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CELLULAR AND MOLECULAR

Expression and Molecular Pharmacology of the Mouse CRTH2 Receptor

Departments of Pharmacology (A.N.H, R.M.B.), Cancer Biology (R.Z.), Medicine (R.Z., M.D.B., R.M.B), and Molecular Physiology and Biophysics (M.D.B.), and Vanderbilt Ingram Cancer Center (R.Z., M.D.B, R.M.B.), Vanderbilt University School of Medicine, Nashville, Tennessee

Received for publication March 3, 2003
Accepted April 16, 2003.

	Abstract

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Prostaglandin D₂ (PGD₂), the predominant prostanoid produced by activated mast cells, is implicated in a variety of allergic diseases. PGD₂ exerts its effects through two G-protein coupled receptors, DP and CRTH2. PGD₂ mediates chemotaxis of eosinophils, basophils, and Th2 cells via CRTH2-evoked signaling, suggesting a role for this receptor in allergic disease. To characterize the mouse CRTH2 ortholog (mCRTH2), we amplified the mCRTH2 receptor gene and expressed it in HEK293 cells. Saturation ligand binding isotherms demonstrated high-affinity binding of [³H]PGD₂, with a K_d of 8.8 ± 0.8 nM. Competition binding assays with a panel unlabeled prostanoids demonstrated an order of affinity of 13,14-dihydro-15-keto-PGD₂ (DK-PGD₂) 15-deoxy-^12,14-PGJ₂ (15d-PGJ₂) PGD₂ PGJ₂. [³H]PGD₂ binding was also displaced by the nonsteroidal anti-inflammatory drug indomethacin, with a K_i value of 1.04 ± 0.13 µM. No [³H]PGD₂ displacement was detected using fluribrofen, ibuprofen, or aspirin as competitors at concentrations of up to 30 µM. PGD₂, DK-PGD₂, 15d-PGJ₂, and indomethacin each inhibited intracellular cAMP generation in stable transfectant ER293/mCRTH2 cells through a pertussis toxin (PTX) sensitive pathway, consistent with mCRTH2 coupling to a G_i heterotrimeric G-protein. Activation of mCRTH2 elicited chemotaxis of ER293/mCRTH2 cells in response to PGD₂, indomethacin, and 15d-PGJ₂. mCRTH2-dependent chemotaxis was inhibited by PTX and wortmannin, indicating dependence on G_i and PI 3-kinase signal transduction pathways. These data provide the first pharmacological and functional characterization of the mouse CRTH2 receptor.

PGD₂ exerts its effects through two G-protein coupled receptors (GPCRs), DP and CRTH2. The DP receptor, a member of the prostanoid subfamily of GPCRs, couples to the G_s-type G protein, and activation of this receptor leads to increases in intracellular cAMP ([cAMP]_i) and calcium (Hirata et al., 1994; Boie et al., 1995). In contrast, CRTH2 shows greatest sequence similarity to chemoattractant GPCRs, and CRTH2-evoked responses include inhibition of [cAMP]_i and increases in intracellular calcium via G_i-dependent pathways (Hirai et al., 2001; Sawyer et al., 2002). CRTH2 has been recently shown to mediate PGD₂-stimulated chemotaxis of Th2 cells, eosinophils, and basophils, suggesting that CRTH2 may play a proinflammatory role in allergic disease (Hirai et al., 2001). Indeed, increased numbers of circulating T cells expressing CRTH2 have been correlated with severity of atopic dermatitis (Cosmi et al., 2000).

In vivo, PGD₂ undergoes degradation to form J-series cyclopentenone prostaglandins such as 15-deoxy-^12,14-PGJ₂ (15d-PGJ₂) (Shibata et al., 2002). 15d-PGJ₂ has been the subject of intense investigation since it was discovered to bind and activate the peroxisome proliferator-activated receptor- (PPAR) and promote adipocyte differentiation, albeit at concentrations in the micromolar range (Kliewer et al., 1995). Several PPAR-independent actions of 15d-PGJ₂ have also been described, including activation of MAP kinase (Lennon et al., 2002), induction of apoptosis (Ward et al., 2002), and up-regulation of IL-8 expression in T cells (Harris et al., 2002). Recently, 15d-PGJ₂ has also been shown to be an agonist at the human CRTH2 receptor (Sawyer et al., 2002) and to activate eosinophils in vitro (Monneret et al., 2002), suggesting that activation of CRTH2 may be responsible for some of the PPAR-independent effects of 15d-PGJ₂. Interestingly, the affinity of 15d-PGJ₂ for CRTH2 is several orders of magnitude greater than for PPAR, with binding and activation occurring at low nanomolar concentrations. At this time, however, the precise role 15d-PGJ₂ plays in inflammatory processes is not clear.

