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The Journal of Immunology, 1999, 162: 2087-2094.
Copyright © 1999 by The American Association of Immunologists

Mitogen-Activated Protein Kinase Activation Through Fc{epsilon} Receptor I and Stem Cell Factor Receptor Is Differentially Regulated by Phosphatidylinositol 3-Kinase and Calcineurin in Mouse Bone Marrow-Derived Mast Cells1

Tamotsu Ishizuka*,{dagger}, Kosuke Chayama*,{dagger}, Katsuyuki Takeda*,{dagger}, Eckard Hamelmann*,{dagger}, Naohiro Terada*,{dagger}, Gordon M. Keller{ddagger}, Gary L. Johnson*,{dagger} and Erwin W. Gelfand2,*,{dagger}

* Division of Basic Sciences, Department of Pediatrics, {dagger} Program in Molecular Signal Transduction, and {ddagger} Division of Immunology, Department of Medicine, National Jewish Medical and Research Center, Denver, CO 80206


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Aggregation of high affinity FcR for IgE (Fc{epsilon}RI) on mast cells activates intracellular signal transduction pathways, including the activation of protein tyrosine kinases, phosphatidylinositol 3-kinase (PI3-kinase), and protein kinase C. Binding of stem cell factor (SCF) to its receptor (SCFR, c-Kit) on mast cells also induces increases in intrinsic tyrosine kinase activity and activation of PI3-kinase. Although ligation of both receptors induces Ras and Raf-1 activation, the downstream consequences of these early activation events are not well defined, except for the activation of extracellular signal-regulated kinases (ERK). Addition of Ag (OVA) to mouse bone marrow-derived mast cells (BMMC) sensitized with anti-OVA IgE triggers the activation of three members of the mitogen-activated protein (MAP) kinase family, c-Jun amino-terminal kinase (JNK), p38 MAP kinase (p38), and extracellular signal-regulated kinases. SCF similarly activates all three MAP kinases. Wortmannin, an inhibitor of PI3-kinase, inhibited both Fc{epsilon}RI- and SCFR-mediated JNK activation and partially inhibited Fc{epsilon}RI, but not SCFR-mediated p38 activation. Cyclosporin A inhibited Fc{epsilon}RI-mediated JNK and p38 activation, but did not affect the activation of these kinases when stimulated through the SCFR. Wortmannin and cyclosporin A inhibited Fc{epsilon}RI-mediated production of TNF-{alpha} and IL-4 in addition to serotonin release in BMMC. These results indicate that both PI3-kinase and calcineurin may contribute to the regulation of cytokine gene transcription and the degranulation response by modulating JNK activity in BMMC.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Mast cells play a central role in inflammatory and immediate allergic responses. Aggregation of the high affinity FcR for IgE (Fc{epsilon}RI) triggers the activation of different signal transduction pathways. Activation of protein tyrosine kinases (PTKs),3 including Syk, Lyn, Btk, and Itk, is one of the earliest signaling events induced by aggregation of the Fc{epsilon}RI on mast cells 1, 2, 3, 4, 5 . PTK activation is thought to be proximal to the activation of phospholipase C{gamma} and protein kinase C, and appears to be essential for mast cell degranulation 6 since PTK inhibitors prevent the liberation of inositol trisphosphate and histamine release 7, 8 . In addition to the release of mast cell granule contents, these pathways lead to later responses, such as the modulation of cytokine gene expression. However, the downstream consequences of these early activation events are not well defined. We have shown recently that three members of the mitogen-activated protein (MAP) kinase family, designated c-Jun amino-terminal kinase (JNK), p38 MAP kinase (p38), and extracellular signal-regulated kinase (ERK), are activated following Fc{epsilon}RI aggregation in a mouse mast cell line, MC/9 9, 10 . Stem cell factor (SCF), the ligand for the Kit tyrosine kinase receptor (SCFR) encoded by the c-Kit proto-oncogene, also plays an important role in the development of mast cells and hemopoiesis 11, 12, 13 . In this study, we show that the same three MAP kinase family members are activated through both Fc{epsilon}RI and SCFR in mouse bone marrow-derived mast cells (BMMC), that the activation of these kinases is differentially regulated by upstream proteins such as PI3-kinase and calcineurin, and both PI3-kinase- and calcineurin-dependent pathways play an important role in the regulation of cytokine production and the degranulation response.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and reagents

Bone marrow was obtained from the femurs of female BALB/c mice (The Jackson Laboratory, Bar Harbor, ME) and cultured in Iscove’s modified Dulbecco’s medium (IMDM; Life Technologies, Grand Island, NY) supplemented with 5% FBS (Summit Biotechnology, Ft. Collins, CO), 50 µM 2-ME (Life Technologies), 2 mM glutamine, 100 µg/ml streptomycin, 100 U/ml penicillin, 0.5 µg/ml amphotericin B, and IL-3 obtained from medium conditioned by X63 AG8-653 myeloma cells transfected with a vector expressing IL-3 14 . After 4 wk of culture, more than 95% of nonadherent cells contained granules that stained positively with toluidine blue. Wortmannin and rapamycin were purchased from Calbiochem (San Diego, CA). Cyclosporin A (CsA) and cyclosporin H (CsH) were provided by Sandoz Pharma (Basel, Switzerland), and FK506 was provided by Fujisawa Pharmaceutical (Osaka, Japan). The MEK1 inhibitor, PD98059, was purchased from New England Biolabs (Beverly, MA). Bovine myelin basic protein was obtained from Upstate Biotechnology (Lake Placid, NY). Goat polyclonal anti-ERK2 (C-14) Ab and anti-Akt1 (C-20) Ab were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Recombinant protein G agarose was purchased from Zymed Laboratories (San Francisco, CA). Kit ligand (SCF) was obtained from medium conditioned by CHO cells transfected with a kit ligand expression vector (kindly provided by Genetics Institute, Cambridge, MA). Medium conditioned by nontransfected CHO cells was used as a control and showed no activity in inducing kinase activation. Mouse rTNF-{alpha}, mouse rIL-4, purified rat anti-mouse TNF-{alpha} mAb, purified rat anti-mouse IL-4 mAb (ELISA capture), biotinylated rabbit anti-mouse TNF-{alpha} polyclonal Ab, and biotinylated rat anti-mouse IL-4 mAb (ELISA detection) were purchased from PharMingen (San Diego, CA). The PKA inhibitor (TTYADFIASGRTGRRNAIHD) and Crosstide (GRPRTSSFAEG) were made in the Molecular Resource Center, National Jewish Medical and Research Center (Denver, CO).

