Advertisement

RasGRP4 Regulates the Expression of Prostaglandin D2in Human and Rat Mast Cell Lines*

      Mast cells (MCs) are a major source of prostaglandin (PG) D2 in connective tissues, and the expression of this eicosanoid has been linked to asthma and other inflammatory disorders. While it is known that the surface receptor c-kit controls PGD2 expression in MCs by regulating the levels of a synthase that converts PGH2 to PGD2, the intracellular signaling proteins that act downstream of c-kit in this cyclooxygenase pathway have not been identified. We recently cloned a new cation-dependent, guanine nucleotide exchange factor/phorbol ester receptor (designated RasGRP4) that is required for the efficient expression of granule proteases in the human MC line HMC-1. GeneChip analysis of ∼12,600 transcripts in RasGRP4 and RasGRP4+ HMC-1 cells revealed a >100-fold difference in the levels of hematopoietic PGD2 synthase mRNA. No other transcript in the eicosanoid pathway was influenced by RasGRP4 in a comparable manner. As assessed by SDS-PAGE immunoblot analysis, RasGRP4+ HMC-1 cells contained substantial amounts of PGD2 synthase protein. RasGRP4+ MCs also produced ∼15-fold more PGD2 than did RasGRP4 MCs when both cell populations were activated by calcium ionophore. The induced transcript is therefore translated, and substantial amounts of functional PGD2 synthase accumulate in RasGRP4+ MCs. In support of the conclusion that RasGRP4 controls PGD2expression in MCs, inhibition of RasGRP4 expression in the rat MC line RBL-2H3 using a siRNA approach resulted in low levels of PGD2 synthase protein.
      Activated human and rodent mast cells (MCs)
      The abbreviations used are: MC, mast cell; mBMMC, mouse bone marrow-derived MC; IL, interleukin; LT, leukotriene; PG, prostaglandin; PGHS, PG endoperoxide H synthase; RT, reverse transcriptase; RBL, rat basophilic leukemia; TNF-α, tumor necrosis factor α; KL, c-kit ligand; ELISA, enzyme-linked immunosorbent assay; siRNA, small interfering RNA
      1The abbreviations used are: MC, mast cell; mBMMC, mouse bone marrow-derived MC; IL, interleukin; LT, leukotriene; PG, prostaglandin; PGHS, PG endoperoxide H synthase; RT, reverse transcriptase; RBL, rat basophilic leukemia; TNF-α, tumor necrosis factor α; KL, c-kit ligand; ELISA, enzyme-linked immunosorbent assay; siRNA, small interfering RNA
      generate and release substantial amounts of prostaglandin (PG) D2 (
      • Lewis R.A.
      • Soter N.A.
      • Diamond P.T.
      • Austen K.F.
      • Oates J.A.
      • Roberts L.J.
      ), and many of the vasodilation and hemodynamic problems that occur in patients with systemic mastocytosis are thought to be caused by the excessive production of this eicosanoid. PGD2 is a neuromodulator/sleep-inducing factor in the central nervous system. In peripheral tissues, PGD2 inhibits platelet aggregation (
      • Smith M.M.
      • Shi L.
      • Navre M.
      ) but activates eosinophils. PGD2 is a potent chemotactic factor for eosinophils (
      • Emery D.L.
      • Djokic T.D.
      • Graf P.D.
      • Nadel J.A.
      ), and PGD2-treated eosinophils increase their calcium mobilization, actin polymerization, and surface expression of CD11b (
      • Raible D.G.
      • Schulman E.S.
      • DiMuzio J.
      • Cardillo R.
      • Post T.J.
      ,
      • Monneret G.
      • Li H.
      • Vasilescu J.
      • Rokach J.
      • Powell W.S.
      ). This eicosanoid also enhances the rate of apoptosis of eosinophils if these granulocytes are cultured for ∼20 h in the absence of a viability-enhancing cytokine such as interleukin (IL) 5 (
      • Ward C.
      • Dransfield I.
      • Murray J.
      • Farrow S.N.
      • Haslett C.
      • Rossi A.G.
      ). Pulmonary MCs play important roles in the initiation and/or progression of asthma, and substantial amounts of PGD2 are released into the lungs during asthma attacks (
      • Murray J.J.
      • Tonnel A.B.
      • Brash A.R.
      • Roberts L.J.
      • Gosset P.
      • Workman R.
      • Capron A.
      • Oates J.A.
      ,
      • Liu M.C.
      • Bleecker E.R.
      • Lichtenstein L.M.
      • Kagey-Sobotka A.
      • Niv Y.
      • McLemore T.L.
      • Permutt S.
      • Proud D.
      • Hubbard W.C.
      ). The observation that patients with asthma undergo bronchoconstriction when they inhale PGD2 (
      • Hardy C.C.
      • Robinson C.
      • Tattersfield A.E.
      • Holgate S.T.
      ) documents the pathologic consequences of high levels of PGD2 in the lung. PGD2 exerts its biological actions via two seven-transmembrane, G protein-coupled receptors (designated PTGDR/DP and GPR44/CRTH2) (
      • Hirata M.
      • Kakizuka A.
      • Aizawa M.
      • Ushikubi F.
      • Narumiya S.
      ,
      • Boie Y.
      • Sawyer N.
      • Slipetz D.M.
      • Metters K.M.
      • Abramovitz M.
      ,
      • Hirai H.
      • Tanaka K.
      • Yoshie O.
      • Ogawa K.
      • Kenmotsu K.
      • Takamori Y.
      • Ichimasa M.
      • Sugamura K.
      • Nakamura M.
      • Takano S.
      • Nagata K.
      ). Targeted disruption of the PTGDR gene in the mouse leads to a marked reduction in antigen-induced airway reactivity to acetylcholine (
