstained in methyl green for 10 min, washed in running tap water, and mounted.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

The Structure of mCysLT₁ and mCysLT₂-- mCysLT₁ and mCysLT₂ were predicted to be polypeptides of 339 and 309 amino acid residues, respectively (Fig. 1A). The identities of the amino acid sequences between 129 mouse and human (16-18) CysLTs are shown in Fig. 1B. mCysLT₁ was longer than human CysLT₁ by two amino acid residues. mCysLT₂ was shorter than human CysLT₂ by 37 amino acid residues, being truncated at both the N and C termini. The sequence of mCysLT₁ was identical among 129, C57BL/6, and BALB/c mice, and there was a mismatch in mCysLT₂ sequences at the 213th amino acid residue between 129 (Val) and C57BL/6 (Ile). The preserved amino acids in the rhodopsin-like G protein-coupled receptor family, including two Cys residues in the first and second extracellular loops, Asp in transmembrane domain 2 (TM2), Trp in TM4, Tyr in TM5, and Pro in TM6, were all present in mCysLT₁ and mCysLT₂. mCysLT₂ had the Asn-Pro-Xaa₂-Tyr motif at the end of the TM7, whereas mCysLT₁ had an Asp residue instead of Asn in the motif. There was no Asp-Arg-Tyr motif at the TM3/intracellular loop 2 transition in mCysLT₁ nor mCysLT₂, although it is a highly conserved motif in the G protein-coupled receptor family. Both mCysLT₁ and mCysLT₂ had possible phosphorylation sites in intracellular loop 3 and the C terminus, and CysLT₁ had several possible N-glycosylation sites in the N terminus and extracellular loops.

View larger version (31K): [in this window] [in a new window]   Fig. 1.   Amino acid sequence of mCysLT1 and mCysLT2. A, structures of mCysLT1 and mCysLT2 are shown. The putative transmembrane segments I-VII of mCysLT1 and mCysLT2 are underlined. The asterisks indicate identical amino acids, and dots indicate similar amino acids. B, amino acid identities between mCysLT1, mCysLT2, human CysLT1, and human CysLT2 are shown. AA, amino acids.

Pharmacological Properties of mCysLT₁ and mCysLT₂-- Mouse orthologues of CysLT₁ and CysLT₂ were identified as functional cysteinyl LT receptors by several methods. CysLT₁ and CysLT₂ are known to increase [Ca²⁺]_i (20, 29). LTD₄ evoked a dose-dependent increase in [Ca²⁺]_i in HEK-293 cell lines stably expressing mCysLT₁ (HEK 7-1 (Fig. 2, A and B) and HEK 7-3 (data not shown)). LTC₄ also evoked a slight increase in [Ca²⁺]_i (Fig. 2B), whereas LTB₄ or LTE₄ did not (data not shown). These cells pretreated with a CysLT₁ antagonist, pranlukast (Fig. 2A) or MK-571 (data not shown), did not respond to LTD₄, whereas they remained responsive to ATP. In a reporter gene assay, B103 cells transiently expressing mCysLT₁ increased luciferase activity in response to LTD₄ in a dose-dependent manner (Fig. 2C). The cells did not respond to either LTB₄ or LTE₄ at a concentration of 10 or 100 nM (data not shown). The LTD₄-induced response was inhibited by pranlukast or MK-571 (Fig. 2D).

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Ca2+ response and reporter gene expression using mCysLT1. A and B, Ca2+ mobilization in HEK-293 cells stably expressing mCysLT1. A, the mCysLT1-expressing cells (HEK 7-1) loaded with Fura2-AM were challenged with 100 nM LTD4 (open arrow) or 10 µM ATP (closed arrow), and the change in [Ca2+]i was measured. The right panel shows the response of the cells treated with 10 µM pranlukast 5 min before the challenge. The results are representatives of three independent experiments. B, HEK 7-1 cells were loaded with Fura2-AM and stimulated with various concentrations of LTC4 () or LTD4 (). Vector-transfected HEK-293 cells stimulated with LTC4 () or LTD4 () were used as negative controls. The differences of [Ca2+]i before and after the challenges are shown (n = 3, means ± S.E.). Statistically significant differences between the vector control and HEK 7-1 are indicated. *, p < 0.01, unpaired t test. C and D, reporter gene assays of B103 cells transiently transfected with mCysLT1 or vector alone. C, Cells transfected with mCysLT1 () or vector alone () were stimulated with various concentrations of LTD4. The data are expressed as fold activation over control (without LTD4) and expressed as the means ± S.E. (n = 3). Statistically significant differences between the control and the LT-stimulated cells are indicated. *, p < 0.05, unpaired t test. D, the effects of two CysLT1 antagonists are shown. The data are expressed as fold activation over the control (without LTD4) and expressed as the means ± S.E. (n = 4). Statistically significant differences between the control and the drug-treated cells are indicated. *, p < 0.05, Bonferroni's multiple t test.