Although the sequence of the mouse CRTH2 receptor ortholog (mCRTH2) has been reported, its pharmacology and function are uncharacterized (Abe et al., 1999). Given the importance of in vivo mouse models such as the OVA-induced experimental asthma model in elucidating the molecular pathogenesis of allergic asthma, characterization of mCRTH2 is essential to understanding its role in allergic airway inflammation. In this study, we describe initial pharmacological and functional characterization of mCRTH2. Radioligand binding experiments reveal that mCRTH2 binds PGD₂ and PGD₂ metabolites with high affinity, as well as indoleacetic acid based nonsteroidal anti-inflammatory drugs (NSAIDs) such as indomethacin. Activation of mCRTH2 expressed in ER293 cells activates the classical G_i-coupled pathway resulting in a reduction of [cAMP]_i levels. Furthermore, mCRTH2 is capable of mediating chemotaxis of ER293/mCRTH2 cells in response to mCRTH2 agonists via G_i and PI 3-kinase dependent pathways.

	Materials and Methods

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Reagents. Prostaglandins and BW245C were purchased from Cayman Chemicals (Ann Arbor, MI). Isoproterenol, isobutylmethylxanthine, indomethacin, sulindac, aspirin, salicylate, and acetaminophen were obtained from Sigma-Aldrich (St. Louis, MO). [³H]PGD₂ was purchased from Amersham Biosciences (Piscataway, NJ). Pertussis toxin and wortmannin were purchased from Calbiochem (La Jolla, CA). DMEM, Opti-MEM I, and hygromycin B were from Invitrogen (Carlsbad, CA). FBS was obtained from Hyclone (Logan, UT). G418 was purchased from Mediatech (Herndon, VA). L-Glutamine and penicillin/streptomycin were from BioWhittaker (Walkersville, MD). Ponasterone A was purchased from Stratagene (La Jolla, CA).

Construction of pEGSH/mCRTH2 and pRc/CMV/mCRTH2 Expression Vectors. The full-length mCRTH2 coding exon was amplified by PCR from mouse embryonic stem cell genomic DNA (129SvEv) using the primers 5'-CATATGGCCAACGTCACACTGAAG-3' (sense) and 5'-CTCCAGGGTGTCTCCCAGACT-3' (antisense) and ligated into the pCRII vector (Invitrogen). The coding region sequence was verified by sequencing and was identical with the previously published sequence (Abe et al., 1999). The mCRTH2 coding exon was sequentially subcloned into NotI/SacI in the pEGSH (Stratagene) and NotI/XbaI in the pRc/CMV (Invitrogen) mammalian expression vectors.

Expression of mCRTH2 in HEK293 and ER293 Cells. Cells were maintained at 37°C in humidified air containing 5.5% CO₂ in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 100 units ml^–1 penicillin, 100 µg ml^–1 streptomycin (medium for ER293 cells also contained 300 µg/ml G418). HEK293 cells were transiently transfected with pRc/CMV/mCRTH2 or pRC/CMV using Lipofectamine 2000 (Invitrogen). ER293 cells (Stratagene) were transfected with pEGSH/mCRTH2 or pEGSH, and cells expressing CRTH2 were selected by addition of medium containing 100 µg/ml hygromycin B at 48 h post-transfection. Clonal cell lines were selected by two rounds of manual colony isolation using cloning rings. Expression of mCRTH2 was induced by addition of 10 µM ponasterone A (ponA) 24 h before harvesting cells and verified by radioligand binding.