Passive sensitization and stimulation of BMMC

BMMC (5 x 106/ml) were cultured with 500 ng/ml anti-OVA IgE 15 for 2 h. This concentration of IgE was shown in initial experiments to be optimal in stimulation of JNK in BMMC. The cells were washed with medium three times and cultured with fresh medium for an additional 2 h. OVA dissolved in PBS was added to the passively sensitized cells or PBS was used as a control. In some experiments, BMMC (3 x 106/ml) were incubated with fresh medium for 2 h and SCF was added to the medium in a final volume of 1%. This concentration of conditioned medium was shown in initial studies to lead to optimal activation of JNK and was comparable with 100 ng/ml rSCF 9 .

Activity of JNK

Glutathione S-transferase-c-Jun 1–79(1–79) fusion protein was prepared as described previously 16 , and kinase activity was measured as described 9, 10 .

Kinase assay of p38 MAP kinase

p38 kinase activity was assayed as described using ATF-2 as substrate and quantified with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) 10 .

Kinase assay of ERK2

In vitro kinase assay of ERK2 was conducted as described previously 17 using myelin basic protein as substrate.

Kinase assay of Akt1

Cells (3 x 106) were lysed in a buffer (10 mM KPO4 (pH 7.4), 0.1% Nonidet P-40, 1 mM EDTA, 5 mM EGTA, 10 mM MgCl2, 20 mM ß-glycerophosphate, 0.5 mM Na3VO4, 2 mM DTT, 1 mM PMSF, 10 µg/ml aprotinin, and 5 mg/ml leupeptin). The lysates were incubated with 0.8 µg goat anti-Akt1 Ab for 2 h at 4°C. Recombinant protein G agarose was added to the lysates and incubated for an additional 1 h at 4°C. The immunoprecipitates were washed twice with lysis buffer, and once with kinase buffer (20 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 0.1 mg/ml BSA, and 1 mM DTT). After the final wash, 50 ml of a kinase assay buffer containing 5 µCi of [{gamma}-32P]ATP (DuPont, Wilmington, DE), 100 µM cold ATP, 1 µg/ml PKA inhibitor, and 5 µg Crosstide 18 was added per sample. The samples were incubated for 15 min at 30°C and the reaction was stopped by adding 10 µl of 0.5 M EDTA. A total of 25 µl of each sample was loaded on phosphocellulose paper (Whatman, Clifton, NJ), and phosphorylation of Crosstide was determined by liquid scintillation counting. The phosphocellulose paper was washed five times with 0.425% (v/v) phosphoric acid.

Assay for serotonin release

BMMC (2 x 106/ml) were cultured in medium containing 1 µCi/ml 5-[1,2-3H](N)-hydroxytryptamine creatinine sulfate (DuPont) and cultured for 15 h. After washing, cells (5 x 106/ml) were cultured with 500 ng/ml anti-OVA IgE for 2 h and washed three times with culture medium. BMMC (1 x 106/ml) were incubated in medium for 2 h and stimulated by addition of OVA. The reaction was stopped by adding ice-cold medium and centrifugation. After centrifugation, supernatants and cell pellets were loaded in the scintillation counter. The percentage of release was calculated by dividing the net supernatant counts by the total counts.

ELISA for TNF-{alpha} and IL-4

Purified rat anti-mouse TNF-{alpha} mAb or purified rat anti-mouse IL-4 mAb was diluted to 2 µg/ml or 1 µg/ml in coating solution (0.1 M NaHCO3, pH 8.2), and 50 µl was added to wells of an ELISA plate (Dynatech Laboratories, Chantilly, VA). After overnight incubation at 4°C, wells were washed twice with washing solution (0.05% Tween-20/PBS) and blocked with PBS containing 10% FCS at room temperature for 2 h. After washing two times, standards (30 pg/ml-2 ng/ml mouse rTNF-{alpha} or 40 pg/ml-2.5 ng/ml mouse rIL-4) and samples were added at 100 µl/well and incubated overnight at 4°C. After washing four times, biotinylated rabbit anti-mouse TNF-{alpha} polyclonal Ab (1 µg/ml) or biotinylated rat anti-mouse IL-4 mAb (0.5 µg/ml) was added to the wells and incubated at room temperature for 45 min, and the wells were washed six times. Avidin-peroxidase (2 µg/ml) was added to the wells and incubated at room temperature for 30 min, and the wells were washed eight times. 2,2'-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS, 30 mg/ml 0.1 M citric acid, pH 4.35) containing 0.03% H2O2 was added at 100 µl/well, and the color reaction was allowed to develop at room temperature for 30 min. The plates were read at OD 410 nm and analyzed by Microplate Manager (Bio-Rad, Hercules, CA).

Statistical analysis

Values were compared by Student’s t test or Welch’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Aggregation of Fc{epsilon}RI and ligation of SCFR on BMMC stimulate JNK, p38, and ERK2 activation

BMMC previously sensitized with anti-OVA IgE were challenged in the presence or absence of OVA. Fig. 1Go, A–C, shows that addition of 10 µg/ml OVA induced the activation of three members of the MAP kinase family, JNK, p38, and ERK2. Similarly, addition of SCF (1%) to BMMC induced the activation of JNK, p38, and ERK2 (Fig. 1Go, D–F). JNK activity reached maximum levels at 15 min, p38 activity at 5 min, and ERK2 activity at 5–15 min after the addition of either OVA or SCF. These data indicate that aggregation of Fc{epsilon}RI receptors or signaling through the SCFR rapidly activates all three MAP kinases in BMMC.