In CHO cells stably expressing mCysLT₂ (CHO-7A1), LTC₄ and LTD₄ exhibited dose-dependent increases in [Ca²⁺]_i (Fig. 3A). The response was inhibited by BAY u9773, a nonselective antagonist of cysteinyl LT receptors (30), in a dose-dependent manner but was not inhibited by a CysLT₁-specific antagonist, MK-571 (Fig. 3B). The response of CHO-8B3 cells, another mCysLT₂-expressing clone, was similar to that of CHO-7A1 (n = 3; data not shown). ATP (10 µM) elicited the same level of increase in [Ca²⁺]_i in CHO-7A1, CHO-8B3, and the vector control (n = 3; data not shown). PC12 cells transiently expressing mCysLT₂ increased luciferase activities in response to LTC₄ and LTD₄ to the same extent in dose-dependent manners (Fig. 3C), and the responses were inhibited by BAY u9773 and not by MK-571 (Fig. 3D). Surprisingly, pranlukast, found to be a CysLT₁-specific antagonist from human studies (16, 17), inhibited the LTC₄-induced increase in [Ca²⁺]_i (Fig. 3B) and the LTD₄-induced luciferase activity (Fig. 3D) in the cells expressing mCysLT₂. Several reports showing that pranlukast does not antagonize human CysLT₂ (18, 20) imply a pharmacological difference of CysLT₂ between human and mouse likely because of significant difference in primary structure (Fig. 1B). BAY u9773 was partially agonistic on mCysLT₂ as is reported in human CysLT₂ (19) (data not shown).

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Ca2+ response and reporter gene expression using mCysLT2. A and B, Ca2+ mobilization in CHO cells stably expressing mCysLT2. The cells were loaded with Fura2-AM and challenged with LTC4 or LTD4. The increase in [Ca2+]i was calculated from the fluorescence ratio (340 nm/380 nm). A, [Ca2+]i increases of mCysLT2-expressing cells (CHO-7A1) challenged with LTC4 () or LTD4 () and vector control cells challenged with LTC4 () or LTD4 () are shown (n = 3, means ± S.E.). Statistically significant differences between the control and CHO-7A1 are indicated. *, p < 0.01, unpaired t test. B, effects of CysLT antagonists were examined. The increase in [Ca2+]i after 100 nM LTC4 stimulation is shown as a percentage to that of the cells without an antagonist (n = 2, each replicate shown). BAY u9773 () and pranlukast () inhibited the response to LTC4, whereas MK-571 () did not affect the response to the LTC4 stimulation. Neither MK-571, pranlukast, nor BAY u9773 affected Ca2+ response to 10 µM ATP (n = 2, data not shown). C and D, reporter gene assay of PC12 cells transiently transfected with mCysLT2 or vector alone. The ratios of firefly luciferase activity to Renilla luciferase activity are shown. C, the responses of mCysLT2-transfected cells challenged with various concentrations of LTC4 () or LTD4 () and vector-transfected cells challenged with LTC4 () or LTD4 () are shown (n = 2, each replicate shown). The experiments using LTC4 were performed in the presence of 5 mM serine and 10 mM borate. D, the responses to 10 nM LTD4 in the presence of various concentrations of MK-571 (), pranlukast (), or BAY u9773 () are shown (n = 2, each replicate shown).

Different Tissue Distribution of CysLT₁ and CysLT₂ mRNA in Two Mouse Inbred Strains-- Hybridization of poly(A)⁺ RNA from various mouse tissues detected transcripts of 3.0 and 5.5 kb for CysLT₁ and CysLT₂, respectively (Fig. 4A). As a whole, the expression levels of CysLTs were higher in C57BL/6 inbred strain than in 129 inbred strain, even though a slight difference in control hybridization (G3PDH) in Northern blotting was observed in some tissues. In C57BL/6 strain, the highest mRNA expression for CysLT₁ was observed in skin, lung, small intestine, and macrophages, and moderate expressions were found in other tissues; the expression of CysLT₂ was ubiquitous with higher expressions in spleen, lung, and small intestine (Fig. 4). Differential tissue expression between two strains suggests that regulatory polymorphism is present.