      • Matsuoka T.
      • Hirata M.
      • Tanaka H.
      • Takahashi Y.
      • Murata T.
      • Kabashima K.
      • Sugimoto Y.
      • Kobayashi T.
      • Ushikubi F.
      • Aze Y.
      • Eguchi N.
      • Urade Y.
      • Yoshida N.
      • Kimura K.
      • Mizoguchi A.
      • Honda Y.
      • Nagai H.
      • Narumiya S.
      ), thereby supporting the earlier inhalation studies in humans and dogs that implicated an adversarial role for PGD2 in the lung.
      In the cyclooxygenase pathway that ultimately leads to PGD2expression, liberated arachidonic acid is converted to PGG2and then to PGH2. PG endoperoxide H synthase (PGHS) 1 (also known as cyclooxygenase 1) and PGHS-2 (also known as cyclooxygenase 2) are both able to carry out this two-step biosynthetic process. The resulting precursor eicosanoid is then metabolized by terminal synthases to form PGD2, PGE2, PGF, PGI2/prostacylin, and thromboxane A2. Two PGD2 synthases have been identified in mice, rats, and humans (
      • Nagata A.
      • Suzuki Y.
      • Igarashi M.
      • Eguchi N.
      • Toh H.
      • Urade Y.
      • Hayaishi O.
      ,
      • Kanaoka Y.
      • Ago H.
      • Inagaki E.
      • Nanayama T.
      • Miyano M.
      • Kikuno R.
      • Fujii Y.
      • Eguchi N.
      • Toh H.
      • Urade Y.
      • Hayaishi O.
      ). The brain enzyme is a glutathione-independent member of the lipocalin family of proteins. The distinct hematopoietic enzyme that is expressed in MCs (
      • Urade Y.
      • Ujihara M.
      • Horiguchi Y.
      • Igarashi M.
      • Nagata A.
      • Ikai K.
      • Hayaishi O.
      ) is a sigma-class, glutathione S-transferase family member.
      PGH2 can be metabolized inside cells to thromboxane A2 and to a variety of PGs. Thus, the amount of PGD2 produced by an FcεRI- or calcium ionophore-activated MC is determined in a large part by the amount of PGD2synthase protein in the cell. MCs are heterogeneous in terms of what eicosanoids they produce. c-kit is a member of the type III receptor tyrosine kinase family. PGD2-expressing MCs contain abundant amounts of c-kit on their surfaces, and Murakami et al. (
      • Murakami M.
      • Matsumoto R.
      • Urade Y.
      • Austen K.F.
      • Arm J.P.
      ) noted that c-kit ligand (KL) somehow regulates the levels of PGD2 synthase in mouse MCs. To a lesser extent, IL-3 and IL-10 also influence the expression of PGD2 synthase in MCs. Treatment of human megakaryocytic cell lines with phorbol esters results in a 2–5-fold increase in the levels of PGD2 synthase mRNA (
      • Suzuki T.
      • Watanabe K.
      • Kanaoka Y.
      • Sato T.
      • Hayaishi O.
      ,
      • Mahmud I.
      • Ueda N.
      • Yamaguchi H.
      • Yamashita R.
      • Yamamoto S.
      • Kanaoka Y.
      • Urade Y.
      • Hayaishi O.
      ). While these findings suggest that one or more diacylglycerol/phorbol ester-responsive proteins play an important role in the expression of PGD2 synthase in hematopoietic cells, the intracellular proteins that act downstream of c-kit and other membrane receptors to control the levels of PGD2 synthase in MCs have not been identified.
      We recently cloned a new member of the Ras guanine nucleotide-releasing protein (RasGRP) family of intracellular signaling proteins (
      • Yang Y.
      • Li L.
      • Wong G.W.
      • Krilis S.A.
      • Madhusudhan M.S.
      • Šali A.
      • Stevens R.L.
      ). In contrast to the other three members of its family, RasGRP4 normally is restricted to mature MCs and their circulating progenitors. RasGRP4 functions as a cation-dependent, guanine nucleotide exchange factor. It also is a diacylglycerol/phorbol ester receptor that appears to act downstream of c-kit. The hRasGRP4 gene resides on chromosome 19q13.1 (
      • Yang Y.
      • Li L.
      • Wong G.W.
      • Krilis S.A.
      • Madhusudhan M.S.
      • Šali A.
      • Stevens R.L.
      ) in the vicinity of a site that has been linked to bronchial hyperresponsiveness (
      • The Collaborative Study on the Genetics of Asthma
      ,
      • Ober C.
      • Tsalenko A.
      • Parry R.
      • Cox N.J.
      ). RasGRP1 is essential for the final stages of T-cell development (
      • Dower N.A.
      • Stang S.L.
      • Bottorff D.A.
      • Ebinu J.O.
      • Dickie P.
      • Ostergaard H.L.
      • Stone J.C.
      ). Although human MCs do not express RasGRP1, RasGRP2, or RasGRP3, transfection studies carried out with the RasGRP4-defective HMC-1 cell line derived from a patient with a MC leukemia suggests that RasGRP4 is required for the final stages of MC development (
      • Yang Y.
      • Li L.
      • Wong G.W.
      • Krilis S.A.
      • Madhusudhan M.S.
      • Šali A.
      • Stevens R.L.
      ). Thus, at least two members of the RasGRP family of signaling proteins appear to control cellular differentiation and maturation. We previously noted that RasGRP4 influences the storage of varied neutral proteases in the secretory granules of a MC line. We now report that RasGRP4 also controls what eicosanoids this immune cell produces.

