Characterization of mouse cysteinyl leukotriene receptors mCysLT1 and mCysLT2: differential pharmacological properties and tissue distribution.

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, mCysLT(1) and mCysLT(2). mCysLT(1) and mCysLT(2) 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 CysLT(2). Pranlukast, a specific inhibitor for human CysLT(1), antagonized mCysLT(2) responses as determined by Ca(2+) elevation and receptor-induced promoter activation. The mRNA expressions of both mCysLTs were higher in C57BL/6 mice than in 129 mice. mCysLT(1) mRNA was expressed mainly in skin, lung, and small intestine. mCysLT(2) was seen more ubiquitously with high expressions in spleen, lung, and small intestine. By in situ hybridization we demonstrated for the first time that mCysLT(1) and mCysLT(2) were expressed in subcutaneous fibroblasts. The different pharmacological characteristics of CysLT(2) 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.


MATERIALS AND METHODS
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 mCysLT 1 and mCysLT 2 -A mouse genome library (129 inbred strain) in Fix II vector (Stratagene, La Jolla, CA) was screened with [␣-32 P]dCTP-labeled partial open reading frame (ORF) of human CysLT 1 (581 nucleotides), and a clone was isolated. The CysLT 1 ORF from C57BL/6 was obtained by PCR with a genome template using sense (5Ј-ATTCCTGGAGAACATGAATGG-3Ј) and antisense (5Ј-CATTGTTCTGCACTGTAGATGAG-3Ј) primers. A mouse expressed sequence tag clone with 88.4% identity in cDNA sequence to the human CysLT 1 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 * 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. This 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.
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 g/ml streptomycin; PC12 cells in Dulbecco's modified Eagle's medium supplemented with 10% horse serum, 10% FCS, 100 IU/ml penicillin, and 100 g/ml streptomycin; and CHO cells in Nutrient Mixture F-12 HAM (Sigma) supplemented with 10% FCS, 100 IU/ml penicillin, and 100 g/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 mCysLT 1 , the cells were transfected with pc4HM-mCysLT 1 and selected with 500 g/ml Zeocin (Invitrogen). Two lines of the cells (named HEK 7-1 and HEK 7-3) were chosen by the increase of intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ) in response to LTD 4 (see below) and maintained in Dulbecco's modified Eagle's medium with 10% FCS, 100 IU/ml penicillin, 100 g/ml streptomycin, and 200 g/ml Zeocin. The expression of mCysLT 1 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 mCysLT 2 , the cells were transfected with pc3.1-mCysLT 2 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 g/ml streptomycin, and 300 g/ml G418. CHO cells transfected with the vector alone were also kept in a medium with G418 and used as a vector control.
[Ca 2ϩ ] i was estimated as described previously (24). A receptor antagonist was applied 5 min before stimulation with LTC 4 or LTD 4 .
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 ϫ 10 4 B103 cells were seeded in collagen-coated 96-well microplates (Asahi Techno Glass, Tokyo, Japan) and transfected with 25 ng of pc4HM-mCysLT 1 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 CysLT 2 , 2 ϫ 10 5 PC12 cells were transfected with 250 ng of pc3.1-mCysLT 2 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.
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 g of total RNA using Superscript II (Invitrogen) and 50 ng of random hexamers according to the manufacturer's protocol, and 2 l of the cDNA was diluted in 38 l 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 l. The reaction components were 1 l of diluted cDNA, 1 ϫ FastStart DNA Master SYBR Green I (Roche Molecular Biochemicals), a final concentration of 3 mM MgCl 2 , and 1 M upstream and downstream primers. pc4HM-mCysLT 1 , pc3.1-mCysLT 2 , 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: mCys1-760ϩ, 5Ј-CAACGAACTATCCACCTTCACC-3Ј; mCys1-923Ϫ, 5Ј-AGCCTTCTCCTAAAGTTTCCAC-3Ј; mCys2-662ϩ, 5Ј-GTCCACGTGCTGCTCATAGG-3Ј; mCys2-843Ϫ, 5Ј-ATTGGCTGCAG-CCATGGTC-3Ј; mG3PDH-879ϩ, 5Ј-AGGTTGTCTCCTGCGACTTC-3Ј; and mG3PDH-1089Ϫ, 5Ј-CTTGCTCAGTGTCCTTGCTG-3Ј. These primer pairs result in PCR products of 164 (mCysLT 1 ), 182 (mCysLT 2 ), 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 mCysLT 1 , and 68°C for mCysLT 2 , 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-m-thin sections, mounted onto silane-coated slides, deparaffinized, and treated with proteinase K (5 g/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 2ϫ SSC with 50% formamide, subjected to hybridization. Fragments of cDNAs for mCysLTs (mCysLT 1 ORF at 687-887 and mCysLT 2 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 g/ml) in a mixture containing 50% formamide, 10% dextran sulfate, 2ϫ 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 mCysLT 1 probe and 50°C for the mCysLT 2 probe. The slides were washed in 2ϫ SSC with 50% formamide for 20 min three times at 42°C. Nonhybridized probes were digested in 20 g/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 0.1ϫ 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
The Structure of mCysLT 1 and mCysLT 2 -mCysLT 1 and mCysLT 2 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. mCysLT 1 was longer than human CysLT 1 by two amino acid residues. mCysLT 2 was shorter than human CysLT 2 by 37 amino acid residues, being truncated at both the N and C termini. The sequence of mCysLT 1 was identical among 129, C57BL/6, and BALB/c mice, and there was a mismatch in mCysLT 2 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 mCysLT 1 and mCysLT 2 . mCysLT 2 had the Asn-Pro-Xaa 2 -Tyr motif at the end of the TM7, whereas mCysLT 1 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 mCysLT 1 nor mCysLT 2 , although it is a highly conserved motif in the G protein-coupled receptor family. Both mCysLT 1 and mCysLT 2 had possible phosphorylation sites in intracellular loop 3 and the C terminus, and CysLT 1 had several possible N-glycosylation sites in the N terminus and extracellular loops.