View larger version (42K): [in this window] [in a new window]   Fig. 4.   Expression of mCysLT1 and mCysLT2 mRNA in various tissues of C57BL/6 and 129 inbred strains. A, Northern blot analysis. Poly(A)+ RNAs (3 µg) were electrophoretically separated, transferred to nylon membranes, and hybridized with [-32P]dCTP-labeled ORFs of mCysLT1, mCysLT2, and human G3PDH. The shown data are representative of three experiments that gave similar results. B, quantitative real time reverse transcriptase-PCR. mRNA levels of C57BL/6 mice (closed bar) and 129 mice (open bar) were obtained as described under "Materials and Methods" and given as fold expression compared with the levels of 129 spleen (n = 3, means ± S.E.). The differences in expression levels between strains were seen in CysLT2 of the spleen (p < 0.05).

Given the importance of cysteinyl LTs in skin diseases including atopic dermatitis (31), we investigated the distribution of CysLTs in mouse skin by in situ hybridization. We chose 129 inbred strain because Goulet et al. (32) had reported the potent inflammatory response of the skin in 129 mice. No signals of CysLTs were detected in epidermis (data not shown). In the subcutaneous connective tissues, however, high expressions of CysLT₁ (Fig. 5a) and CysLT₂ mRNA (Fig. 5c) were seen mostly in fibroblasts. No signal was obtained using the sense control (Fig. 5, b and d). It has been reported that cysteinyl LTs increase collagen synthesis in fibroblasts (33, 34), and our report is the first to demonstrate the expression of CysLTs in fibroblasts. Further study is needed to uncover yet unknown roles of cysteinyl LTs in wound healing and pathological collagen synthesis.

View larger version (136K): [in this window] [in a new window]   Fig. 5.   In situ hybridization of CysLT1 and CysLT2 mRNA in 129 mouse skin. a and b, in situ hybridization of CysLT1. a, antisense probe, showing expression in subcutaneous fibroblasts (arrows). b, sense control probe. c and d, in situ hybridization of CysLT2. c, antisense probe, showing expression in subcutaneous fibroblasts (arrows). d, sense control probe. Scale bars are 50 µm.

In conclusion, we have cloned mCysLT₁ and mCysLT₂ and found differences in the pharmacological characteristics between mouse and human CysLT₂. There are differences in mRNA expression of CysLT₁ and CysLT₂ between mouse strains, suggesting the importance of choosing a proper mouse strain for a disease model. We also discovered expression of both CysLTs in subcutaneous fibroblasts. These data are useful in interpreting and understanding the physiological and pathological roles of CysLTs in animal models of human diseases.

	ACKNOWLEDGEMENTS

We thank Drs. D. A. Wong, S. Sato, and K. Kishimoto for suggestions and M. Ito for technical assistance.

	FOOTNOTES

^* The work was supported in part by Grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan and grants from the Yamanouchi Foundation for Metabolic Disorders and the ONO Medical Research Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AB044087 and AB058930.

To whom correspondence should be addressed. Tel.: 81-3-5802-2925; Fax: 81-3-3813-8732; E-mail: tshimizu@m.u-tokyo.ac.jp.

Published, JBC Papers in Press, February 19, 2002, DOI 10.1074/jbc.M109447200

	ABBREVIATIONS

The abbreviations used are: LT, leukotriene; CHO, Chinese hamster ovary; FCS, fetal calf serum; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; HEK, human embryonic kidney; ORF, open reading frame; PBS, phosphate-buffered saline; m, mouse; AM, acetoxymethyl; TM, transmembrane domain.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				M. Lopez-Parra, E. Titos, R. Horrillo, N. Ferre, A. Gonzalez-Periz, M. Martinez-Clemente, A. Planaguma, J. Masferrer, V. Arroyo, and J. Claria Regulatory effects of arachidonate 5-lipoxygenase on hepatic microsomal TG transfer protein activity and VLDL-triglyceride and apoB secretion in obese mice J. Lipid Res., December 1, 2008; 49(12): 2513 - 2523. [Abstract] [Full Text] [PDF]

				M. P. W. Moos, J. D. Mewburn, F. W. K. Kan, S. Ishii, M. Abe, K. Sakimura, K. Noguchi, T. Shimizu, and C. D. Funk Cysteinyl leukotriene 2 receptor-mediated vascular permeability via transendothelial vesicle transport FASEB J, December 1, 2008; 22(12): 4352 - 4362. [Abstract] [Full Text] [PDF]