      EXPERIMENTAL PROCEDURES

       Transcript Analysis of RasGRP4 and RasGRP4+ HMC-1 Cells

      RasGRP4+ and RasGRP4 HMC-1 cells (
      • Yang Y.
      • Li L.
      • Wong G.W.
      • Krilis S.A.
      • Madhusudhan M.S.
      • Šali A.
      • Stevens R.L.
      ) were cultured in enriched medium (Iscove's modified Dulbecco's medium (BioWhittaker) containing 10% heat-inactivated fetal calf serum (Sigma), 2 mml-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, and 10 μm monothioglycerol (Sigma) with or without 200–500 μg/ml G418) in the absence of human cytokines. Total RNA was isolated from the two populations of cells with TRIzol (Invitrogen), and comparative transcript profiling was carried out at the Gene Array Technology Center (Brigham and Women's Hospital, Boston, MA) with HG-U95A GeneChips (Affymetrix, Santa Clara, CA) and the experimental protocol recommended by Affymetrix. Each GeneChip contains ∼12,600 probe sets. In these analyses, 8 μg of total RNA from RasGRP4 and RasGRP4+ HMC-1 cells were reverse-transcribed using the GeneChip T7-oligo(dT) promoter primer kit. Biotinylated complementary RNAs, generated from the resulting cDNAs, were fragmented and incubated with the GeneChips for 16 h. The resulting GeneChips were incubated with streptavidin-phycoerythrin staining solution. The obtained signals were then amplified by sequentially incubating the GeneChips with goat IgG, biotinylated goat anti-streptavidin antibody, and staining solution. Hybridization to the array was quantified with a Hewlett-Packard gene array laser scanner. In separate studies, the generated RT-PCR products were subjected to gel electrophoresis to confirm that they were derived from the authentic PGD2 synthase transcript.

       Quantitation of PGD2 Synthase mRNA Levels in Cells by Real-time RT-PCR

      The GeneChip data obtained with the PGD2 synthase probe set were confirmed by real-time RT-PCR. The PCR primers and fluorogenic probes for measuring PGD2synthase mRNA levels were designed with the use of “Primer Express” (Applied Biosystems, CA). TaqMan's 18 S rRNA control reagents were used to normalize RNA levels in each HMC-1 sample. Fluorescent probes were selected such that their T mwas ∼10 °C higher than the matching primer pair. Each high performance liquid chromatography-purified fluorescent probe contained a 6-carboxyfluorescein (FAM) reporter dye covalently attached at its 5′ end and a black hole quencher 1 quencher dye covalently attached at its 3′ end. The forward primer 5′-GGGCAGAGAAAAAGCAAGATGT-3′, the reverse primer 5′-CCCCCCTAAATATGTGTCCAAG-3′, and the dual-labeled fluorescent probe 5′-(FAM)-CAATGAGCTGCTCACGTATAATGCGCC-(BHQ-1)-3′ were used to quantitate PGD2 synthase mRNA levels in these assays. Reactions were carried out using an iCycler IQ real-time detection system (Bio-Rad). SuperScript one-step RT-PCR with PlatinumTaq kits (Invitrogen) were used. Each 50-μl reaction contained 200 ng of total RNA, 5 mm MgSO4, 500 nm forward and reverse primers, and 200 nmfluorescent probe. Samples were analyzed in triplicate. Negative-control reactions were carried out on replicate samples that had not been subjected to the reverse transcriptase step. Additional negative-control reactions were carried out in wells lacking HMC-1 cellular RNA. The reaction conditions were as follows: 15 min at 50 °C and 5 min at 95 °C, followed by 45 two-temperature cycles (15 s at 95 °C and 1 min at 60 °C). The standard curve method (
      • Heid C.A.
      • Stevens J.
      • Livak K.J.
      • Williams P.M.
      ,
      • Winer J.
      • Jung C.K.
      • Shackel I.
      • Williams P.M.
      ) was used to analyze the obtained data.