Preparation of Membranes. Membranes for radioligand binding experiments were harvested 48 h post-transfection. Cells were rinsed once with ice-cold PBS containing 1 mM EDTA and lysed by scraping in lysis buffer (15 mM HEPES, pH 7.6, 5 mM EDTA, 5 mM EGTA, and 2 mM phenylmethylsulfonyl fluoride) and passage through a 21-gauge needle five times. To collect membranes, the cell lysate was layered on a 60% sucrose cushion and centrifuged at 150,000g for 1 h at 4°C. The membrane fraction was passed through a 26-gauge needle five times and frozen at –80°C. Membranes from stable transfectant cell lines ER293/mCRTH2 and ER293/pEGSH were prepared following incubation of the cells with 10 µM ponA for 24 h.

Radioligand Binding Assay. Membranes were incubated with [³H]PGD₂ at 4°C for 1.5 h in binding buffer [25 mM HEPES (pH7.4), 1 mM EDTA, 5 mM MgCl₂, 140 mM NaCl, 5 mM KCl]. The binding reaction was terminated by the addition of 3 ml of ice-cold binding buffer and rapidly filtered under vacuum over Whatman GF/F filters (Clifton, NJ). Filters were washed three times with 3 ml of ice-cold binding buffer, dried, and counted in 4 ml of Ultima Gold scintillation fluid (Packard Biosciences, Groningen, The Netherlands). For saturation binding experiments, nonspecific binding was determined in the presence of 10 µM 13,14-dihydro-15-keto-PGD₂ (DK-PGD₂). Competition binding experiments were performed in the presence of 3 nM [³H]PGD₂ and varying concentrations of competing ligands.

Intracellular Ca²⁺ Assay. ER293/mCRTH2 cells were plated in 96-well plates (50,000 cells/well) and mCRTH2 expression was induced with 10 µM ponA for 24 h. Mobilization of intracellular calcium was measured on a FLEXstation system (Molecular Devices, Sunnyvale, CA) using the FLEXstation calcium assay kit according to the manufacturers instructions (Molecular Devices). Briefly, cells were labeled with calcium assay reagent resuspended in Hanks' balanced salt solution/20 mM HEPES, pH 7.4, for 1 h at 37°C before measurement. PGD₂, indomethacin, and carbachol were added to parallel wells in a volume equivalent to 10% of the final well volume while fluorescence was monitored, _ex = 485 nm, _em = 525 nm. In each case, the experiment was terminated by addition of 10 µM ionomycin to determine maximum Ca²⁺ response.

[cAMP]_i Assay. ER293/mCRTH2 cells were grown to 80% confluence in six-well plates and incubated for 24 h in the presence of 10 µM ponA. Thirty minutes before the addition of ligands, medium was replaced with Opti-MEM I containing 0.5 mM isobutylmethylxanthine. Cells were incubated with ligands for 15 min, washed once with PBS, and the reaction was terminated by the addition of 0.1 M HCl. Cells were scraped free and the resulting cell suspension was centrifuged for 10 min at 1000g. Supernatants were assayed for protein content by BCA assay (Pierce, Rockford, IL). After normalization to protein content, [cAMP]_i levels were determined by an enzyme-linked immunoassay according to the manufacturer's instructions (Cayman Chemical).

Cell Migration Assay. ER293/mCRTH2 cells were incubated with 10 µM ponA for 24 h before harvesting. Cells were trypsinized, washed three times in PBS, and resuspended in DMEM. Cells (100,000) were added to the upper chamber of 24-well 0.8-µm polycarbonate transwell inserts (Costar, Cambridge, MA) that had been previously treated overnight with 5 µg/ml Matrigel (BD Biosciences, Bedford, MA) in PBS at 4°C and blocked in the presence of 2% BSA in PBS for one h at 37°C. Ligands were diluted in DMEM and added to the lower chamber. After incubating for 4 h at 37°C, inserts were removed, and the cells adhering to the top of the membrane were removed with a cotton swab. Cells on the bottom of the membrane were fixed with 3.7% formaldehyde for 1 h, washed twice with PBS, and stained overnight with crystal violet. For each insert, five independent fields were counted in blinded fashion at 200x magnification. In some studies, cells were incubated for 12 h with 100 ng/ml pertussis toxin before harvesting. In other studies, cells were treated with 100 nM wortmannin for 10 min, which was maintained at the indicated concentration throughout the chemotaxis assay. For chemokinesis control experiments, 100 nM PGD₂ was added to both sides of the transwell membrane, and transwell inserts were processed and counted as above.