Pharmacological Properties of mCysLT 1 and mCysLT 2 -Mouse orthologues of CysLT 1 and CysLT 2 were identified as functional cysteinyl LT receptors by several methods. CysLT 1 and CysLT 2 are known to increase [Ca 2ϩ ] i (20,29). LTD 4 evoked a dose-dependent increase in [Ca 2ϩ ] i in HEK-293 cell lines stably expressing mCysLT 1 (HEK 7-1 (Fig. 2, A and B) and HEK 7-3 (data not shown)). LTC 4 also evoked a slight increase in [Ca 2ϩ ] i (Fig. 2B), whereas LTB 4 or LTE 4 did not (data not shown). These cells pretreated with a CysLT 1 antagonist, pranlukast ( Fig. 2A) or MK-571 (data not shown), did not respond to LTD 4 , whereas they remained responsive to ATP. In a reporter gene assay, B103 cells transiently expressing mCysLT 1 increased luciferase activity in response to LTD 4 in a dose-dependent manner (Fig. 2C). The cells did not respond to either LTB 4 or LTE 4 at a concentration of 10 or 100 nM (data not shown). The LTD 4 -induced response was inhibited by pranlukast or MK-571 (Fig. 2D).
In CHO cells stably expressing mCysLT 2 (CHO-7A1), LTC 4 and LTD 4 exhibited dose-dependent increases in [Ca 2ϩ ] 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 CysLT 1 -specific antagonist, MK-571 (Fig. 3B). The response of CHO-8B3 cells, another mCysLT 2 -expressing clone, was similar to that of CHO-7A1 (n ϭ 3; data not shown). ATP (10 M) elicited the same level of increase in [Ca 2ϩ ] i in CHO-7A1, CHO-8B3, and the vector control (n ϭ 3; data not shown). PC12 cells transiently expressing mCysLT 2 increased luciferase activities in response to LTC 4 and LTD 4 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 CysLT 1 -specific antagonist from human studies (16,17), inhibited the LTC 4 -induced increase in [Ca 2ϩ ] i (Fig. 3B) and the LTD 4 -induced luciferase activity (Fig. 3D) in the cells expressing mCysLT 2 . Several reports showing that pranlukast does not antagonize human CysLT 2 (18,20) imply a pharmacological difference of CysLT 2 between human and mouse likely because of significant difference in primary structure (Fig. 1B). BAY u9773 was partially agonistic on mCysLT 2 as is reported in human CysLT 2 (19) (data not shown).
Different Tissue Distribution of CysLT 1 and CysLT 2 mRNA in Two Mouse Inbred Strains-Hybridization of poly(A) ϩ RNA from various mouse tissues detected transcripts of 3.0 and 5.5 kb for CysLT 1 and CysLT 2 , 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 CysLT 1 was observed in skin, lung, small intestine, and macrophages, and moderate expressions were found in other tissues; the expression of CysLT 2 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.
Given the importance of cysteinyl LTs in skin diseases including atopic dermatitis (31) CysLTs were detected in epidermis (data not shown). In the subcutaneous connective tissues, however, high expressions of CysLT 1 (Fig. 5a) and CysLT 2 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.
In conclusion, we have cloned mCysLT 1 and mCysLT 2 and found differences in the pharmacological characteristics between mouse and human CysLT 2 . There are differences in mRNA expression of CysLT 1 and CysLT 2 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.