       SDS-PAGE Immunoblot Analysis

      RasGRP4 and RasGRP4+ HMC-1 cells were analyzed for their expression of four enzymes that participate in the cyclooxygenase and/or 5-lipoxygenase pathways. Both populations of cells were resuspended at a density of 4 × 105 cells/ml in enriched medium alone or medium supplemented with varied combinations of IL-3 (10 ng/ml), IL-4 (10 ng/ml), IL-10 (10 ng/ml) (Chemicon, Temecula, CA), IL-13 (10 ng/ml), KL (50 ng/ml), and tumor necrosis factor α (TNF-α; 20 ng/ml) (R & D Systems, Minneapolis, MN). After 5 days of cytokine exposure, the cultured cells were washed with phosphate-buffered saline, boiled in SDS sample buffer containing β-mercaptoethanol, and subjected to gel electrophoresis. The resolved proteins were blotted onto polyvinylidene difluoride membranes (Bio-Rad) and probed with rabbit antibodies that recognize human PGHS-1, 5-lipoxygenase, PGE2 synthase, and hematopoietic-type PGD2 synthase (Cayman Chemical, Ann Arbor, MI). The latter antibody also recognizes rat hematopoietic-type PGD2 synthase. After each blot was washed three times with Tris-buffered saline containing 0.1% Tween 20, the treated blot was exposed to Tris-buffered saline (15 ml) containing 5% nonfat milk, 0.1% Tween 20, 0.5% goat serum, and a 1:1000 dilution of a stock solution of horseradish peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad) for 1 h at room temperature. Immunoreactive proteins were visualized using a chemiluminescence kit (Genotech, St. Louis, MO) and BioMax MR film (Eastman Kodak Co.).

       Calcium Ionophore Activation of RasGRP4 and RasGRP4+ HMC-1 Cells

      RasGRP4 and RasGRP4+ HMC-1 cells were washed, suspended at a concentration of 106 cells/ml in calcium/magnesium-free phosphate-buffered saline, and stimulated with 0.5 μmcalcium ionophore A23187 (Sigma) at 37 °C for 30 min as done in other eicosanoid studies of MCs (
      • Razin E.
      • Mencia-Huerta J.M.
      • Lewis R.A.
      • Corey E.J.
      • Austen K.F.
      ). The generated eicosanoids PGD2, PGE2, and leukotriene C4(LTC4) in the supernatants were quantitated using the relevant ELISA kits (Cayman Chemical). Each reaction was read at 450 nm using an ELISA plate reader (Molecular Device). Data are given as mean ± S.D. Significance was defined as p < 0.05 by the Student's t test.

       siRNA-mediated Inhibition of RasGRP4 Expression in RBL-2H3 Cells

      A siRNA approach similar to that described by Elbashiret al. (
      • Elbashir S.M.
      • Harborth J.
      • Lendeckel W.
      • Yalcin A.
      • Weber K.
      • Tuschl T.
      ) was used to evaluate the consequences of decreased expression of RasGRP4 in the rat MC line RBL-2H3. The coding sequence of rat RasGRP4 (
      • Li L.
      • Yang Y.
      • Stevens R.L.
      ) was scanned to identify a gene-specific 21-nucleotide sequence downstream of an “AA” sequence that possesses a 55% GC content. A BLAST search confirmed that the selected sequence (corresponding to residues 27–47 in GenBankTMaccession number AF465263) is not present in another transcript in GenBankTM data bases. The RasGRP4-specific oligonucleotide 5′-GUCUCAUCAGGAAUGCUCUGGdTdT-3′ and its corresponding oligonucleotide 5′-CCAGAGCAUUCCUGAUGAGACdTdT-3′ were synthesized and purified (Dharmacon Research, Lafayette, CO) and then annealed to form the final siRNA duplex with its TT overhangs. The resulting siRNA duplex was introduced into RBL-2H3 cells (line CRL-2256; American Type Culture Collection, Manassas, VA) using a liposome transfection approach. Liposome/siRNA complexes were formed at room temperature using 3 μl of 20 μm siRNA, 2 μl of LipofectAMINETM 2000 (Invitrogen), and 100 μl of Opti-MEM I serum-free culture medium (Invitrogen). The resulting solution was added dropwise to each culture dish containing ∼5 × 104 adherent MCs. The cells were incubated 3–4 h at 37 °C. One ml of serum-enriched medium was then added, and the cells were cultured for an additional 24–48 h. The transiently transfected cells were harvested, and the levels of PGD2 synthase and β-actin protein were measured using the above SDS-PAGE immunoblot approach. In these assays, each protein blot was incubated ∼17 h with anti-PGD2 synthase antibody and then for 1 h with the anti-β-actin antibody (Sigma) before final development.