Data Analysis. All data are presented as the mean ± S.E.M. K_d and B_max values for saturation isotherm radioligand binding experiments were calculated based on a one-site binding model using Prism 3.0 software (GraphPad Software, Inc., San Diego, CA). Competition binding curves and IC₅₀ values were calculated using a one-site competition model: Y (fraction bound) = (max + (max – min))/(1 + 10^(X – log(IC₅₀))), where X = log[competitor] (Prism). K_i values were calculated according to the method of Cheng and Prusoff (1973). EC₅₀ values for [cAMP]_i dose-response experiments were calculated using a fixed slope sigmoidal dose-response model: Y (% max stimulation) = (max + (max – min))/(1 + 10^(log(EC₅₀) – X)), where X = log[agonist] (Prism). Differences between means were tested for statistical significance using Dunnett's multiple comparison test (analysis of variance) for inhibitor and dose-response chemotaxis experiments and two-tailed unpaired t test for all other comparisons, with P < 0.5 considered significant (InStat 3 software; GraphPad).

	Results

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Cloning of mCRTH2. To characterize the mouse CRTH2 receptor, we amplified the coding region from mouse genomic DNA by PCR using 5' sense and 3' antisense primers based on the published sequence of mCRTH2 (Abe et al., 1999). The resulting fragment contained a 1149-base pair intronless open reading frame that was identical in sequence to the previously published sequence (GenBank NM 009962), as well as an equivalent region of sequence in the Celera mouse genome database.

Radioligand Binding. To characterize mCRTH2 ligand binding, membranes were prepared from HEK293 cells that had been transiently transfected with pRc/CMV/mCRTH2. Saturation isotherm binding experiments revealed single-site high-affinity specific binding of [³H]PGD₂, with a K_d of 8.8 ± 0.8 nM and a B_max of 2.6 ± 0.6 pmol/mg of membrane protein (Fig. 1). Membranes from HEK293 cells transfected with the empty pRc/CMV vector exhibited no specific [³H]PGD₂ binding (data not shown).

View larger version (16K): [in this window] [in a new window]   Fig. 1. Saturation isotherm analysis of [3H]PGD2 binding to mCRTH2 expressed in HEK293 cells. Membranes isolated from HEK293 cells transiently transfected with mCRTH2 were incubated with varying concentrations of [3H]PGD2 in the absence (total binding) or presence (nonspecific binding) of 10 µM DK-PGD2, as described under Materials and Methods. Specific binding was determined to be the difference between total and nonspecific binding and is shown above. The Kd and Bmax values were determined using a one-site binding model (Prism 3.0). A Scatchard plot (inset) is shown for reference. Each data point was determined in triplicate; error bars represent S.E.M. These data are representative of three independent experiments.

Binding affinities (K_i) for a variety of prostanoids were evaluated by their ability to displace [³H]PGD₂ in competition binding experiments. PGD₂ bound to mCRTH2 with the highest affinity, with an order of affinity of PGD₂ >> PGF₂ > PGE₂ (Fig. 2a). Several PGD₂ metabolites and analogs also bound to CRTH2 with high affinity, with an order of affinity of DK-PGD₂ 15d-PGJ₂ PGD₂ PGJ₂ 15-deoxy-^12,14-PGD₂ (Table 1). Indomethacin has been reported to bind and activate the human CRTH2 receptor (Hirai et al., 2002; Sawyer et al., 2002). We tested the ability of indomethacin and other commonly used NSAIDs to bind to mCRTH2. Of those tested, only indomethacin and sulindac displaced [³H]PGD₂ at concentrations less than 30 µM in competition binding experiments, with indomethacin exhibiting the highest affinity (Fig. 2b; Table 1).