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  		FIGURE 1. Aggregation of Fc{epsilon}RI and ligation of SCFR stimulates MAP kinases in BMMC. BMMC sensitized with anti-OVA IgE (3 x 106 cells) were stimulated by addition of 10 µg/ml OVA. JNK (A), p38 (B), and ERK2 (C) activities were measured at 0, 1, 5, 15, 30, 45, and 60 min after addition of OVA. BMMC were stimulated by addition of SCF (1%), and JNK (D), p38 (E), and ERK2 (F) activities were measured 0, 1, 5, 15, 30, 45, and 60 min later. Representative autoradiographs from three independent experiments are shown. Equal loading of the gels was verified by immunoblotting with anti-p38 or anti-ERK2.

 
Akt1 is activated by aggregation of Fc{epsilon}RI and ligation of SCFR in BMMC

The Akt proto-oncogene encodes a serine/threonine kinase, Akt1 19 , which is rapidly and specifically activated by growth factors such as platelet-derived growth factor (PDGF) 20 . It is well known that Akt1 activation is mediated through PI3-kinase signaling 21 and Akt1 activation is predicted to parallel PI3-kinase activity. Akt1 was activated both by aggregation of Fc{epsilon}RI and ligation of the SCFR (Fig. 2Go). SCFR-induced Akt1 activation was stronger than Fc{epsilon}RI-mediated activation in BMMC. Activation was maximal 1–5 min after addition of the ligand (Fig. 2GoA). Under both conditions, Akt1 activation was inhibited by wortmannin, a PI3-kinase inhibitor 22 , in a dose-dependent manner (Fig. 2GoB). These results confirm that PI3-kinase is activated following aggregation of Fc{epsilon}RI and ligation of the SCFR in mast cells 23 .



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  		FIGURE 2. Aggregation of Fc{epsilon}RI and ligation of SCFR stimulate Akt1 in BMMC. A, BMMC sensitized with anti-OVA IgE (3 x 106 cells) were stimulated by addition of 10 µg/ml OVA. Similarly, BMMC (3 x 106 cells) were stimulated by addition of 1% (v/v) SCF. Akt1 activities were measured at 0, 1, 5, 15, and 30 min after stimulation. Akt1 activities are shown as fold increases over basal activation (at 0 min). B, BMMC sensitized with anti-OVA IgE (3 x 106 cells) or nonsensitized BMMC (3 x 106 cells) were stimulated for 5 min by addition of 10 µg/ml OVA or 1% (v/v) SCF after 15-min preincubation with wortmannin or control vehicle (0.01% DMSO). Results are from four independent experiments (mean ± SE). *, p < 0.05; **, p < 0.01.

 
Effects of wortmannin on JNK, p38, and ERK2 activation in BMMC

Addition of wortmannin demonstrated the sensitivity of JNK activation via Fc{epsilon}RI in BMMC; inhibition of JNK was dose dependent, and in the presence of 100 nM wortmannin, JNK activation was inhibited by 90% (Fig. 3GoA). Wortmannin also inhibited p38 activation via Fc{epsilon}RI in BMMC in a dose-dependent manner, although the degree of inhibition in p38 activity was less (50%) than observed for JNK activation (Fig. 3GoB). Wortmannin failed to inhibit ERK2 activation via Fc{epsilon}RI in BMMC (Fig. 3GoC). These results indicate that Fc{epsilon}RI-mediated PI3-kinase activation is involved in JNK activation, and to some extent, p38 activation in BMMC, whereas ERK activation is independent. Virtually identical results were obtained previously in MC/9 cells 9, 10 . Preincubation of BMMC with wortmannin before the addition of SCF revealed that JNK activation was inhibited in a dose-dependent manner, similar to activation of JNK through Fc{epsilon}RI, but SCF-induced activation of p38 and ERK2 was insensitive in these mast cells (Fig. 3Go, D–F).



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  		FIGURE 3. Effects of wortmannin on Fc{epsilon}RI or SCFR-mediated activation of MAP kinases in BMMC. BMMC sensitized with anti-OVA IgE (3 x 106 cells) were incubated for 15 min in the presence of 1–1000 nM wortmannin or control vehicle (0.01% DMSO), and stimulated for 5 min (p38, ERK2) or 15 min (JNK) after addition of 10 µg/ml OVA. A, Wortmannin inhibited Fc{epsilon}RI-mediated JNK activation in a dose-dependent manner (90% inhibition at 100 nM). B, Wortmannin inhibited Fc{epsilon}RI-mediated p38 activation in a dose-dependent manner (50% inhibition at 100 nM). C, Wortmannin did not inhibit Fc{epsilon}RI-mediated ERK2 activation. BMMC were stimulated by addition of SCF in the presence of wortmannin. D, Wortmannin inhibited SCF-mediated JNK activation in a dose-dependent manner (90% inhibition at 100 nM). Wortmannin did not inhibit SCF-mediated p38 (E) or ERK2 (F) activation. Representative autoradiographs from three independent experiments are shown. Equal loading of the gels was verified by immunoblotting with anti-p38 or anti-ERK2.

 
Effects of CsA on JNK, p38, and ERK2 activation in BMMC

CsA is known to affect mast cell function, particularly the degranulation response and cytokine production 24, 25, 26, 27 . Addition of CsA (1 µg/ml) strongly inhibited JNK activation (80% inhibition) and partially inhibited p38 activation (40% inhibition) through Fc{epsilon}RI; ERK2 activation via Fc{epsilon}RI was not affected. CsH, a derivative of CsA that does not bind cyclophilin A or B and is not immunosuppressive 28, 29, 30, 31 , did not affect JNK activation (Fig. 4GoA). FK506 and RAP similarly did not affect JNK activation. It is known that BMMC lack FKBP12 32 , and since binding to FKBP12 is required for FK506 to inhibit calcineurin 33 , the absence of the protein accounts for the lack of FK506-inhibitory activity. By contrast, CsA did not affect SCF-induced JNK, p38, or ERK2 activation (Fig. 4Go, D–F).