      RESULTS AND DISCUSSION

      All nontransformed rodent and human MCs that have been examined to date preferentially metabolize arachidonic acid via the cyclooxygenase pathway to PGD2 rather than to PGE2. Nevertheless, Macchia et al. (
      • Macchia L.
      • Hamberg M.
      • Kumlin M.
      • Butterfield J.H.
      • Haeggstrom J.Z.
      ) discovered that HMC-1 cells produce ∼20-fold more PGE2 than PGD2. This surprising finding allowed us to use the c-kit + HMC-1 cell line to further elucidate the intracellular signaling pathways that control PGD2production in MCs. Transcript analysis (Fig. 1) revealed that the failure of HMC-1 cells to generate large amounts of PGD2 is a consequence of a low rate of transcription of the PGD2 synthase gene and/or a high rate of catabolism of its transcript.
      Figure thumbnail gr1
      Figure 1Evaluation of PGD2 synthase mRNA levels in RasGRP4 and RasGRP4+ HMC-1 cells. A, real-time RT-PCR was used to quantitate the levels of PGD2 synthase mRNA in RasGRP4 HMC-1 cells (lane 1) and in HMC-1 cells that were transfected with a RasGRP4-expressing construct on three separate occasions over a six month period (lanes 2–4). The PGD2 synthase (PGDS) mRNA levels in each sample were normalized to that of 18S rRNA. The RNA used in the GeneChip experiment () was obtained from the first transfection experiment (lane 2). B, a separate semiquantitative RT-PCR approach also was used to evaluate the levels of PGD2 synthase mRNA in these same four populations of HMC-1 cells. Gel electrophoresis confirmed that the 100-bp product generated in each case corresponds to PGD2 synthase. Size markers are shown on the left.
      The RasGRP4 transcript was initially cloned from IL-3-developed mouse bone marrow-derived MCs (mBMMCs). While all mouse, rat, and human MCs appear to express RasGRP4 mRNA and/or protein, the amount of RasGRP4 protein in a mouse peritoneal MC greatly exceeds that in a mBMMC as assessed by SDS-PAGE immunoblot analysis.
      L. Li, Y. Yang, and R. L. Stevens, unpublished data.
      Calcium ionophore- or FcεRI-activated mBMMCs produce ∼25-fold more LTC4than PGD2, whereas peritoneal MCs activated in a similar manner produce >40-fold more PGD2 than LTC4(
      • Lewis R.A.
      • Soter N.A.
      • Diamond P.T.
      • Austen K.F.
      • Oates J.A.
      • Roberts L.J.
      ,
      • Razin E.
      • Mencia-Huerta J.M.
      • Lewis R.A.
      • Corey E.J.
      • Austen K.F.
      ). The cumulative data raised the possibility that RasGRP4 regulates arachidonic acid metabolism in MCs. Thus, we evaluated whether or not RasGRP4 controls PGD2 and/or LTC4 expression in HMC-1 and RBL-2H3 cells.
      Comparative transcript analysis of RasGRP4 and RasGRP4+ HMC-1 cells using an Affymetrix GeneChip approach revealed a dramatic difference in the steady-state levels of the transcript that encodes hematopoietic PGD2 synthase in the two populations of cells (Table I). RasGRP4+ HMC-1 cells contained >100-fold more PGD2 synthase mRNA than did the starting population of HMC-1 cells that express nonfunctional forms of RasGRP4. No transcript was induced to a comparable level, including the transcripts that encode brain-type PGD2 synthase and LTC4synthase. Table I shows profile data relating to the levels of the transcripts that encode different proteins that participate in arachidonic acid metabolism. The PGD2 synthase GeneChip data were confirmed by real-time RT-PCR (Fig. 1 A) and by semiquantitative RT-PCR (Fig. 1 B) analyses in three separate populations of RasGRP4-expressing cells. In a control experiment, HMC-1 cells transfected with the expression vector pcDNA3.1 lacking the RasGRP4 cDNA contained barely detectable amounts of PGD2 synthase transcript (data not shown).
      Table IComparative expression of the transcripts that encode 20 proteins that participate in arachidonic acid metabolism in cells