View larger version (22K): [in this window] [in a new window]   Fig. 2. Competition binding analysis of mCRTH2 ligands. Membranes isolated from HEK293 cells transiently transfected with mCRTH2 were incubated with 3 nM [3H]PGD2 in the presence of varying concentrations of prostanoids (A) or NSAIDs (B), as described under Materials and Methods. PGD2 binding is plotted for comparison. Binding curves were generated using a one-site competition model (Prism 3.0). Each data point was determined in triplicate; error bars represent S.E.M. These data are representative of three independent experiments.

mCRTH2 Intracellular Signaling. To characterize the intracellular signaling pathways activated by mCRTH2, we generated a stable transfectant ER293 cell line expressing mCRTH2. In this cell line, expression of mCRTH2 is under the control of a modified ecdysone receptor promoter system and is induced by pretreatment with ponA. After a 24-h incubation with 10 µM ponA, mCRTH2 expression was determined to be 0.7 ± 0.4 pmol/mg of membrane protein by radioligand binding. We tested the ability of mCRTH2 to activate the classical G_i-mediated pathway leading to inhibition of [cAMP]_i in ER293/mCRTH2 cells, as has been observed for the human CRTH2 receptor (Sawyer et al., 2002). PGD₂, indomethacin, and PGD₂ metabolites inhibited increases in [cAMP]_i in isoproterenol-stimulated cells in a dose-dependent manner (Fig. 3a; Table 2). This response was abolished following pretreatment of the cells with pertussis toxin (PTX), demonstrating that mCRTH2 couples to a G_i-type G protein (Fig. 3b). Vector transfected cells showed no response to mCRTH2 agonists (data not shown).

View larger version (15K): [in this window] [in a new window]   Fig. 3. mCRTH2-evoked inhibition of [cAMP]i. A, ER293/mCRTH2 cells were stimulated with 500 nM isoproterenol/50 nM sodium ascorbate in the presence of varying concentrations of mCRTH2 agonists and [cAMP]i levels were determined as described under Materials and Methods. B, cells were additionally treated with or without 100 ng/ml PTX for 12 h before stimulation with 500 nM isproterenol and 100 nM PGD2. Each data point was determined in duplicate for dose-response studies and triplicate for PTX studies (error bars in B represent S.E.M.); the data are representative of three to four independent experiments.

In addition to inhibition of [cAMP]_i, activation of CRTH2 has been demonstrated to lead to increases in intracellular calcium (Hirai et al., 2001). Therefore, we investigated if mCRTH2 ligands could stimulate increases in intracellular calcium in ER293/CRTH2 cells. Despite the ability to bind to mCRTH2, PGD₂ and indomethacin were unable to stimulate increases in intracellular calcium in this cell line. In control experiments, stimulation of endogenous muscarinic cholinergic receptors in ER293/CRTH2 and ER293/vector cells with carbachol led to a robust calcium response (data not shown).

Cell Migration. Because activation of the human CRTH2 receptor has been shown to mediate chemotaxis of Th2 cells, basophils, and eosinophils (Gervais et al., 2001; Hirai et al., 2001, 2002), we tested whether mCRTH2 was able to mediate a chemotactic response of ER293/mCRTH2 cells to mCRTH2 agonists. In transwell cell migration assays, nanomolar concentrations of both PGD₂ and indomethacin were able to stimulate migration of ER293/mCRTH2 cells in a dose-dependent manner (Fig. 4a). The PGD₂ metabolites PGJ₂ and 15d-PGJ₂ also stimulated migration (Fig. 4c). No migration was observed for vector-transfected cells (Fig. 4b) or ER293/mCRTH2 cells incubated with ibuprofen (data not shown). In chemokinesis control experiments, no significant increase in migration of ER293/CRTH2 cells was observed, demonstrating that the observed migration is true chemotaxis (data not shown). These data demonstrate that mCRTH2 is capable of functionally coupling to signal transduction pathways that mediate chemotaxis in ER293 cells.

View larger version (19K): [in this window] [in a new window]   Fig. 4. mCRTH2 mediates chemotaxis of ER293/mCRTH2 cells. Migration of ER293/mCRTH2 (A and C) or ER293/pEGSH (B) cells in response to mCRTH2 agonists was assessed as described under Materials and Methods. A, PGD2 and indomethacin elicit a characteristic bell-shaped dose response. B, PGD2 (100 nM) and indomethacin (100 nM) were unable to stimulate migration of vector-transfected cells; however, these cells migrate in response 5% FBS, a nonspecific chemoattractant. C, PGD2 and metabolites PGJ2 and 15d-PGJ2 (100 nM) are equipotent in stimulating chemotaxis of ER293/mCRTH2 cells. Data are expressed as percentage of cells migrating in the absence of added ligand (control). Each data point represents the average of five high-power fields of a single experimental condition; error bars represent S.E.M. These data are representative of three to four independent experiments. Data points within brackets are significantly different from baseline control (A); , P < 0.01 compared with control (B and C).

Chemotaxis induced by activation of GPCRs has been shown to involve G_i- and inositol phosphate-dependent signal transduction (Neptune and Bourne, 1997; Hirsch et al., 2000). To investigate which signal transduction pathways mediate migration of ER293/mCRTH2 cells in response to PGD₂, we treated cells with PTX or wortmannin, which inhibit the G_i-type G-protein and PI 3-kinase, respectively. While cell migration was completely abolished by pretreatment with PTX, treatment with wortmannin resulted in partial inhibition of PGD₂-stimulated migration (Fig. 5). Taken together these results demonstrate that mCRTH2-evoked chemotaxis is mediated by G_i and PI 3-kinase dependent pathways.

View larger version (12K): [in this window] [in a new window]   Fig. 5. PGD2-stimulated chemotaxis of ER293/mCRTH2 cells is inhibited by PTX and wortmannin. Cells were pretreated with or without 100 ng/ml PTX for 12 h or 100 nM wortmannin for 10 min before performing migration assay. Migration of cells in response to 100 nM PGD2 was determined as described under Materials and Methods. Data are expressed as percentage of untreated cells migrating in the absence of added ligand (control). Each data point represents the average of five high-power fields of a single experimental condition; error bars represent S.E.M. These data are representative of three independent experiments.

	Discussion

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PGD₂ elicits a variety of physiological responses including modulation of smooth muscle tone, renin secretion, sleep induction, and the inflammatory response (Morrow and Roberts, 2001). PGD₂ is the predominant prostanoid produced by activated mast cells and has been implicated in Th2-mediated atopic and inflammatory diseases such as allergic asthma (Lewis et al., 1982; Murray et al., 1986). The murine OVA-induced experimental asthma model has become widely used to elucidate the molecular pathogenesis of allergic asthma (Foster et al., 1996; Wills-Karp et al., 1998). Although the molecular mechanisms in the pathogenesis of asthma are complex, the emerging picture suggests that PGD₂ may play a central role in this disease. Transgenic mice overexpressing lipocalin-type prostaglandin D synthase produced increased levels of PGD₂ and Th2 cytokines and exhibited greater bronchoalveolar infiltration of lymphocytes and eosinophils upon OVA challenge compared with wild-type mice (Fujitani et al., 2002). In vitro chemotaxis assays have demonstrated that chemotaxis of human Th2 cells, eosinophils, and basophils in response to PGD₂ is mediated by CRTH2 (Hirai et al., 2001); however, it has not been established if mCRTH2 plays a role in mediating the effects of PGD₂ in murine asthma models. Indeed, mCRTH2 has been reported to have a much wider pattern of mRNA expression than that observed for the human CRTH2 receptor (Abe et al., 1999), and it is unclear if they play the same physiological roles in vivo.

As a first step in defining the role of mCRTH2 in mouse physiology, we have cloned and characterized the pharmacology of the receptor. mCRTH2 binds PGD₂ and PGD₂ metabolites with high affinity. The mouse DP receptor, a member of the prostanoid subfamily of G-protein coupled receptors, also binds PGD₂ with high affinity (K_d = 40 nM) (Hirata et al., 1994). DP-null mice have an attenuated asthmatic response when challenged with intermediate levels of OVA, but at higher levels, their response is similar to wild-type (Matsuoka et al., 2000). This suggests that, while the DP receptor plays a role in OVA-induced airway hyperreactivity, activation of this receptor may not account for all of the effects of PGD₂ in this model.

Several metabolites of PGD₂ bind to mCRTH2 with similar affinity to PGD₂. DK-PGD₂ is the product of the NADP-linked 15-hydroxyprostaglandin dehydrogenase pathway (Giles and Leff, 1988), and its biological role, if any, has not been established. In contrast, the cyclopentenone prostaglandins PGJ₂ and 15d-PGJ₂ are capable of activating the PPAR nuclear receptor and promote adipocyte differentiation (Kliewer et al., 1995). Numerous PPAR-independent effects of 15d-PGJ₂ have also been described including activation of MAP kinase (Lennon et al., 2002), induction of apoptosis (Ward et al., 2002), and up-regulation of IL-8 expression in activated T cells (Harris et al., 2002). In vivo, 15d-PGJ₂ has been detected in the cytoplasm of foamy macrophages in human aortic atherosclerotic plaques, and LPS-stimulation of macrophages in vitro leads to accumulation of both intracellular and extracellular 15d-PGJ₂ (Shibata et al., 2002). The role of 15d-PGJ₂ in inflammatory processes appears to be complex. 15d-PGJ₂ has been shown to exert anti-inflammatory effects in the acute inflammatory carrageenan-induced pleurisy and chronic collage-induced arthritis murine models (Cuzzocrea et al., 2002). In contrast, the observed up-regulation of IL-8 in activated T cells in response to 15d-PGJ₂ would be expected to be proinflammatory. 15d-PGJ₂ binds to both human (Sawyer et al., 2002) and mouse CRTH2 receptors with an affinity several orders of magnitude greater than that observed for PPAR (Kliewer et al., 1995), raising the possibility that 15d-PGJ₂ may play a proinflammatory role through activation of CRTH2. Consistent with this possibility, nanomolar concentrations of 15d-PGJ₂ have been shown to lead to calcium mobilization, actin polymerization, and CD-11b expression in human eosinophils (Monneret et al., 2002). In addition, data presented here provide the first direct evidence that 15d-PGJ₂ can also stimulate chemotaxis via mCRTH2.

mCRTH2 is closely related to peptide chemoattractant receptors such as FPR and C5aR (Abe et al., 1999), which mediate neutrophil chemotaxis (Pellas et al., 1998; Gao et al., 1999). Nanomolar concentrations of mCRTH2 agonists stimulated chemotaxis of ER293/mCRTH2 cells, which was inhibited by pretreatment with the G_i inhibitor PTX or the PI 3-kinase inhibitor wortmannin. Involvement of G_i and PI 3-kinase in the chemotactic response mediated by GPCRs has been well established in a number of systems, including neutrophils (Niggli and Keller, 1997; Hirsch et al., 2000), Dictyostelium (Meili et al., 1999), and HEK293 cells (Neptune and Bourne, 1997). Our studies further confirm these observations and demonstrate that mCRTH2 couples to the classic signaling pathways that mediate chemotaxis.

In addition to PGD₂ and PGD₂-derived prostanoids, mCRTH2 is capable of binding nonprostaglandin molecules such as indomethacin, a commonly prescribed NSAID. Indomethacin is a nonspecific cyclooxygenase (COX) inhibitor (Mitchell et al., 1993) that also possesses COX-independent activity such as activation of PPAR (Lehmann et al., 1997) and CRTH2 (Hirai et al., 2002). In the present studies, we found that indomethacin is a potent activator of mCRTH2, approximately 2.5-fold less potent than PGD₂. In contrast indomethacin bound to the mCRTH2 receptor with an affinity 25-fold lower than that of PGD₂. One possible explanation for this discrepancy is that indomethacin has a very high intrinsic activity at mCRTH2 compared with PGD₂. In this case, a smaller fraction of receptor occupancy would be required for a given response. Alternatively, differential transport or metabolism of the endogenous PGD₂ ligand versus indomethacin may effectively lower the PGD₂ concentration in live cell assays such as [cAMP]_i signaling, although not in binding assays on membranes. This would result in a right-ward shift in the PGD₂ dose-response curve relative to the indomethacin curve. The ability of indomethacin to bind and activate CRTH2 leading to chemotaxis of Th2 cells, eosinophils, and basophils, as well as stimulation of shape change, CD11b expression, and respiratory burst in eosinophils, is not shared by other NSAIDs (Hirai et al., 2002; Stubbs et al., 2002). In the present studies, both indomethacin and sulindac were capable of binding mCRTH2, and indomethacin acted as an agonist at mCRTH2. Indomethacin and sulindac share a common indoleacetic acid core molecular structure, which appears to be fundamental for mCRTH2 recognition. Other NSAIDs, such as ibuprofen that are not indoleacetic acid derivatives, do not bind or activate mCRTH2. Indomethacin is commonly used in a number of mouse models because of its potent action in inhibiting both COX-1 and COX-2. In these models, the effects of indomethacin as a COX inhibitor may be confounded by its potent agonist activity at the mCRTH2 receptor, and other NSAIDs, such as ibuprofen that do not activate mCRTH2, may be a more advantageous choice.