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  		FIGURE 4. Effects of cyclosporin A on Fc{epsilon}RI or SCFR-mediated activation of MAP kinases in BMMC. BMMC sensitized with anti-OVA IgE (3 x 106 cells) were incubated for 15 min in the presence of 1 µg/ml CsA, 1 µg/ml CsH, 10 ng/ml FK506, 10 ng/ml RAP, or control vehicle (0.01% ethanol) and stimulated for 5 min (p38, ERK2) or 15 min (JNK) after addition of 10 µg/ml OVA. A, CsA, but not CsH, FK506, or RAP, significantly inhibited Fc{epsilon}RI-mediated JNK activation (80% inhibition). B, 1 µg/ml CsA partially inhibited Fc{epsilon}RI-mediated p38 activation (40% inhibition) (C), but not ERK2 activation. BMMC were stimulated by addition of SCF in the presence of 1 µg/ml CsA or control vehicle. CsA did not affect SCF-mediated JNK (D), p38 (E), or ERK2 (F) activation. Representative autoradiographs from three independent experiments are shown. Equal loading of the gels was verified by immunoblotting using anti-p38 and anti-ERK2.

 
Serotonin release from Ag-stimulated BMMC is inhibited by wortmannin, CsA, and PD98059

It has been shown that wortmannin inhibits mast cell degranulation at the same concentration in which inhibition of PI3-kinase is observed 34, 35 . Wortmannin inhibited Fc{epsilon}RI-mediated serotonin release from BMMC in a dose-dependent manner; at a concentration of 100 nM wortmannin, serotonin release was inhibited by more than 75% (Fig. 5GoA). CsA also inhibits Fc{epsilon}RI-mediated degranulation in rat basophilic leukemia cells and basophils 24, 25, 26 , and a role for calcineurin in the degranulation process has been proposed 36 . CsA (1 µg/ml) partially (56%) but significantly inhibited Fc{epsilon}RI-mediated serotonin release in these BMMC. In concert with the data on kinase activation, FK506 and RAP did not affect mast cell degranulation in BMMC (Fig. 5GoB). The MEK1 inhibitor PD98059 37, 38 also inhibited serotonin release and ERK2 activation in Ag-stimulated BMMC in a dose-dependent fashion (Fig. 5Go, C and D). These data imply that MEK1 and ERK2 activation may be involved in mast cell degranulation; however, the exact mechanism whereby PD98059 inhibits mast cell degranulation is not defined.



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  		FIGURE 5. Serotonin release from Ag-stimulated BMMC is inhibited by wortmannin, cyclosporin A, and PD98059. A, BMMC sensitized with anti-OVA IgE (1 x 106 cells) were stimulated by addition of 10 µg/ml OVA after a 15-min preincubation with wortmannin or control vehicle. Serotonin release was measured at 30 min after stimulation. Wortmannin inhibited Fc{epsilon}RI-mediated serotonin release in a dose-dependent manner (mean ± SD from four independent experiments; *, p < 0.05; **, p < 0.01). B, BMMC sensitized with anti-OVA IgE (1 x 106 cells) were stimulated by addition of 10 µg/ml OVA after a 15-min preincubation with 1 µg/ml CsA, 10 ng/ml FK506, 10 ng/ml RAP, or control vehicle (0.01% ethanol). Serotonin release was measured at 30 min after stimulation. CsA, but not FK506 or RAP, significantly inhibited Fc{epsilon}RI-mediated serotonin release (50% inhibition) (mean ± SD from four independent experiments; **, p < 0.01). C, BMMC sensitized with anti-OVA IgE (1 x 106 cells) were stimulated by addition of 10 µg/ml OVA after a 60-min preincubation with PD98059 or control vehicle (0.1% DMSO). Serotonin release was measured at 30 min after stimulation. PD98059 significantly inhibited Fc{epsilon}RI-mediated serotonin release (mean ± SD from four independent experiments; *, p < 0.01). D, BMMC sensitized with anti-OVA IgE were stimulated for 5 min by addition of 10 µg/ml OVA after a 60-min preincubation with PD98059 or control vehicle (0.1% DMSO). In vitro kinase assay of ERK2 was performed as described in Materials and Methods. PD98059 (30 µM) inhibited Fc{epsilon}RI-mediated ERK2 activation. A representative autoradiograph from three independent experiments is shown. Equal loading of the gel was verified by immunoblotting using anti-ERK2.

 
CsA and wortmannin, but not PD98059, inhibit both TNF-{alpha} and IL-4 production following the aggregation of Fc{epsilon}RI on BMMC

BMMC sensitized with anti-OVA IgE were incubated with 10 µg/ml OVA for 3 h, and cytokine secretion in the medium was measured by ELISA. Aggregation of Fc{epsilon}RI on BMMC induced both TNF-{alpha} and IL-4 production (TNF-{alpha}, 1.44 ± 0.44 ng/106 cells; IL-4, 1.14 ± 0.80 ng/106 cells). As shown in Fig. 6GoA, wortmannin inhibited both TNF-{alpha} and IL-4 production in a dose-dependent manner; the production of IL-4 appeared to be more sensitive to the drug. CsA, but not FK506 and RAP, inhibited both TNF-{alpha} and IL-4 production in a similar manner (Fig. 6Go, B and C). The inhibition by CsA was complete at 1 µg/ml, suggesting that inhibition of calcineurin completely blocks cytokine production in BMMC. In contrast, addition of the MEK1 inhibitor failed to alter TNF-{alpha} or IL-4 production (Fig. 6GoD), although it significantly inhibited ERK2 activation (Fig. 5GoD). These data not only indicate the role of calcineurin in BMMC cytokine production, but also imply the absence of a role for ERK or MEK1 activation in TNF-{alpha} or IL-4 production in these cells. Addition of SCF to BMMC did not trigger any release of IL-4 or TNF-{alpha}.