      Transcript and its GenBank™ accession numberAffymetrix ID numberFold change
      The “fold change” values represent the levels of the indicated transcripts in RasGRP4+HMC-1 cells relative to that in RasGRP4− HMC-1 cells. In each case, data were normalized to the ubiquitously expressed transcripts that encode β-actin and glyceraldehyde-3-phosphate dehydrogenase.
      PGD2synthase, hematopoietic (AF150241)35,523107.0
      15-Lipoxygenase, type 1 (M23892)34,6366.2
      Thromboxane A synthase 1 (D34625)33,7774.5
      15-Lipoxygenase, type 2 (U78294)37,4303.4
      12-Lipoxygenase (M62982)35,1241.8
      PGE2 synthase 1 (AF010316)38,1311.8
      PGF synthase/AKR1C3 (D17793)37,3991.4
      Hydroxyprostaglandin dehydrogenase 15-(NAD) (X82460)37,3221.4
      PGH2synthase 2/COX2 (U04636)10691.4
      PGH2 synthase 1/COX1 (M59979)37,9691.3
      5-Lipoxygenase (J03600)3071.2
      LTC4 synthase (U50136)39,9681.2
      PGD2 synthase, brain (M98539)2161.1
      PGI2/prostacylin synthase (D38145)7591.0
      LTB4 hydroxylase (D12620)13051.0
      ATP-binding cassette, subfamily C (CFTR/MRP), member 1 (L05628)18960.9
      5-Lipoxygenase-activating protein/FLAP (AI806222)37,0990.9
      12-Lipoxygenase, 12R type (AF038461)33,0290.9
      LTA4 hydrolase (J03459)38,0810.8
      Microsomal glutathione S-transferase 2 (U77604)820−1.6
      a The “fold change” values represent the levels of the indicated transcripts in RasGRP4+HMC-1 cells relative to that in RasGRP4 HMC-1 cells. In each case, data were normalized to the ubiquitously expressed transcripts that encode β-actin and glyceraldehyde-3-phosphate dehydrogenase.
      Because the levels of a transcript do not always correlate with the levels of its translated product, an SDS-PAGE immunoblot approach was used to compare the levels of PGD2 synthase protein in RasGRP4 and RasGRP4+ HMC-1 cells. The amount of PGD2 synthase protein in RasGRP4 HMC-1 cells was nearly below detection (Fig. 2). In contrast, RasGRP4+HMC-1 cells contained substantial amounts of an intracellular 25-kDa protein that was recognized by the anti-PGD2 synthase antibody. The induced PGD2 synthase transcript is therefore translated and the appropriately sized biosynthetic enzyme accumulates in the transfectants. As assessed by SDS-PAGE immunoblot analysis, RasGRP4 did not induce HMC-1 cells to increase their accumulation of PGHS1, PGE2 synthase, or 5-lipoxgenase protein (Fig. 2). Thus, RasGRP4 induces a selective accumulation of PGD2synthase mRNA and protein in this MC line.
      Figure thumbnail gr2
      Figure 2PGD2 synthase, PGE2synthase, PGHS-1, and 5-lipoxygenase protein levels in RasGRP4 and RasGRP4+HMC-1 cells. SDS-PAGE immunoblots, prepared from the lysates of RasGRP4 and RasGRP4+ HMC-1 cells, were probed with antibodies specific for human PGD2 synthase (PGDS), PGHS-1, PGE2 synthase (PGES), and 5-lipoxygenase. Recombinant human PGD2 synthase (rhPGDS) was used in the former analysis as a positive control.
      As assessed by ELISA, calcium ionophore-activated RasGRP4+HMC-1 cells produced 12–20-fold more PGD2(p < 0.05) than did RasGRP4 HMC-1 cells (Fig. 3). The levels of PGE2 and LTC4 were modestly increased and decreased, respectively, in the calcium ionophore-treated RasGRP4+ cells. However, the variations in the amounts of these eicosanoids were not statistically significant. The fact that HMC-1 cells express nonfunctional forms of RasGRP4 indicates that RasGRP4 is not essential in the early stages of MC development, including the c-kit/KL-mediated proliferation of its progenitors. Nevertheless, the observation that HMC-1 cells are unable to granulate (
      • Yang Y.
      • Li L.
      • Wong G.W.
      • Krilis S.A.
      • Madhusudhan M.S.
      • Šali A.
      • Stevens R.L.
      ) and to produce substantial amounts of PGD2 (Fig. 3) implies that RasGRP4 is required for the efficient expression of the cassette of genes that encode a number of the granule and lipid mediators of MC. The siRNA data obtained from transiently transfected RBL cells (Fig. 4) support this conclusion. RBL cells contain PGD2 synthase protein, and these rat MCs (
      • Seldin D.C.
      • Adelman S.
      • Austen K.F.
      • Stevens R.L.
      • Hein A.
      • Caulfield J.P.
      • Woodbury R.G.
      ) produce substantial amounts of PGD2 when exposed to calcium ionophore (
      • Steinhoff M.M.
      • Lee L.H.
      • Jakschik B.A.
      ). RBL-2H3 cells also contain RasGRP4 mRNA.2 Thus, a siRNA approach was used to evaluate the consequences of decreased expression of RasGRP4 in RBL-2H3 cells. As noted in Fig. 4, inhibition of RasGRP4 expression in the MC line resulted in a transient (12–48 h) inhibition of PGD2synthase expression. As far as we are aware, no one has examined eicosanoid production in transgenic mice that lack RasGRP1 or in cultured cells that have been induced to express varied forms of the other RasGRP family members. Nevertheless, the finding that RasGRP4 regulates PGD2 expression in two populations of cultured MCs raises the possibility that RasGRP1, RasGRP2, and/or RasGRP3 regulate eicosanoid production in other cell types.
      Figure thumbnail gr3
      Figure 3Generation of PGD2, PGE2, and LTC4 in calcium ionophore-activated RasGRP4 and RasGRP4+HMC-1 cells. RasGRP4 and RasGRP4+ HMC-1 cells were exposed to calcium ionophore A23187 for 30 min. The amounts of PGD2 (left bars), PGE2(middle bars), and LTC4 (right bars) generated in each experiment were determined by separate ELISAs.
      Figure thumbnail gr4
      Figure 4PGD2 synthase levels in control and siRNA-treated RBL-2H3 cells. SDS-PAGE immunoblots, prepared from the lysates of RBL-2H3 cells before and after these cells were transfected with a RasGRP4-specific siRNA for 48 h, were probed for with anti-PGD2 synthase (PGDS) and anti-β-actin antibodies. Similar data were obtained in a second siRNA experiment.
      Earlier in vitro studies suggested that KL is required for maximal expression of PGD2 synthase in mouse MCs. HMC-1 cells are able to proliferate in the absence of exogenous human cytokines, because these transformed cells possess an activating mutation in c-kit (
      • Furitsu T.
      • Tsujimura T.
      • Tono T.
      • Ikeda H.
      • Kitayama H.
      • Koshimizu U.
      • Sugahara H.
      • Butterfield J.H.
      • Ashman L.K.
      • Kanayama Y.
      ). The inability of HMC-1 cells to produce large amounts of PGD2 supports the conclusion that RasGRP4 acts downstream of c-kit. Murakami et al. (
      • Murakami M.
      • Matsumoto R.
      • Urade Y.
      • Austen K.F.
      • Arm J.P.
      ) identified a number of cytokines that influence the KL-mediated expression of PGD2 synthase in cultured mouse MCs either in a positive or negative manner. As assessed by GeneChip analysis (data not shown), HMC-1 cells express the transcripts that encode the surface receptors for IL-4, IL-10, IL-13, and KL. This MC line also expresses three distinct receptors that recognize TNF-α and its family members. RasGRP4 and RasGRP4+ HMC-1 cells were therefore cultured for 5 days in the presence of varied combinations of IL-3, IL-4, IL-10, IL-13, KL, and TNF-α. None of these cytokines were able to induce PGD2 synthase expression in RasGRP4 HMC-1 cells (data not shown). In addition, none of these cytokines were able to inhibit the expression of PGD2 synthase in RasGRP4+ HMC-1 cells. These data imply that RasGRP4 is the dominant intracellular signaling protein that controls PGD2 expression in MCs no matter what extracellular cytokine environment this immune cell encounters in tissues.

      Acknowledgments

      We thank Dr. Richard Pratt (Brigham and Women's Hospital, Boston, MA) for helpful discussions regarding GeneChip and real-time RT-PCR analyses.

      REFERENCES

        • Lewis R.A.
        • Soter N.A.
        • Diamond P.T.
        • Austen K.F.
        • Oates J.A.
        • Roberts L.J.
        J. Immunol. 1982; 129: 1627-1631
        • Smith M.M.
        • Shi L.
        • Navre M.
        J. Biol. Chem. 1995; 270: 6440-6449
        • Emery D.L.
        • Djokic T.D.
        • Graf P.D.
        • Nadel J.A.
        J. Appl. Physiol. 1989; 67: 959-962
        • Raible D.G.
        • Schulman E.S.
        • DiMuzio J.
        • Cardillo R.
        • Post T.J.
        J. Immunol. 1992; 148: 3536-3542
        • Monneret G.
        • Li H.
        • Vasilescu J.
        • Rokach J.
        • Powell W.S.
        J. Immunol. 2002; 168: 3563-3569
        • Ward C.
        • Dransfield I.
        • Murray J.
        • Farrow S.N.
        • Haslett C.
        • Rossi A.G.
        J. Immunol. 2002; 168: 6232-6243
        • Murray J.J.
        • Tonnel A.B.
        • Brash A.R.
        • Roberts L.J.
        • Gosset P.
        • Workman R.
        • Capron A.
        • Oates J.A.
        N. Engl. J. Med. 1986; 315: 800-804
        • Liu M.C.
        • Bleecker E.R.
        • Lichtenstein L.M.
        • Kagey-Sobotka A.
        • Niv Y.
        • McLemore T.L.
        • Permutt S.
        • Proud D.
        • Hubbard W.C.
        Am. Rev. Respir. Dis. 1990; 142: 126-132
        • Hardy C.C.
        • Robinson C.
        • Tattersfield A.E.
        • Holgate S.T.
        N. Engl. J. Med. 1984; 311: 209-213
        • Hirata M.
        • Kakizuka A.
        • Aizawa M.
        • Ushikubi F.
        • Narumiya S.
        Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11192-11196
        • Boie Y.
        • Sawyer N.
        • Slipetz D.M.
        • Metters K.M.
        • Abramovitz M.
        J. Biol. Chem. 1995; 270: 18910-18916
        • Hirai H.
        • Tanaka K.
        • Yoshie O.
        • Ogawa K.
        • Kenmotsu K.
        • Takamori Y.
        • Ichimasa M.
        • Sugamura K.
        • Nakamura M.
        • Takano S.
        • Nagata K.
        J. Exp. Med. 2001; 193: 255-261
        • Matsuoka T.
        • Hirata M.
        • Tanaka H.
        • Takahashi Y.
        • Murata T.
        • Kabashima K.
        • Sugimoto Y.
        • Kobayashi T.
        • Ushikubi F.
        • Aze Y.
        • Eguchi N.
        • Urade Y.
        • Yoshida N.
        • Kimura K.
        • Mizoguchi A.
        • Honda Y.
        • Nagai H.
        • Narumiya S.
        Science. 2000; 287: 2013-2017
        • Nagata A.
        • Suzuki Y.
        • Igarashi M.
        • Eguchi N.
        • Toh H.
        • Urade Y.
        • Hayaishi O.
        Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4020-4024
        • Kanaoka Y.
        • Ago H.
        • Inagaki E.
        • Nanayama T.
        • Miyano M.
        • Kikuno R.
        • Fujii Y.
        • Eguchi N.
        • Toh H.
        • Urade Y.
        • Hayaishi O.
        Cell. 1997; 90: 1085-1095
        • Urade Y.
        • Ujihara M.
        • Horiguchi Y.
        • Igarashi M.
        • Nagata A.
        • Ikai K.
        • Hayaishi O.
        J. Biol. Chem. 1990; 265: 371-375
        • Murakami M.
        • Matsumoto R.
        • Urade Y.
        • Austen K.F.
        • Arm J.P.
        J. Biol. Chem. 1995; 270: 3239-3246
        • Suzuki T.
        • Watanabe K.
        • Kanaoka Y.
        • Sato T.
        • Hayaishi O.
        Biochem. Biophys. Res. Commun. 1997; 241: 288-293
        • Mahmud I.
        • Ueda N.
        • Yamaguchi H.
        • Yamashita R.
        • Yamamoto S.
        • Kanaoka Y.
        • Urade Y.
        • Hayaishi O.
        J. Biol. Chem. 1997; 272: 28263-28266
        • Yang Y.
        • Li L.
        • Wong G.W.
        • Krilis S.A.
        • Madhusudhan M.S.
        • Šali A.
        • Stevens R.L.
        J. Biol. Chem. 2002; 277: 25756-25774
        • The Collaborative Study on the Genetics of Asthma
        Nat. Genet. 1997; 15: 389-392
        • Ober C.
        • Tsalenko A.
        • Parry R.
        • Cox N.J.
        Am. J. Hum. Genet. 2000; 67: 1154-1162
        • Dower N.A.
        • Stang S.L.
        • Bottorff D.A.
        • Ebinu J.O.
        • Dickie P.
        • Ostergaard H.L.
        • Stone J.C.
        Nat. Immunol. 2000; 1: 317-321
        • Heid C.A.
        • Stevens J.
        • Livak K.J.
        • Williams P.M.
        Genome Res. 1996; 6: 986-994
        • Winer J.
        • Jung C.K.
        • Shackel I.
        • Williams P.M.
        Anal. Biochem. 1999; 270: 41-49
        • Razin E.
        • Mencia-Huerta J.M.
        • Lewis R.A.
        • Corey E.J.
        • Austen K.F.
        Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 4665-4667
        • Elbashir S.M.
        • Harborth J.
        • Lendeckel W.
        • Yalcin A.
        • Weber K.
        • Tuschl T.
        Nature. 2001; 411: 494-498
        • Li L.
        • Yang Y.
        • Stevens R.L.
        Mol. Immunol. 2002; 38: 1283-1288
        • Macchia L.
        • Hamberg M.
        • Kumlin M.
        • Butterfield J.H.
        • Haeggstrom J.Z.
        Biochim. Biophys. Acta. 1995; 1257: 58-74
        • Seldin D.C.
        • Adelman S.
        • Austen K.F.
        • Stevens R.L.
        • Hein A.
        • Caulfield J.P.
        • Woodbury R.G.
        Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 3871-3875
        • Steinhoff M.M.
        • Lee L.H.
        • Jakschik B.A.
        Biochim. Biophys. Acta. 1980; 618: 28-34
        • Furitsu T.
        • Tsujimura T.
        • Tono T.
        • Ikeda H.
        • Kitayama H.
        • Koshimizu U.
        • Sugahara H.
        • Butterfield J.H.
        • Ashman L.K.
        • Kanayama Y.
        J. Clin. Invest. 1993; 92: 1736-1744