Stimulation of CRTH2 has been demonstrated to lead to both increases in intracellular calcium and inhibition of [cAMP]_i via PTX-sensitive mechanisms (Hirai et al., 2001; Sawyer et al., 2002). Activation of mCRTH2 in ER293 cells inhibited isoproterenol-induced increases in [cAMP]_i but did not elicit an observable change in intracellular calcium. One possibility is that mCRTH2 is inherently incapable of coupling to the required signal transduction machinery for raising intracellular calcium. It is likely, however, that ER293 cells, a derivative of HEK293 cells, do not possess the appropriate G proteins for this response. Differences in the complement of heterotrimeric G-proteins expressed in a particular cell type have been observed to lead to differences in the ability for a given GPCR to activate a particular signal transduction pathway. For instance, activation of the G_i-coupled sphingosine-1-phosphate receptor Edg-1 leads to an increase in intracellular calcium in Chinese hamster ovary but not HEK293 cells (Okamoto et al., 1998; Van Brocklyn et al., 1998). In accordance with this possibility, PGD₂ stimulation of HEK293 cells transfected with the human CRTH2 causes only a slight increase in intracellular calcium; this response was greatly enhanced upon transfection of the G-protein G₁₅ (Sawyer et al., 2002).

In this study, we have described pharmacological characterization of the mouse CRTH2 receptor and demonstrated that mCRTH2 is G_i-coupled, can be activated by PGD₂, PGD₂ metabolites, and indoleacetic acid NSAIDs, and mediates chemotaxis of ER293/CRTH2 cells via G_i and PI 3-kinase signal transduction pathways. In contrast to the human receptor, which is expressed in Th2 but not Th1 cells (Nagata et al., 1999), mCRTH2 mRNA has been detected at low levels in both Th1 and Th2 cells (Abe et al., 1999). Although the significance of this expression difference is not clear, it has been shown that G_i-coupled chemoattractant receptors expressed on T cells play an important role in the pathogenesis of allergic airway inflammation in mice. When mice received allo-transfer of PTX-treated Th2 cells, they exhibited greatly reduced infiltration of lymphocytes and eosinophils in the OVA-induced experimental asthma model (Mathew et al., 2002). Eosinophilic inflammation did occur when the cells were directly instilled into the airway, indicating the importance of G_i-coupled chemoattractant signaling and resulting migration of Th2 cells in the pathogenesis of allergic airway inflammation. Based on the pharmacology of mCRTH2, it is expected that future studies using mCRTH2 knock-out mice will provide insight into the molecular pathogenesis of allergic diseases.

	Footnotes

 
This work was supported by grants from the National Institutes of Health GM15431 (R.M.B.), DK46205 (R.M.B.), DK37097 (M.D.B.) as well as the Vanderbilt-Ingram Cancer Center. A.N.H. is supported by a PhRMA Foundation predoctoral fellowship. R.Z. is a clinician scientist of the National Kidney Foundation (NKF) and has an Advanced Career Development Award from the Veterans Administration.

DOI: 10.1124/jpet.103.050955.

ABBREVIATIONS: PGD₂, prostaglandin D₂; OVA, ovalbumin; GPCR, G-protein coupled receptor; [cAMP]_i, intracellular cAMP; DK-PGD₂, 13,14-dihydro-15-keto-PGD₂; 15d-PGJ₂, 15-deoxy-^12,14-PGJ₂; PPAR, peroxisome proliferator-activated receptor-; IL, interleukin; NSAID, nonsteroidal anti-inflammatory drug; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; PCR, polymerase chain reaction; ponA, ponasterone A; PTX, pertussis toxin; PI 3-kinase, phosphatidylinositol 3-kinase; COX, cyclooxygenase; BW245C, (4S)-(3-[(R,S)-3-cyclohexyl-3-hydroxypropyl]-2,5-dioxo)-4-imidazolidineheptonoic acid; G418, geneticin.

Address correspondence to: Richard M. Breyer, Vanderbilt University, Division of Nephrology S3223MCN, 1161 21st Ave. South, Nashville, Tennessee 37232-2372. E-mail: rich.breyer{at}vanderbilt.edu

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