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  		FIGURE 6. Wortmannin and CsA inhibit Fc{epsilon}RI-mediated TNF-{alpha} and IL-4 production in BMMC. A, BMMC sensitized with anti-OVA IgE (1 x 106 cells) were stimulated by addition of 10 µg/ml OVA after a 15-min preincubation with wortmannin or control vehicle (0.01% DMSO). Supernatants were harvested 3 h after stimulation, and TNF-{alpha} and IL-4 in the medium were measured by ELISA. Measurements were normalized to levels of stimulated cells cultured in the presence of 0.01% DMSO. Wortmannin inhibited both TNF-{alpha} and IL-4 production in Ag-stimulated BMMC in a dose-dependent manner (mean ± SD from six independent experiments; *, p < 0.05; **, p < 0.01). B and C, BMMC sensitized with anti-OVA IgE (1 x 106 cells) were stimulated by addition of 10 µg/ml OVA after a 15-min preincubation with 1 µg/ml CsA, 10 ng/ml FK506, 10 ng/ml RAP, or control vehicle (0.01% ethanol). Supernatants were harvested 3 h after stimulation. Measurements were normalized to the values of stimulated cells cultured in medium containing 0.01% ethanol. CsA, but not FK506 or RAP, strongly inhibited Fc{epsilon}RI-mediated TNF-{alpha} and IL-4 production in BMMC (mean ± SD from six independent experiments; *, p < 0.05; **, p < 0.01). D, BMMC sensitized with anti-OVA IgE (1 x 106 cells) were stimulated by addition of 10 µg/ml OVA after a 60-min preincubation with PD98059 or control vehicle (0.1% DMSO). Supernatants were harvested 3 h after stimulation. Measurements were normalized to values of stimulated cells cultured in the presence of 0.1% DMSO. PD98059 did not affect Fc{epsilon}RI-mediated TNF-{alpha} and IL-4 production in BMMC (mean ± SD from six independent experiments).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Mast cells are responsible for the initiation of immediate hypersensitivity responses in allergic diseases in large part because they release preformed mediators such as histamine and generate lipid mediators. It is also known that mast cells produce cytokines in rapid response to Ag cross-linking of IgE bound to Fc{epsilon}RI, and that release of a number of cytokines, including TNF-{alpha} and IL-4, may play major roles in triggering and sustaining the allergic inflammatory response 39 . However, little is known about the signal transduction pathways, other than the role of calcineurin/NF-AT, that regulate cytokine gene expression in mast cells. Clues are evident from the promoters for the TNF-{alpha} and IL-4 genes, which contain nuclear factor-{kappa}B-, AP-1-, AP-2-, NF-AT-, Ets-, AP-1/ATF-, NF-AT/AP-1, and c-Maf-related elements, implicating additional signal transduction pathways that regulate cytokine gene expression 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 .

In our previous studies, we demonstrated aggregation of Fc{epsilon}RI resulted in the rapid activation of JNK, p38, and ERK2 in the mouse mast cell line, MC/9 9, 10 . In MC/9 cells, wortmannin, at concentrations that inhibit PI3-kinase activity 19, 50, 51, 52, 53, 54 , strongly inhibited JNK and partially inhibited p38 activation, but not ERK2 activation 9, 10 . As shown in this study, these three members of the MAP kinase family were also activated following aggregation of Fc{epsilon}RI of BMMC. The effects of wortmannin on Fc{epsilon}RI-mediated JNK, p38, and ERK2 activation in BMMC were virtually identical to those observed in MC/9 cells 9, 10 . Mechanistically, these results indicate that there is an early separation in the signaling pathways activated through Fc{epsilon}RI to differentially regulate the ERK, p38, and JNK sequential protein kinase pathways.

Signaling through the tyrosine kinase receptor, SCFR, also resulted in activation of these three MAP kinase family members in BMMC. Wortmannin inhibited SCFR-induced JNK activation, but did not affect p38 or ERK2 activation in BMMC (Figs. 2Go and 7Go). The effects of wortmannin on SCFR-induced JNK activation in BMMC contrast with the resistance demonstrated in MC/9 cells 55 . These results suggest that in BMMC, PI3-kinase activation is required for JNK activation through either Fc{epsilon}RI or SCFR, and that, at least in MC/9 cells, the SCFR can utilize a wortmannin-insensitive pathway to activate JNK. One of the downstream events following PI3-kinase is the activation of Akt1 20, 21 . As shown in this study, addition of Ag to passively sensitized cells or SCF stimulated Akt1 activity, and this activity was completely blocked in the presence of wortmannin in BMMC.



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  		FIGURE 7. Effects of wortmannin and CsA on Fc{epsilon}RI- and SCFR-mediated activation of MAP kinases in BMMC.

 
Addition of the immunosuppressant CsA inhibited Fc{epsilon}RI-mediated JNK activation and partially inhibited p38 activation without affecting ERK2 activation. Another inhibitor of calcineurin activation, FK506, did not affect Fc{epsilon}RI-mediated JNK activation in BMMC, almost certainly due to our demonstration that BMMC do not express the binding protein FKBP12 (data not shown). In T cells, CsA has been demonstrated to inhibit cytokine production by inhibiting calcineurin activity and the nuclear translocation of NF-AT 56 . However, based on the data presented in this work and our studies of MC/9 cells 55 , CsA can also inhibit the Fc{epsilon}RI-mediated induction of JNK activation and JNK-activated transcription factors. These results indicate an additional mechanism whereby CsA can regulate cytokine production in mast cells. In contrast to Fc{epsilon}RI signaling, SCFR-induced activation of JNK and p38 was insensitive to CsA, further indicating that signaling through Fc{epsilon}RI and SCFR can utilize different upstream pathways for the activation of JNK and p38.

CsA inhibits the Fc{epsilon}RI-mediated degranulation of rat basophilic leukemia cells and human basophils without affecting phosphatidylinositol hydrolysis or Ca2+ fluxes 30 . Similarly, CsA, but not FK506, inhibited serotonin release in BMMC. CsA did not affect Fc{epsilon}RI-mediated Ca2+ fluxes in these BMMC (data not shown). Although the mechanism underlying the inhibition of the degranulation response remains to be defined, the inhibition of calcineurin is suspected to play a role 36 . The inhibitor PD98059 also abolished Fc{epsilon}RI-mediated degranulation, suggesting that MEK1 and ERK activation are involved in the degranulation response. PD98059, on the other hand, had no effect on cytokine production, indicating that MEK1 or ERK2 activation is not required for this response. These data clearly distinguish the downstream signaling requirements for the degranulation response and cytokine production following Fc{epsilon}RI aggregation.

We previously showed that the wortmannin-sensitive pathways play an important role in TNF-{alpha} production in MC/9 cells 10 . It appears that calcineurin and NF-AT, the MEK kinase/JNK kinase/JNK pathway, and PI3-kinase may play a role in the synthesis and secretion of cytokines such as TNF-{alpha} and IL-4 in response to the activation through Fc{epsilon}RI in BMMC. In addition to cytokine production, wortmannin also inhibited Fc{epsilon}RI-mediated degranulation, as previously reported 34, 35 . Thus, the activation of PI3-kinase is important for both the secretion of preformed mediators and the induction of cytokine synthesis. However, neither activation of PI3-kinase alone nor JNK alone is sufficient to induce cytokine production, since addition of SCF, which can trigger both of these responses, cannot induce TNF-{alpha} or IL-4 production (data not shown). SCF induced little serotonin release (2% net release) or consistent arachidonic acid release (6% net release) at the concentrations used to activate JNK. Our findings define the importance of the calcineurin and PI3-kinase pathways in IgE-mediated signaling through Fc{epsilon}RI in BMMC.


   Acknowledgments
 
We thank Drs. Akihiro Oshiba, Gregory Cieslewicz, Anthony Joetham, and Saiphone Webb for their assistance.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants HL-36577 (E.W.G.), AI-42246 and DK-37871 (G.L.J.). Back

2 Address correspondence and reprint requests to Dr. Erwin W. Gelfand, Department of Pediatrics, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206. E-mail address: Back

3 Abbreviations used in this paper: PTK, protein tyrosine kinase; BMMC, bone marrow-derived mast cell; CsA, cyclosporin A; CsH, cyclosporin H; ERK, extracellular signal-regulated kinase; JNK, c-Jun amino-terminal kinase; MAP, mitogen-activated protein; MEK, MAP/ERK kinase; NF-AT, NF of activated T cells; PI3, phosphatidylinositol 3; RAP, rapamycin; SCF, stem cell factor. Back

Received for publication July 9, 1998. Accepted for publication November 11, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Eiseman, E., J. B. Bolen. 1992. Engagement of the high-affinity IgE receptor activates src protein-related tyrosine kinase. Nature 355:78.[Medline]
  2. Hutchcroft, J. E., R. L. Geahlen, G. G. Deanin, J. M. Oliver. 1992. Fc{epsilon}RI-mediated tyrosine phosphorylation and activation of the 72-kDa protein-tyrosine kinase, PTK72, in RBL-2H3 rat tumor mast cells. Proc. Natl. Acad. Sci. USA 89:9107.[Abstract/Free Full Text]
  3. Shiue, L., J. Green, O. M. Green, J. L. Karas, J. P. Morgenstern, M. K. Ram, M. K. Taylor, M. J. Zoller, L. D. Zydowsky, J. B. Bolen, J. S. Brugge. 1995. Interaction of p72syk with the {gamma} and ß subunits of the high-affinity receptor for immunoglobulin E, Fc{epsilon}RI. Mol. Cell. Biol. 15:272.[Abstract]
  4. Kawakami, Y., L. Yao, T. Miura, S. Tsukada, O. N. Witte, T. Kawakami. 1994. Tyrosine phosphorylation and activation of Bruton tyrosine kinase upon Fc{epsilon}RI cross-linking. Mol. Cell. Biol. 14:5108.[Abstract/Free Full Text]
  5. Kawakami, Y., L. Yao, M. Tashiro, S. Gibson, G. M. Mills, T. Kawakami. 1997. Activation and interaction with protein kinase C of a cytoplasmic tyrosine kinase, Itk/Tsk/Emt, on Fc{epsilon}RI cross-linking on mast cells. J. Immunol. 155:3556.[Abstract]
  6. Ozawa, K., Z. Szallasi, M. G. Kazanietz, P. M. Blumberg, H. Mischak, J. F. Mushinski, M. A. Beaven. 1993. Ca2+-dependent and Ca2+-independent isozymes of protein kinase C mediate exocytosis in antigen-stimulated rat basophilic RBL-2H3 cells: reconstitution of secretory responses with Ca2+ and purified isozymes in washed permeabilized cells. J. Biol. Chem. 268:1749.[Abstract/Free Full Text]
  7. Kawakami, T., N. Inagaki, M. Takei, H. Fukamachi, K. M. Coggeshall, K. Ishizaka, T. Ishizaka. 1992. Tyrosine phosphorylation is required for mast cell activation by Fc{epsilon}RI cross-linking. J. Immunol. 148:3513.[Abstract]
  8. Oliver, J. M., D. L. Burg, B. S. Wilson, J. L. McLaughlin, R. L. Geahlen. 1994. Inhibition of mast cell Fc{epsilon}RI-mediated signaling and effector function by the Syk-selective inhibitor, piceatannol. J. Biol. Chem. 269:29697.[Abstract/Free Full Text]
  9. Ishizuka, T., A. Oshiba, N. Sakata, N. Terada, G. L. Johnson, E. W. Gelfand. 1996. Aggregation of the Fc{epsilon}RI on mast cells stimulates c-Jun amino-terminal kinase activity: a response inhibited by wortmannin. J. Biol. Chem. 271:12762.[Abstract/Free Full Text]
  10. Ishizuka, T., N. Terada, P. Gerwins, E. Hamelmann, A. Oshiba, G. R. Fanger, G. L. Johnson, E. W. Gelfand. 1997. Mast cell tumor necrosis factor {alpha} production is regulated by MEK kinases. Proc. Natl. Acad. Sci. USA 94:6358.[Abstract/Free Full Text]
  11. Tsai, M., T. Takeishi, H. Thompson, K. E. Langley, K. M. Zsebo, D. D. Metcalfe, E. N. Geissler, S. J. Galli. 1991. Induction of mast cell proliferation, maturation, and heparin synthesis by the rat c-kit ligand, stem cell factor. Proc. Natl. Acad. Sci. USA 88:6382.[Abstract/Free Full Text]
  12. Tsai, M., L.-S. Shih, G. F. J. Newlands, T. Takeishi, K. E. Langley, K. M. Zsebo, H. R. P. Miller, E. N. Geissler, S. J. Galli. 1991. The rat c-kit ligand, stem cell factor, induces the development of connective tissue-type and mucosal mast cells in vivo: analysis by anatomical distribution, histochemistry, and protease phenotype. J. Exp. Med. 174:125.[Abstract/Free Full Text]
  13. Zsebo, K. M., D. A. Williams, E. N. Geissler, V. C. Broudy, F. H. Martin, H. L. Atkins, R.-Y. Hsu, N. C. Birkett, K. H. Okino, D. C. Murdock, et al 1990. Stem cell factor is encoded at the SI locus of the mouse and is the ligand for the c-kit tyrosine kinase receptor. Cell 63:213.[Medline]
  14. Karasuyama, H., F. Melchers. 1988. Establishment of mouse cell lines which constitutively secrete large quantities of interleukin 2, 3, 4, or 5, using modified cDNA expression vectors. Eur. J. Immunol. 18:97.[Medline]
  15. Oshiba, A., E. Hamelmann, K. Takeda, K. L. Bradley, J. E. Loader, G. L. Larsen, E. W. Gelfand. 1996. Passive transfer of immediate hypersensitivity and airway hyperresponsiveness by allergen-specific immunoglobulin (Ig) E and IgG1 in mice. J. Clin. Invest. 97:1398.[Medline]
  16. Minden, A., A. Lin, M. McMahon, C. Lange-Carter, B. Derijard, R. J. Davis, G. L. Johnson, M. Karin. 1994. Differential activation of ERK and JNK mitogen-activated protein kinases by Raf-1 and MEKK. Science 266:1719.[Abstract/Free Full Text]
  17. Cook, S. J., F. McCormick. 1993. Inhibition by cAMP of Ras-dependent activation of Raf. Science 262:1069.[Abstract/Free Full Text]
  18. Cross, D. A. E., D. R. Alessi, P. Cohen, M. Andjelkovich, B. A. Hemmings. 1995. Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature 378:785.[Medline]
  19. Bellacosa, A., J. R. Testa, S. P. Staal, P. N. Tsichlis. 1991. A retroviral oncogene, akt, encoding a serine-threonine kinase containing an SH2-like region. Science 254:274.[Abstract/Free Full Text]
  20. Franke, T. F., S.-I. Yang, T. O. Chan, K. Datta, A. Kazlauskas, D. K. Morrison, D. R. Kaplan, P. N. Tsichlis. 1995. The protein kinase encoded by the akt proto-oncogene is a target of the PDGF-activated phosphatidylinositol 3-kinase. Cell 81:727.[Medline]
  21. Franke, T. F., D. R. Kaplan, L. C. Cantley, A. Toker. 1997. Direct regulation of the akt proto-oncogene product by phosphatidylinositol-3,4-bisphosphate. Science 275:665.[Abstract/Free Full Text]
  22. Okada, T., L. Sakuma, Y. Fukui, O. Hazeki, M. Ui. 1994. Blockage of chemotactic peptide-induced stimulation of neutrophils by wortmannin as a result of selective inhibition of phosphatidylinositol 3-kinase. J. Biol. Chem. 269:3563.[Abstract/Free Full Text]
  23. Rottapel, R., M. Reedijk, D. E. Williams, S. D. Lyman, D. M. Anderson, T. Pawson, A. Bernstein. 1991. The Steel/W transduction pathway: Kit autophosphorylation and its association with a unique subset of cytoplasmic signaling proteins is induced by the Steel factor. Mol. Cell. Biol. 11:3043.[Abstract/Free Full Text]
  24. Trenn, G., R. Taffs, R. Hohman, R. Kincaid, E. M. Shevach, M. Sitkovsky. 1989. Biochemical characterization of the inhibitory effect of CsA on cytolytic T lymphocyte effector functions. J. Immunol. 142:3796.[Abstract]
  25. Hultsch, T., J. L. Rodriguez, M. A. Kaliner, R. J. Hohman. 1990. Cyclosporin A inhibits degranulation of rat basophilic leukemia cells and human basophils: inhibition of mediator release without affecting PI hydrolysis or Ca2+ fluxes. J. Immunol. 144:2659.[Abstract]
  26. Cirillo, R., M. Triggiani, L. Siri, A. Ciccarelli, G. R. Pettit, M. Condorelli, G. Marone. 1990. Cyclosporin A rapidly inhibits mediator release from human basophils presumably by interacting with cyclophilin. J. Immunol. 144:3891.[Abstract]
  27. Hatfield, S. M., N. W. Roehm. 1991. Cyclosporin and FK506 inhibition of murine mast cell cytokine production. J. Pharmacol. Exp. Ther. 260:680.[Abstract/Free Full Text]
  28. Ryffel, B., G. Woerly, M. Murray, H.-P. Eugster, B. Car. 1993. Binding of active cyclosporins to cyclophilin A and B, complex formation with calcineurin A. Biochem. Biophys. Res. Commun. 194:1074.[Medline]
  29. Handschumacher, R. E., M. W. Harding, J. Rice, R. J. Drugge. 1984. Cyclophilin: a specific cytosolic binding protein for cyclosporin A. Science 226:544.[Abstract/Free Full Text]
  30. LeGrue, S. J., R. Turner, N. Weisbrodt, J. R. Dedman. 1986. Does the binding of cyclosporin to calmodulin result in immunosuppression?. Science 234:68.[Abstract/Free Full Text]
  31. Gelfand, E. W., R. K. Cheung, G. B. Mills. 1987. The cyclosporins inhibit lymphocyte activation at more than one site. J. Immunol. 138:1115.[Abstract/Free Full Text]
  32. Kaye, R. E., D. A. Fruman, B. E. Bierer, M. W. Albers, L. D. Zydowsky, S. I. Ho, Y.-J. Jin, M. C. Castells, S. L. Schreiber, C. T. Walsh, et al 1992. Effects of cyclosporin A and FK506 on Fc{epsilon} receptor type I-initiated increases in cytokine mRNA in mouse bone marrow-derived progenitor mast cells: resistance to FK506 is associated with a deficiency in FK506-binding protein FKBP12. Proc. Natl. Acad. Sci. USA 89:8542.[Abstract/Free Full Text]
  33. Liu, J., Jr J. D. Farmer, W. S. Lane, J. Friedman, I. Weissmann, S. L. Schreiber. 1991. Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506 complexes. Cell 66:807.[Medline]
  34. Yano, H., S. Nakanishi, K. Kimura, N. Hanai, Y. Saitoh, Y. Fukui, Y. Nonomura, Y. Matsuda. 1993. Inhibition of histamine secretion by wortmannin through the blockade of phosphatidylinositol 3-kinase in RBL-2H3 cells. J. Biol. Chem. 268:25846.[Abstract/Free Full Text]
  35. Marquardt, D. L., J. L. Alongi, L. L. Walker. 1996. The phosphatidylinositol 3-kinase inhibitor wortmannin blocks mast cell exocytosis but not IL-6 production. J. Immunol. 156:1942.[Abstract]
  36. Kincaid, R. L.. 1995. The role of calcineurin in immune system responses. J. Allergy Clin. Immunol. 96:1170.[Medline]
  37. Alessi, D. R., A. Cuenda, P. Cohen, D. T. Dudley, A. R. Saltiel. 1995. PD 098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo. J. Biol. Chem. 46:27489.
  38. Pang, L., T. Sawada, S. J. Decker, A. R. Saltiel. 1995. Inhibition of MAP kinase kinase blocks the differentiation of PC-12 cells induced by nerve growth factor. J. Biol. Chem. 270:13585.[Abstract/Free Full Text]
  39. Galli, S. J.. 1993. New concepts about the mast cell. N. Engl. J. Med. 328:257.[Free Full Text]
  40. Rao, A.. 1994. NF-ATp: a transcription factor required for the co-ordinate induction of several cytokine genes. Immunol. Today 15:274.[Medline]
  41. Rao, A.. 1995. NF-ATp, a cyclosporin-sensitive transcription factor implicated in cytokine gene induction. J. Leukocyte Biol. 57:536.[Abstract]
  42. Shakhov, A. N., M. A. Collart, P. Vassalli, S. A. Nedospasov, C. V. Jongeneel. 1990. {kappa}B-type enhancers are involved in lipopolysaccharide-mediated transcriptional activation of the tumor necrosis factor {alpha} gene in primary macrophages. J. Exp. Med. 171:35.[Abstract/Free Full Text]
  43. Drouet, C., A. N. Shakhov, C. V. Jongeneel. 1991. Enhancers and transcription factors controlling the inducibility of the tumor necrosis factor-{alpha} promoter in primary macrophages. J. Immunol. 147:1694.[Abstract]
  44. Economou, J. S., K. Rhoades, R. Essner, W. H. McBride, J. C. Gasson, D. L. Morton. 1989. Genetic analysis of the human tumor necrosis factor {alpha}/cachectin promoter region in a macrophage cell line. J. Exp. Med. 170:321.[Abstract/Free Full Text]
  45. Newell, C. L., A. B. Deisseroth, G. Lopez-Berestein. 1994. Interaction of nuclear proteins with an AP-1/CRE-like promoter sequence in the human TNF-{alpha} gene. J. Leukocyte Biol. 56:27.[Abstract]
  46. Krämer, B., K. Wiegmann, M. Kronke. 1995. Regulation of the human TNF promoter by the transcription factor Ets. J. Biol. Chem. 270:6577.[Abstract/Free Full Text]
  47. Ho, I. C., M. R. Hodge, J. W. Rooney, L. H. Glimcher. 1996. The protooncogene c-maf is responsible for tissue-specific expression of interleukin-4. Cell 85:973.[Medline]
  48. Wenner, C. A., S. J. Szabo, K. M. Murphy. 1997. Identification of IL-4 promoter elements conferring Th2-restricted expression during T helper cell subset development. J. Immunol. 158:765.[Abstract]
  49. Aune, T. M., R. A. Flavell. 1997. Differential expression of transcription directed by a discrete NF-AT binding element from IL-4 promoter in naive and effector CD4 T cells. J. Immunol. 159:36.[Abstract]
  50. Kimura, K., S. Hattori, Y. Kabuyama, Y. Shizawa, J. Takayanagi, S. Nakamura, S. Toki, Y. Matsuda, K. Onodera, Y. Fukui. 1994. Neurite outgrowth of PC12 cells is suppressed by wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase. J. Biol. Chem. 269:18961.[Abstract/Free Full Text]
  51. Kotani, K., K. Yonezawa, K. Hara, H. Ueda, Y. Kitamura, H. Sakaue, A. Ando, A. Chavanieu, B. Calas, F. Grigorescu, M. Nishiyama, M. D. Waterfield, M. Kasuga. 1994. Involvement of phosphoinositide 3-kinase in insulin- or IGF-1-induced membrane ruffling. EMBO J. 13:2313.[Medline]
  52. Okada, T., Y. Kawano, T. Sakakibara, O. Hazeki, M. Ui. 1994. Essential role of phosphatidylinositol 3-kinase in insulin-induced glucose transport and antilipolysis in rat adipocytes. J. Biol. Chem. 269:3568.[Abstract/Free Full Text]
  53. Arcaro, A., M. P. Wymann. 1993. Wortmannin is a potent phosphatidylinositol 3-kinase inhibitor: the role of phosphatidylinositol 3,4,5-trisphosphate in neutrophil responses. Biochem. J. 296:297.
  54. Ninomiya, N., K. Hazeki, Y. Fukui, T. Seya, T. Okada, O. Hazeki, M. Ui. 1994. Involvement of phosphatidylinositol 3-kinase in Fc{gamma} receptor signaling. J. Biol. Chem. 269:22732.[Abstract/Free Full Text]
  55. Ishizuka, T., H. Kawasome, N. Terada, K. Takeda, P. Gerwins, G. M. Keller, G. L. Johnson, E. W. Gelfand. 1998. Stem cell factor augments Fc {epsilon} RI-mediated TNF-{alpha} production and stimulates MAP kinases via a different pathway in MC/9 mast cells. J. Immunol. 161:3642.
  56. Flanagan, W. M., B. Corthesy, R. J. Bram, G. R. Crabtree. 1991. Nuclear association of a T-cell transcription factor blocked by FK-506 and cyclosporin A. Nature 352:803.[Medline]



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