				Y. Kaetsu, Y. Yamamoto, S. Sugihara, T. Matsuura, G. Igawa, K. Matsubara, O. Igawa, C. Shigemasa, and I. Hisatome Role of cysteinyl leukotrienes in the proliferation and the migration of murine vascular smooth muscle cells in vivo and in vitro Cardiovasc Res, October 1, 2007; 76(1): 160 - 166. [Abstract] [Full Text] [PDF]

				Y. Iizuka, T. Yokomizo, K. Terawaki, M. Komine, K. Tamaki, and T. Shimizu Characterization of a Mouse Second Leukotriene B4 Receptor, mBLT2: BLT2-DEPENDENT ERK ACTIVATION AND CELL MIGRATION OF PRIMARY MOUSE KERATINOCYTES J. Biol. Chem., July 1, 2005; 280(26): 24816 - 24823. [Abstract] [Full Text] [PDF]

				Y. Hui, Y. Cheng, I. Smalera, W. Jian, L. Goldhahn, G. A. FitzGerald, and C. D. Funk Directed Vascular Expression of Human Cysteinyl Leukotriene 2 Receptor Modulates Endothelial Permeability and Systemic Blood Pressure Circulation, November 23, 2004; 110(21): 3360 - 3366. [Abstract] [Full Text] [PDF]

				K. Okunishi, M. Dohi, K. Nakagome, R. Tanaka, and K. Yamamoto A Novel Role of Cysteinyl Leukotrienes to Promote Dendritic Cell Activation in the Antigen-Induced Immune Responses in the Lung J. Immunol., November 15, 2004; 173(10): 6393 - 6402. [Abstract] [Full Text] [PDF]

				T. C. Beller, A. Maekawa, D. S. Friend, K. F. Austen, and Y. Kanaoka Targeted Gene Disruption Reveals the Role of the Cysteinyl Leukotriene 2 Receptor in Increased Vascular Permeability and in Bleomycin-induced Pulmonary Fibrosis in Mice J. Biol. Chem., October 29, 2004; 279(44): 46129 - 46134. [Abstract] [Full Text] [PDF]

				Y. Kanaoka and J. A. Boyce Cysteinyl Leukotrienes and Their Receptors: Cellular Distribution and Function in Immune and Inflammatory Responses J. Immunol., August 1, 2004; 173(3): 1503 - 1510. [Abstract] [Full Text] [PDF]

				T. C. Beller, D. S. Friend, A. Maekawa, B. K. Lam, K. F. Austen, and Y. Kanaoka Cysteinyl leukotriene 1 receptor controls the severity of chronic pulmonary inflammation and fibrosis PNAS, March 2, 2004; 101(9): 3047 - 3052. [Abstract] [Full Text] [PDF]

				K. Noguchi, S. Ishii, and T. Shimizu Identification of p2y9/GPR23 as a Novel G Protein-coupled Receptor for Lysophosphatidic Acid, Structurally Distant from the Edg Family J. Biol. Chem., July 3, 2003; 278(28): 25600 - 25606. [Abstract] [Full Text] [PDF]

				P. Liu, D. A. Misurski, and V. Gopalakrishnan Cysteinyl leukotriene-dependent [Ca2+]i responses to angiotensin II in cardiomyocytes Am J Physiol Heart Circ Physiol, April 1, 2003; 284(4): H1269 - H1276. [Abstract] [Full Text] [PDF]

				C. Brink, S.-E. Dahlen, J. Drazen, J. F. Evans, D. W. P. Hay, S. Nicosia, C. N. Serhan, T. Shimizu, and T. Yokomizo International Union of Pharmacology XXXVII. Nomenclature for Leukotriene and Lipoxin Receptors Pharmacol. Rev., March 1, 2003; 55(1): 195 - 227. [Abstract] [Full Text] [PDF]

				A. Maekawa, K. F. Austen, and Y. Kanaoka Targeted Gene Disruption Reveals the Role of Cysteinyl Leukotriene 1 Receptor in the Enhanced Vascular Permeability of Mice Undergoing Acute Inflammatory Responses J. Biol. Chem., May 31, 2002; 277(23): 20820 - 20824. [Abstract] [Full Text] [PDF]

< This Article Abstract Full Text (PDF) All Versions of this Article: 277/21/18763    most recent M109447200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed