Fyn kinase is a key contributor in coupling FcεRI to mast cell degranulation. A limited macroarray analysis of FcεRI-induced gene expression suggested potential defects in lipid metabolism, eicosanoid and glutathione metabolism, and cytokine production. Biochemical analysis of these responses revealed that Fyn-deficient mast cells failed to secrete the inflammatory eicosanoid products leukotrienes B4 and C4, the cytokines IL-6 and TNF, and chemokines CCL2 (MCP-1) and CCL4 (MIP-1β). FcεRI-induced generation of arachidonic acid and normal induction of cytokine mRNA were defective. Defects in JNK and p38 MAPK activation were observed, whereas ERK1/2 and cytosolic phospholipase A2 (S505) phosphorylation was normal. Pharmacological studies revealed that JNK activity was associated with generation of arachidonic acid. FcεRI-mediated activation of IκB kinase β and IκBα phosphorylation and degradation was defective resulting in a marked decrease of the nuclear NF-κB DNA binding activity that drives IL-6 and TNF production in mast cells. However, not all cytokine were affected, as IL-13 production and secretion was enhanced. These studies reveal a major positive role for Fyn kinase in multiple mast cell inflammatory responses and demonstrate a selective negative regulatory role for certain cytokines.

Aggregation of FcεRI on mast cells results in release of preformed inflammatory mediators (e.g., histamine, vasoactive amines, proteases, serotonin) from cytoplasmic granules and de novo production of inflammatory lymphokines and leukotrienes (LT)3 that serve to mobilize and activate additional circulating leukocytes, thus promoting inflammation (1). The Src family protein tyrosine kinase (SrcPTK) Lyn, which phosphorylates the ITAM motifs of the FcεRI β- and γ-chains (2) initiates the propagation of signals through the subsequent activation of Syk kinase and the formation of a macromolecular “signalsome” anchored by the adapter linker for activation of T cells (LAT) (3). We found that, in addition to Lyn kinase, the SrcPTK Fyn was also activated upon FcεRI aggregation and that it was required for optimum mast cell degranulation (4). Others have demonstrated that Fyn kinase can contribute to the activation of phospholipase D (PLD) by phosphorylating this enzyme (5, 6) and we recently found that PLD activity is defective in Fyn-deficient mast cells (A. Olivera and J. Rivera, submitted for publication). This is important because PLD has been demonstrated to contribute to mast cell degranulation (7, 8). Nonetheless, the extent of our knowledge about the Fyn-dependent pathway is limited. In previous work, we demonstrated that Fyn kinase was important in the phosphorylation of the adapter protein, Grb2-associated binder protein 2, the phosphorylation of which is critical for activation of PI3K and for membrane targeting of the SH2 domain-containing protein tyrosine phosphatase 2 (4, 9). The findings also demonstrated a defect in protein kinase B (Akt) phosphorylation as well as a loss in PI3K-dependent kinase 1 (PDK1) phosphorylation of protein kinase C δ (PKCδ).

Despite the similarities in structure and function among SrcPTKs, Lyn and Fyn appear to have opposing roles in regulating the degranulation of mast cells (10). Lyn-deficient mast cells showed a hyperdegranulation phenotype that is mediated, at least in part, as a consequence of increased Fyn kinase activity (10). Lyn was found to be important for the phosphorylation of the adapter C-terminal Src kinase-binding protein, which functions as a scaffold for recruitment of C-terminal Src kinase to the membrane where it exerts negative regulatory control on SrcPTKs, like Fyn, by phosphorylation of a negative regulatory tyrosine at the C terminus (10). These findings provide evidence for a dominant-negative regulatory role of Lyn kinase in mast cell responses. Given this conclusion and our prior demonstration that fyn−/− mice were resistant to anaphylactic challenge, and that bone marrow-derived mast cells (BMMCs) derived from these mice were markedly defective in degranulation, we sought to learn whether Fyn kinase serves as a positive regulator of mast cell responses. We also wished to explore the role of Fyn kinase in delayed responses, which govern the chronic state of allergic inflammation.

The studies in this report analyzed the role of Fyn kinase in signaling events controlling gene expression, arachidonic acid production leading to de novo synthesis and release of LTs, and cytokine production and secretion. These responses are essential for IgE-mediated host defense through effective recruitment of an inflammatory immune response (11). Thus, dysregulation of the production and secretion of these products is a major contributory factor to the persistent inflammation that characterizes chronic allergic inflammation. We now describe that, beyond its role in degranulation, Fyn kinase is required for various signaling pathways leading to gene expression. In the absence of Fyn, defective activation of selected MAP kinase signaling pathways was observed. In addition, the gene expression of various FcεRI-stimulated inflammatory transcription factors and secreted products was defective. These findings demonstrate a key role for Fyn as a positive regulator of multiple mast cell effector responses. They also reveal a negative regulatory role for Fyn in selected cytokine responses. This provides a basis to further explore the therapeutic potential of this kinase in allergy and asthma.

DNP-specific mouse IgE was produced essentially as previously described (12). Abs to IκBα, p44/42, p38, JNK, cytosolic phospholipase A2 (cPLA2), phospho-Akt (Thr308 and Ser473), phospho-IκBα (Ser32/36), phospho-IKKαβ (Ser180/Ser181), phospho-p44/42 (Thr202/Tyr182), phospho-p38 (Thr180/Tyr182), phospho-JNK (Thr183/185), phospho-cPLA2 (Ser505), and ubiquitin were purchased from Cell Signaling Technology. Abs to Akt and Vav1 were purchased from Santa Cruz Biotechnology. Anti-phosphotyrosine Abs and Abs to LAT and phospho-LAT (Tyr191) were from Upstate Biotechnologies. Secondary Abs used were Alexa Fluor 680-conjugated goat anti-rabbit IgG (Molecular Probes) and IRDye800-conjugated goat anti-mouse IgG (Rockland Immunochemicals). The proteosome inhibitor MG-132, p38 inhibitor SB20358, and JNK inhibitor SP600125 were purchased from Calbiochem. Cell culture medium (RPMI 1640) was from Mediatech. FCS was purchased from Invitrogen Life Technologies. Murine IL-3 and stem cell factor (SCF) were from PeproTech. DNP36-HSA (Ag), Triton X-100, n-octyl-β-d-glucopyranoside, indomethacin, and all other chemicals were purchased from Sigma-Aldrich. TNF, IL-6, LTB4, LTC4, CCL4, and IL-13 ELISAs were from R&D Systems and CCL2 ELISA was from Biosource. [14C]Arachidonic acid was purchased from PerkinElmer Life Sciences.

Bone marrow was isolated from femurs and tibias of sex and age-matched (8- to 12-wk-old) fyn+/+, fyn−/−, lyn−/−, and fyn−/−/lyn−/− mice (SV129 × C57/BL6, (N5)) that were maintained and used in accordance with National Institutes of Health guidelines and a National Institute of Arthritis and Musculoskeletal and Skin Diseases-approved animal study proposal. Bone marrow cells were cultured in complete RPMI 1640 supplemented with 20% FBS and 20 ng/ml each of SCF and IL-3. Cultures were monitored by flow cytometry for surface expression of FcεRI and used for experiments when >95% of the cells were FcεRI+. BMMCs were sensitized with IgE before all experiments by incubating in Tyrode’s-BSA buffer (20 mM HEPES, 135 mM NaCl, 1 mM MgCl2, 5 mM KCl, 1.8 mM CaCl2, 5.6 mM glucose, 0.05% BSA (pH 7.4)) containing anti-DNP IgE (0.1 μg/106 cells) for 3 h at 37°C.

Cells (i.e., 3 × 107 per sample) were first deprived of SCF overnight in complete RPMI 1640 culture medium and sensitized with IgE. After washing to remove unbound IgE, BMMCs were stimulated with 25 ng/ml Ag in Tyrodes-BSA for the indicated times at 37°C. For the experiments in which p38, JNK, and proteosomal activities were inhibited, the cells were preincubated for 20 min at 37°C and activated in Tyrodes-BSA ± pharmacological inhibitor at the indicated concentration. Immediately after stimulation, cells were placed in an ice-water bath and ice-cold PBS was added to stop the reaction. Pelleted cells were lysed in 1.0 ml of borate-buffered saline that contained 1% Triton X-100, 60 mM n-octyl-β-d-glucopyranoside, 2 μg/ml leupeptin and pepstatin, 10 μg/ml aprotinin, 2 mM PMSF, 5 mM sodium pyrophosphate, 1 mM sodium orthovanadate, and 50 mM sodium fluoride for 20 min on ice. Lysates were centrifuged for 10 min (4°C) at 12,000 × g, and the supernatant was collected. Total protein concentration was determined using the DC Protein Assay (Bio-Rad). For immunoprecipitation, 0.5 mg of total protein in 1.0 ml of lysis buffer was incubated for 3 h with Abs prebound to protein G-Sepharose (mAbs) or protein A-Sepharose (polyclonal Abs). Proteins were recovered with equal volume (50 μl) of 2× SDS sample buffer containing 1% 2-ME and 1 mM sodium orthovanadate and resolved by SDS-PAGE. In the case of immunoprecipitation of tyrosine phosphorylated proteins for analysis of FcεRIγ phosphorylation, a mixture of the Abs (20 μg of total) to phosphotyrosine (4G10 and PY20) was used. The lysates were normalized to equal receptor numbers before immunoprecipitation based on 125I-γIgE labeling of receptors. For whole cell lysates (WCL), 50 μg of total protein in an equal volume of 2× SDS sample buffer was resolved by SDS-PAGE. Proteins were electrophoretically transferred onto nitrocellulose membranes (Invitrogen Life Technologies). Membranes were blocked with Odyssey Blocking Buffer (Li-Core Biosciences), probed with the desired primary Ab and appropriate infrared-labeled secondary Ab and visualized using the Odyssey Infrared Imaging System (Li-Cor Biosciences). Quantitation was done using Odyssey software.

The mouse 1.2k BD Atlas nylon macroarray, carrying a diverse array of signaling genes, was performed by BD Biosciences/Clontech Custom Atlas Array Hybridization and Analysis services. RNA purification and cDNA probe synthesis used the Atlas Pure Total RNA Labeling System and hybridization was done using the ExpressHyb solution. Gene expression profiles were obtained from RNA extracted from IgE-sensitized wild-type (wt) and fyn−/− BMMCs. Activation was with 10 ng/ml Ag (DNP-HSA) for 1 h based on our previous observation of maximal cytokine gene responses (13). After normalization to housekeeping genes present on the array, the gene expression profiles of FcεRI-activated wt and fyn−/− BMMCs were compared using AtlasImage software. Two individual analyses of gene expression were conducted. The cumulative data are reported as the ratio of the spot intensities using a cutoff ratio of 2.00 with a r2 coefficient of at least 0.93. Genes listed in Table I had a variance of no >36% and a minimum cutoff ratio of 3.0.

Table I.

Expression profile of inhibited genes in FcεRI-stimulated fyn−/− BMMC

GeneaFold DecreaseGenBank No.
Cell surface Ag   
 BST-1, (CD157) 4.6 D31788 
 ICAM1 3.3 X52264 
 IL-7R α 3.5 M29697 
 uPAR1 (CD87) 3.3 X62700 
 Syndecan 3 3.0 U52826 
 Thymus cell Ag 1, θ (Thy 1) 5.8 X03151 
 TNFR2 3.3 M59378 
 Transferrin receptor (CD71) 4.9 X57349 
Transcription factor/regulator   
 cAMP-dependent transcription factor 3 (ATF3) 5.6 U19118 
 AP-1 3.0 J04115 
 Anti-proliferative B-cell translocation gene 2 3.0 M64292 
 IκBα 3.2 U36277 
 Microsomal GST1 3.4 J03752 
 Ski-related oncogene, snoN 5.5 U36203 
 T cell death-associated protein (TDAG51) 5.3 U44088 
Cell signaling/metabolic   
 Gem (Ras family member) 3.4 U10551 
d-3-phosphoglycerate dehydrogenase 3.4 L21027 
 Socs 3 3.7 U88328 
Secreted product   
 Cathepsin L precursor 4.7 X06086 
 Fibronectin 1 precursor 11.4 X82402 
 Follistatin precursor 3.2 Z29532 
 Gelatinase B 3.8 X72795 
 IL-1β 5.7 M15131 
 IL-4 3.0 M25892 
 IL-6 precursor 3.2 X06203 
 MIP-1β 7.8 M35590 
 Nerve growth factor α subunit 5.5 M11434 
 Osteopontin precursor 8.5 J04806 
 Prothrombin precursor 3.0 X52308 
GeneaFold DecreaseGenBank No.
Cell surface Ag   
 BST-1, (CD157) 4.6 D31788 
 ICAM1 3.3 X52264 
 IL-7R α 3.5 M29697 
 uPAR1 (CD87) 3.3 X62700 
 Syndecan 3 3.0 U52826 
 Thymus cell Ag 1, θ (Thy 1) 5.8 X03151 
 TNFR2 3.3 M59378 
 Transferrin receptor (CD71) 4.9 X57349 
Transcription factor/regulator   
 cAMP-dependent transcription factor 3 (ATF3) 5.6 U19118 
 AP-1 3.0 J04115 
 Anti-proliferative B-cell translocation gene 2 3.0 M64292 
 IκBα 3.2 U36277 
 Microsomal GST1 3.4 J03752 
 Ski-related oncogene, snoN 5.5 U36203 
 T cell death-associated protein (TDAG51) 5.3 U44088 
Cell signaling/metabolic   
 Gem (Ras family member) 3.4 U10551 
d-3-phosphoglycerate dehydrogenase 3.4 L21027 
 Socs 3 3.7 U88328 
Secreted product   
 Cathepsin L precursor 4.7 X06086 
 Fibronectin 1 precursor 11.4 X82402 
 Follistatin precursor 3.2 Z29532 
 Gelatinase B 3.8 X72795 
 IL-1β 5.7 M15131 
 IL-4 3.0 M25892 
 IL-6 precursor 3.2 X06203 
 MIP-1β 7.8 M35590 
 Nerve growth factor α subunit 5.5 M11434 
 Osteopontin precursor 8.5 J04806 
 Prothrombin precursor 3.0 X52308 
a

The RNA purification and cDNA probe synthesis used the Atlas Pure Total RNA Labeling System and hybridization on the 1.2k BD Atlas nylon macroarray was done using the ExpressHyb Solution. Gene expression profiles were obtained from RNA extracted from IgE/Ag-stimulated (10 ng/ml-Ag, 1 h) wt and fyn−/− BMMCs normalized to nonstimulated BMMCs and to housekeeping genes present on the array. The gene expression profiles of two individual analyses were compared using AtlasImage software. The cumulative data interpreted to be significant was determined from the ratio of the spot intensities using a cutoff ratio of 2.00 with an r2 coefficient of at least 0.93. Genes reported in this table had no >36% variance among membranes and a minimum cutoff ratio of 3.0.

FcεRI-mediated release of cytokine and LTs was measured by specific ELISA. For lymphokine measurements, IgE-sensitized cells (2.0 × 106 per sample) were stimulated with the indicated concentration of Ag in Tyrodes-BSA for 3 h at 37°C. For LT measurements, IgE-sensitized cells (106 per sample) were stimulated with the indicated concentrations of Ag in Tyrode’s-BSA containing 10 μg/ml indomethacin (Sigma-Aldrich) for 30 min at 37°C. After incubation, the cells were centrifuged (2000 × g, 2 min, 4°C) and TNF, IL-6, IL-13, CCL2, CCL4, LTB4, and LTC4 were measured in the collected cell-free medium according to the manufacturer’s instructions. Arachidonic acid release was quantitated using 14C-labeled arachidonic acid. BMMCs (106 per sample) were loaded with 1 μCi 14C-labeled arachidonic acid overnight at 37°C. After incubation, the cells were washed twice with Tyrode’s-BSA, sensitized with IgE, and activated with 25 ng/ml Ag for the indicated time. After activation, cells were pelleted and the supernatant was removed. The cell pellet was lysed with 1% Triton X-100 detergent on ice for 20 min. Samples (20 μl) from supernatant and lysate were counted and the percentage of arachidonic acid released into the medium from total cellular content was calculated.

RPA was performed as previously described (13). Briefly, custom-made templates were used (BD Biosciences), and probe synthesis was with RiboQuant In vitro Transcription Kit (BD Biosciences) using 100 μCi [α-33P]UTP (ICN Biomedicals) following the manufacturer’s suggested protocol. Hybridization was conducted with 10 μg of total RNA and 106 cpm of probe in 13 μl of hybridization buffer at 56°C overnight. RNaseI digestion and subsequent steps were conducted according to the manufacturer’s instruction. Protected fragments were precipitated in the presence of 1.5 μg of the glycoblue carrier (Ambion) and resolved on a denaturing 6% polyacrylamide gel, which was autoradiographed and developed in Kodak Biomax Transcreen-LE using Kodak BioMax MS film (Eastman Kodak). Quantitation of the autoradiograph was by densitometry using ImageQuant from Molecular Dynamics. Data was normalized to the control genes L32 or GAPDH. To detect genes whose mRNA expression was not detected by RPA, we used RT-PCR. BMMC were incubated with IgE as above, washed once with Tyrode’s-BSA and stimulated with the indicated concentrations of Ag. After 1 h, cells were harvested and total RNA was extracted using Tri-Reagent. Total RNA was used for the cDNA synthesis. PCR and gel analysis was performed using the conditions and primers previously described (14).

Nuclear NF-κB DNA binding activity was determined using the TransAM NF-κB p65 Transcription Factor ELISA kit (Active Motif) according to the manufacturer’s instruction. Nuclear lysates were prepared from IgE-sensitized BMMCs that had been activated with 25 ng/ml Ag for the indicated times. NF-κB p65 binding activity was determined by incubating nuclear lysates in wells containing the NF-κB consensus binding site oligonucleotide. The plates were washed to remove unbound p65 and DNA-bound p65 was detected with Ab to p65. After washing, a secondary HRP-conjugated Ab was added to the wells and a colorimetric reaction was performed. Absorbance was read at 450 nm.

To further understand the role of Fyn kinase in FcεRI-dependent mast cell function, we initiated a limited screening of 1176 murine genes by membrane array analysis. The RNA purification, hybridization, and data analysis on the mouse 1.2k BD Atlas nylon macroarray was performed by BD Biosciences Clontech Custom Atlas Array Hybridization and Analysis services.

Table I reports FcεRI-induced genes the expression of which was reduced in Fyn-deficient mast cells, expressed as fold reduction relative to activated wt cells. Although a 2-fold difference in mRNA expression is considered significant in this screening based on AtlasImage quantitative analysis, we used a cutoff of at least 3-fold and corroborated the observed results for a number of these genes by independent methods. The FcεRI-inducible genes affected by Fyn deficiency could be classified into four classes, cell surface receptors/Ags, transcription factors/regulators, cell signaling/metabolic genes, and secreted factors. Of the cell surface Ag genes, Thy-1 (5.8-fold), transferrin receptor (4.9-fold), and the ADP-ribosyl cyclase-bone stromal cell Ag (BST)-1 (4.6-fold) were most significantly reduced in expression. Of the transcription factors, activated transcription factor (ATF)-3 (5.6-fold), a member of the CREB/ATF family of transcription factors induced by JNK (15), and SnoN (5.5-fold), a negative regulator of TGF-β signaling (16), were most affected by Fyn deficiency. The induction of IκBα, which is required for sequestering NF-κB in the cytosol (17), was also found to be defective (3.2-fold). Of the cell signaling/metabolic genes, the T cell death-associated protein (TDAG51), which regulates Fas expression and is Akt regulated (18, 19), and Socs 3 were reduced 5.3- and 3.7-fold, respectively. For genes of secreted products, fibronectin precursor (11.4-fold), osteopontin precursor (8.5-fold), and CCL4 (7.8-fold) were most dramatically reduced. Moreover, defects in induction of IL-1β, IL-4, IL-6, and CCL4 mRNAs suggested the requirement for Fyn in cytokine gene expression. Interestingly, a reduction in the expression of microsomal glutathione S-transferase (a member of the membrane-associated proteins in eicosanoid and glutathione metabolism (Ref.20) and of d-3-phosphoglycerate dehydrogenase (a key enzyme in l-serine biosynthesis and thus in lipid and sphingolipid biosynthesis (21)), suggested a possible defect in the production of sphingolipids, phospholipids, and possibly eicosanoids by Fyn-deficient mast cells. In general, these preliminary findings pointed to a key role for Fyn kinase in FcεRI-dependent induction of a variety of genes that contribute to the overall inflammatory ability of mast cells. In this study, we focused on a biochemical analysis of FcεRI-activated signaling pathways that lead to cytokine gene expression and in determining the consequence of Fyn deficiency on the late-phase responses of mast cells.

Signaling competence of the FcεRI is primarily provided by the γ subunit, whereas the β subunit functions as an amplifier (22, 23). We previously demonstrated that the FcεRIβ appeared to be phosphorylated normally in Fyn-deficient mast cells (4); thus, we directly assessed the consequence of Fyn deficiency on FcεRIγ phosphorylation. This was important because deletion or mutations of the FcεRIγ, which prevent its phosphorylation, resulted in inhibition of mast cell degranulation and cytokine production (24, 25). To assess this possibility, phosphorylated FcεRI from activated BMMCs was immunoprecipitated using anti- phosphotyrosine Abs and equal numbers (as determined by 125I-labeledIgE binding) of receptors were resolved by SDS-PAGE. As shown in Fig. 1,A, the amount of FcεRIγ phosphorylated in the absence of Fyn was comparable to that of wt cells. Although the representative example shown in Fig. 1,A suggests a modest increase in phosphorylated receptors in the absence of Fyn, this trend (104 ± 9% of wt levels, n = 4) was not statistically significant. In contrast, loss of Lyn had a pronounced inhibitory effect on the recovery of phosphorylated FcεRIγ, with the inhibition averaging ∼96 ± 3% (n = 5). To determine whether the small amount of FcεRIγ phosphorylated in the absence of Lyn was due to the previously described increase in Fyn kinase activity (10), we analyzed mast cells derived from Fyn/Lyn double-deficient mice. Fig. 1 A demonstrates that Fyn was not responsible for the small amount of phosphorylated FcεRIγ recovered from lyn−/− mast cells. The loss of both Lyn and Fyn kinases caused a slight but consistent enhancement in the amount of phosphorylated receptor, relative to Lyn-deficient mast cells, suggesting that removal of both kinases makes FcεRI available as a substrate for a yet unidentified kinase.

FIGURE 1.

Phosphorylation of FcεRIγ, Vav1, and LAT in IgE/Ag-activated fyn−/− BMMCs. IgE-sensitized BMMCs were activated with Ag for the indicated times and the phosphorylation of FcεRIγ (A), LAT (B, upper panel), and Vav1 (C, lower panel) was assessed by SDS-PAGE and Western blot analysis. FcεRIγ phosphorylation of equal numbers of receptors (see Materials and Methods) was detected by immunoprecipitating tyrosine phosphorylated proteins from 3.0 × 107 wt, lyn−/−, lyn/fyn−/−, and fyn−/− BMMCs using a combination of Abs to phosphotyrosine (clone 4G-10 and PY20) and probing with Ab to FcRγ (A). WCL (50 μg of total protein) was probed with Ab to phospho-LAT (Tyr191) and LAT (B, upper panels). Phosphorylated Vav1 was detected by immunoprecipitating Vav1 from 0.5 mg of total WCL protein and probing blots with Ab to phosphotyrosine (clone 4G-10) and Vav1 Abs (B, lower panels). Blots are representative of at least three separate experiments with three individual BMMC cultures. Phosphorylated and total proteins were detected using the fluorescence-based Odyssey. Fold induction of phosphorylated protein normalized to the amount of each individual protein in a given lane was determined by the relative ratio of fluorescence intensity and compared with 0 min (arbitrarily set to 1).

FIGURE 1.

Phosphorylation of FcεRIγ, Vav1, and LAT in IgE/Ag-activated fyn−/− BMMCs. IgE-sensitized BMMCs were activated with Ag for the indicated times and the phosphorylation of FcεRIγ (A), LAT (B, upper panel), and Vav1 (C, lower panel) was assessed by SDS-PAGE and Western blot analysis. FcεRIγ phosphorylation of equal numbers of receptors (see Materials and Methods) was detected by immunoprecipitating tyrosine phosphorylated proteins from 3.0 × 107 wt, lyn−/−, lyn/fyn−/−, and fyn−/− BMMCs using a combination of Abs to phosphotyrosine (clone 4G-10 and PY20) and probing with Ab to FcRγ (A). WCL (50 μg of total protein) was probed with Ab to phospho-LAT (Tyr191) and LAT (B, upper panels). Phosphorylated Vav1 was detected by immunoprecipitating Vav1 from 0.5 mg of total WCL protein and probing blots with Ab to phosphotyrosine (clone 4G-10) and Vav1 Abs (B, lower panels). Blots are representative of at least three separate experiments with three individual BMMC cultures. Phosphorylated and total proteins were detected using the fluorescence-based Odyssey. Fold induction of phosphorylated protein normalized to the amount of each individual protein in a given lane was determined by the relative ratio of fluorescence intensity and compared with 0 min (arbitrarily set to 1).

Close modal

The immediate consequence downstream of FcεRI phosphorylation is the activation of Syk kinase and the formation of a macromolecular signaling complex scaffolded by the adapter LAT (3), both of which contribute to cytokine responses. We previously demonstrated that Syk and LAT phosphorylation appeared intact in the absence of Fyn. However, it was possible that membrane targeting of Syk was altered or that phosphorylation of a specific tyrosine residues on LAT (such as Y191), which is one of four known to contribute to mast cell degranulation and lymphokine production (26), might be selectively impaired. We investigated the phosphorylation of LAT (Y191) and Vav1 because this adapter and this guanine nucleotide exchange factor, respectively, interact with, and are substrates of, Syk and form a complex with LAT (26). As shown in Fig. 1,B, Fyn deficiency did not alter the ability of these proteins to become phosphorylated in response to FcεRI stimulation. Tyrosine 191 of LAT is a key contributor to the stability of the macromolecular complex assembled by LAT (26); thus, its phosphorylation suggests that this complex is intact. This was further supported by normal ERK activation (Fig. 2,B, lower panels), which we previously demonstrated to be dependent on LAT (27) and to be part of a complex that included Vav1 (28). These findings extend our previous observations by demonstrating that Fyn does not participate in FcεRI phosphorylation and that phosphorylation of proteins like LAT, Vav1, and ERKs are not affected by Fyn deficiency. Thus, the apparent defects in FcεRI-mediated gene induction noted in Table I appear to be independent of the Lyn-Syk-LAT axis.

FIGURE 2.

Activation of Akt and MAPK in IgE/Ag-activated fyn−/− BMMCs. IgE-sensitized wt and fyn−/− BMMCs were activated with 25 ng/ml Ag for the indicated times, and the phosphorylation of Akt (A), p38MAPK (B, top panels), ERK1/2 (B, bottom panels), and JNK1/2 (C) was assessed by SDS-PAGE and Western blotting with Abs directed against total and phospho-specific Akt (Ser473 and Thr308), p38 (Thr180/181), ERK1/2 (Thr202/Tyr204), and JNK1/2 (Thr183/185). Fold induction of phosphorylation of ERK1/2 and JNK1/2 is the mean of all experiments. Fold induction was determined as in Fig. 1. Blots are representative of three or more individual experiments with different BMMC cultures.

FIGURE 2.

Activation of Akt and MAPK in IgE/Ag-activated fyn−/− BMMCs. IgE-sensitized wt and fyn−/− BMMCs were activated with 25 ng/ml Ag for the indicated times, and the phosphorylation of Akt (A), p38MAPK (B, top panels), ERK1/2 (B, bottom panels), and JNK1/2 (C) was assessed by SDS-PAGE and Western blotting with Abs directed against total and phospho-specific Akt (Ser473 and Thr308), p38 (Thr180/181), ERK1/2 (Thr202/Tyr204), and JNK1/2 (Thr183/185). Fold induction of phosphorylation of ERK1/2 and JNK1/2 is the mean of all experiments. Fold induction was determined as in Fig. 1. Blots are representative of three or more individual experiments with different BMMC cultures.

Close modal

Our prior studies (4) also demonstrated that Akt (S473) phosphorylation was defective in Fyn-deficient mast cells. Serine 473 on Akt was recently shown to be a target of PKCβII in mast cells (29). However, the key site for Akt activation is T308, which is a target of PDK1. Fig. 2 A demonstrates that FcεRI-dependent Akt phosphorylation at both T308 and S473 is defective (with 47–68% inhibition at 1 min postactivation, respectively) in the absence of Fyn. The inhibition at the T308 site is consistent with our prior report of defective PDK1 activation in fyn−/− mast cells, which is required for full activation of multiple PKC isozymes (4). Given that PKCβII phosphorylates Ser473 of Akt in mast cells (29), these results provide evidence of a broader defect in PKC activation in the absence of Fyn that is likely due to diminished PDK1 activity. Thus, two signaling pathways (PKCs and Akt) that are known to be involved in gene expression in many cell types were found to be defective in the absence of Fyn.

We subsequently analyzed the phosphorylation (activation) of MAP kinases (p38MAPK, JNK, ERK), the function of which is key for activation of various transcription factors, like ATF3 and AP-1, and whose activity has been linked to NF-κB (30, 31, 32). Phosphorylation of p38 MAPK and ERK in wt cells was detectable as early as 1 min postactivation, whereas JNK phosphorylation was apparent at 5 min postactivation (Fig. 2, B and C). It was qualitatively and quantitatively apparent that phosphorylation of both p38 MAPK and JNK1/2 was defective in fyn−/− BMMCs relative to wt cells with the most severe defect (77 ± 9% inhibition, n = 4) in JNK phosphorylation (Fig. 2, B and C). In contrast to p38 MAPK and JNK, and consistent with normal LAT phosphorylation and function (27), ERK1/2 phosphorylation was intact in fyn−/− BMMCs (Fig. 2 B). Collectively, the findings demonstrate that the activities of PKC, Akt, JNK, and p38MAPK were defective in the absence of Fyn.

Depending on the cell type, ERK, JNK, and p38MAPK have all been implicated in the activation of cPLA2 (33, 34), the major enzyme responsible for the production of arachidonate (precursor of eicosanoids) in mast cells (35, 36). JNK and p38MAPK have also been implicated in the expression of eicosanoid-related enzymes (37, 38). Moreover, because the genetic profiling data in Table I suggested a defect in the induction of genes in the membrane-associated proteins in eicosanoid and glutathione metabolism family as well as in l-serine biosynthesis, it seemed reasonable to determine whether cPLA2 and/or arachidonic acid production was defective. Phosphorylation of S505 of cPLA2 induces increased enzymatic activity and membrane targeting affinity (39). Fig. 3,A shows that S505 phosphorylation appears to be intact in the absence of Fyn. Although a slight loss of cPLA2 protein was noted, this was not significant. This demonstrates that cPLA2 phosphorylation of S505 in fyn−/− BMMC is normal and thus independent of Fyn kinase, JNK, or p38MAPK. This is consistent with the previous demonstration of a major role for ERK kinase in the activation of cPLA2 in mast cells (40), an enzyme whose activity is intact in the absence of Fyn. However, the release of arachidonic acid was not induced in fyn−/− BMMCs even up to 4 h post-FcεRI stimulation (Fig. 3,B). In contrast, arachidonic acid release from wt BMMC peaked at 30 min and was considerably sustained for up to 4 h. As might be expected from the loss of arachidonic acid production, the release of LTs, LTB4 and cysLTs (LTC4), from fyn−/− BMMCs was defective over a wide range of Ag concentrations when compared with wt cells (Fig. 3, C and D). Of note, LTB4 release was optimal at lower Ag concentrations than cysLTs from wt cells.

FIGURE 3.

Assessment of cPLA2 phosphorylation, arachidonic acid production, and LT secretion in IgE/Ag-activated fyn−/− BMMCs. A, Western blot analysis of cPLA2 phosphorylation at S505 from IgE-sensitized BMMCs activated with 25 ng/ml Ag. Shown is a representative blot. Fold induction was calculated as in Fig. 1. B, Percent of arachidonic acid released after IgE/Ag activation. IgE-sensitized BMMCs were loaded with 14C-labeled arachidonic acid and activated with 25 ng/ml Ag. Arachidonic acid released into the extracellular medium was calculated at 30-min intervals up to 4 h and is expressed as the percent of total 14C-labeled arachidonic acid incorporated in the cell. IgE-mediated release of LTB4 (C) and LTC4 (D) was determined by ELISA from BMMCs activated for 30 min with varying concentrations of Ag in Tyrodes buffer containing 10 μg/ml indomethacin. Mean ± SEM from at least three individual experiments is shown.

FIGURE 3.

Assessment of cPLA2 phosphorylation, arachidonic acid production, and LT secretion in IgE/Ag-activated fyn−/− BMMCs. A, Western blot analysis of cPLA2 phosphorylation at S505 from IgE-sensitized BMMCs activated with 25 ng/ml Ag. Shown is a representative blot. Fold induction was calculated as in Fig. 1. B, Percent of arachidonic acid released after IgE/Ag activation. IgE-sensitized BMMCs were loaded with 14C-labeled arachidonic acid and activated with 25 ng/ml Ag. Arachidonic acid released into the extracellular medium was calculated at 30-min intervals up to 4 h and is expressed as the percent of total 14C-labeled arachidonic acid incorporated in the cell. IgE-mediated release of LTB4 (C) and LTC4 (D) was determined by ELISA from BMMCs activated for 30 min with varying concentrations of Ag in Tyrodes buffer containing 10 μg/ml indomethacin. Mean ± SEM from at least three individual experiments is shown.

Close modal

Whether the defective p38MAPK and JNK activation seen in Fyn-deficient BMMC had any role in the decreased arachidonic acid production was further explored by use of pharmacological agents that selectively inhibit these kinases. As shown in Fig. 4,A, the use of the p38MAPK-selective inhibitor SB203580 in wt mast cells failed to significantly suppress arachidonic acid release. In contrast, the JNK-selective inhibitor, SP600125, showed effective inhibition (66 ± 7%, n = 3) of arachidonic acid release (Fig. 4,B). Fig. 4, C and D, shows that these inhibitors (at the concentrations used) selectively inhibited the intended target but had no significant effect on the S505 phosphorylation of cPLA2. These findings demonstrate an important role for Fyn-dependent JNK activity in regulating arachidonic acid release in BMMC that is independent of cPLA2 (S505) phosphorylation.

FIGURE 4.

Arachidonic acid release from wt BMMCs in the presence of pharmacological inhibitors of p38 and JNK1/2. 14C-Labeled arachidonic acid release was measured from IgE-sensitized wt and fyn−/− BMMCs activated for 30 min with 25 ng/ml Ag and preincubated for 20 min in the presence or absence of 5 μM p38 inhibitor (SB203580) (A) or 10 μM JNK inhibitor (SP600125) (B). Western blot analysis was done to assess specificity of SB203580 for p38 (C) and SP600125 for JNK (D) with Abs directed against total and phospho-specific p38 and JNK1/2. p38MAPK and cPLA2 phosphorylation was determined at 1 min and JNK phosphorylation was determined at 12 min. No differences were observed in cPLA2 phosphorylation with SP or SB. Data in A and B is mean ± SEM of at least three individual experiments with different BMMC cultures. Significance was determined by unpaired t test; ∗, p < 0.05.

FIGURE 4.

Arachidonic acid release from wt BMMCs in the presence of pharmacological inhibitors of p38 and JNK1/2. 14C-Labeled arachidonic acid release was measured from IgE-sensitized wt and fyn−/− BMMCs activated for 30 min with 25 ng/ml Ag and preincubated for 20 min in the presence or absence of 5 μM p38 inhibitor (SB203580) (A) or 10 μM JNK inhibitor (SP600125) (B). Western blot analysis was done to assess specificity of SB203580 for p38 (C) and SP600125 for JNK (D) with Abs directed against total and phospho-specific p38 and JNK1/2. p38MAPK and cPLA2 phosphorylation was determined at 1 min and JNK phosphorylation was determined at 12 min. No differences were observed in cPLA2 phosphorylation with SP or SB. Data in A and B is mean ± SEM of at least three individual experiments with different BMMC cultures. Significance was determined by unpaired t test; ∗, p < 0.05.

Close modal

p38 MAPK and JNK have also been implicated in regulating NF-κB activity in several cellular systems (30, 31, 32). Moreover, the down-regulation of IκBα gene expression (Table I) upon FcεRI stimulation suggested a possible defect in NF-κB function, given the regulatory control of IκBα on the activation of this transcription factor (17). The common pathway leading to NF-κB activation requires the phosphorylation and degradation of the inhibitor of NF-κB, IκBα, by phosphorylated IκB kinase (IKKαβ). This is a key step in release of NF-κB/Rel subunits (p50, p65), which then translocate from the cytosol to the nucleus and initiate cytokine gene transcription. Degradation of IκBα requires its ubiquitination, which marks it for a proteosomal pathway. Recovery of IκBα protein is dependent on nuclear NF-κB activity, which initiates transcription of this gene (17). Because our gene array data showed a defect in FcεRI-induced IκBα gene expression, this suggested a possible decrease in nuclear NF-κB activity. We found that FcεRI-induced phosphorylation of IKKα and β subunits was defective in the absence of Fyn (Fig. 5,A). IKKβ phosphorylation was most severely inhibited (90 ± 6%) by Fyn deficiency. Although IκBα was moderately phosphorylated in fyn−/− BMMCs, it was not degraded as observed in wt cells (Fig. 5 A).

FIGURE 5.

Assessment of NF-κB pathway activation in fyn−/− BMMCs. To analyze the phosphorylation of IKKαβ and IκBα, BMMCs from wt and fyn−/− were activated with 25 ng/ml Ag for the indicated times, and WCL (50 μg of total protein) was probed in a Western blot analysis with phospho-specific Abs (A). Western blot analysis was performed on WCLs from wt and fyn−/− BMMCs activated with 25 ng/ml Ag in the presence of 5 μM MG-132 proteosomal inhibitor to determine whether the observed defect in IκBα degradation in fyn−/− BMMCs was due to inefficient phosphorylation of the protein or defective degradation process (B, upper panel). The ratio of phosphorylated to nonphosphorylated IκBα in the presence of MG-132 was calculated (B, lower panel). C, Ubiquitination of IκBα was determined by immunoprecipitation of IκBα from 0.5 mg of total WCL protein and Western blot analysis with Ab to ubiquitin. D, Nuclear NF-κB p65 DNA binding activity was measured by ELISA. Nuclear lysates were incubated in wells containing the NF-κB consensus binding site oligonucleotide followed by binding of secondary HRP-conjugated Ab and colorimetric reaction (see Materials and Methods). Activity is reported as the mean absorbance at 450 nm from four individual experiments. Blots are representative of at least three individual experiments. Mean ± SEM is shown.

FIGURE 5.

Assessment of NF-κB pathway activation in fyn−/− BMMCs. To analyze the phosphorylation of IKKαβ and IκBα, BMMCs from wt and fyn−/− were activated with 25 ng/ml Ag for the indicated times, and WCL (50 μg of total protein) was probed in a Western blot analysis with phospho-specific Abs (A). Western blot analysis was performed on WCLs from wt and fyn−/− BMMCs activated with 25 ng/ml Ag in the presence of 5 μM MG-132 proteosomal inhibitor to determine whether the observed defect in IκBα degradation in fyn−/− BMMCs was due to inefficient phosphorylation of the protein or defective degradation process (B, upper panel). The ratio of phosphorylated to nonphosphorylated IκBα in the presence of MG-132 was calculated (B, lower panel). C, Ubiquitination of IκBα was determined by immunoprecipitation of IκBα from 0.5 mg of total WCL protein and Western blot analysis with Ab to ubiquitin. D, Nuclear NF-κB p65 DNA binding activity was measured by ELISA. Nuclear lysates were incubated in wells containing the NF-κB consensus binding site oligonucleotide followed by binding of secondary HRP-conjugated Ab and colorimetric reaction (see Materials and Methods). Activity is reported as the mean absorbance at 450 nm from four individual experiments. Blots are representative of at least three individual experiments. Mean ± SEM is shown.

Close modal

Because IκBα was rapidly degraded in wt BMMC, its level of phosphorylation is likely under-represented relative to Fyn-deficient BMMC, in which this protein is stable. To address this possibility, we assessed IκBα phosphorylation and degradation in BMMCs activated in the presence of the proteosomal inhibitor MG-132. We predicted that inhibiting IκBα degradation in wt cells should reveal the extent of the defect in its phosphorylation in fyn−/− BMMCs. As the results demonstrate, 5 μM MG-132 was sufficient to effectively inhibit IκBα degradation (Fig. 5,B). IκBα phosphorylation in fyn−/− BMMCs was defective (69 ± 7% inhibition, n = 3) as indicated by the more transient and hypophosphorylated state of this protein. Additional support for defective phosphorylation and degradation of IκBα came from the observation that FcεRI-inducible ubiquitination of IκBα in fyn−/− BMMCs was reduced compared with wt cells (Fig. 5,C). Moreover, the DNA binding activity of nuclear NF-κB(p65) was remarkably reduced in fyn−/− BMMCs (Fig. 5 D). This was consistent with the finding that IκBα phosphorylation and degradation was defective, thus sequestering NF-κB (p65) in the cytosol and inhibiting further IκBα gene expression in response to FcεRI stimulation.

Given the above results, and those in Table I, we set out to investigate the impact of Fyn deficiency on cytokine gene expression induced by FcεRI stimulation. As shown in Fig. 6, A and B, fyn−/− BMMC were defective in production of mRNA for several cytokines including IL-4 and IL-6, confirming the data in Table I. Quantitation of the IL-6 response by densitometry found a decrease of 59 ± 7% at suboptimal and optimal concentrations of Ag, which was consistent with the decrease of IL-6 precursor seen in the gene array analysis (Table I). TNF mRNA was also greatly reduced in fyn−/− BMMCs with as much as 70% inhibition; however, this gene was not on the macroarray. Both of these genes are known targets of NF-κB activity in mast cells (41, 42). Interestingly, IL-13 mRNA was normally induced in fyn−/− BMMCs and showed a trend for increased mRNA levels relative to wt cells. Chemokine mRNAs were also reduced in the absence of Fyn, especially at suboptimal concentrations of Ag, suggesting decreased sensitivity of these genes in the absence of Fyn kinase. This is consistent with our previous finding that chemokine production can occur under conditions of suboptimal occupancy with IgE or Ag and that these conditions favor the activation of Fyn, Grb2-associated binder protein 2, and PI3K (13). Thus, for genes like CCL2, a significant reduction of mRNA expression was observed at 1 and 3 ng/ml Ag (∼55 ± 9%, n = 3), whereas these differences were narrowed at higher concentrations (10 ng/ml) of Ag (∼27 ± 6%, n = 3) (Fig. 6,A). In contrast, quantitation of CCL4 revealed significant inhibition at suboptimal (∼85 ± 3%, n = 3) and optimal doses (∼64 ± 7%, n = 3) of Ag. To corroborate these data with protein secretion, we measured FcεRI-mediated release of IL-6, IL-13, TNF, CCL2, and CCL4 in the extracellular medium (Fig. 7). Release of both TNF and IL-6 from fyn−/− BMMCs was severely impaired at all Ag concentrations, measuring <25 ± 4% (n = 4) of that released by wt cells when stimulated at optimal Ag concentrations (Fig. 7, A and B). CCL4 secretion from fyn−/− BMMCs was essentially undetectable even though mRNA production was still observed (Fig. 7,C vs 6A). Release of CCL2 from fyn−/− BMMCs was also defective, with the largest differences observed at low Ag concentrations (Fig. 7,D), consistent with the more modest differences in mRNA expression with increasing Ag dose. Mirroring the mRNA studies, IL-13 secretion was significantly enhanced (Fig. 7 E). At optimal doses of Ag, a 3-fold increase in secreted IL-13 was observed, suggesting a negative regulatory role for Fyn in IL-13 expression. Collectively, the findings demonstrate an important role for Fyn in generating the signals required for normal mast cell cytokine production and reveal that Fyn kinase exerts a negative regulatory role for selected cytokines.

FIGURE 6.

FcεRI-stimulated cytokine mRNA induction in fyn−/− BMMCs. A, RPA analysis of cytokine (left panel) and chemokine (right panel) mRNA responses from IgE-sensitized wt and fyn−/− BMMCs activated with varying concentrations of Ag. B, RT-PCR analysis was used to detect IL-2, IL-3, and IL-4 mRNA levels in fyn−/− BMMCs. Panels show a representative experiment of three individual experiments.

FIGURE 6.

FcεRI-stimulated cytokine mRNA induction in fyn−/− BMMCs. A, RPA analysis of cytokine (left panel) and chemokine (right panel) mRNA responses from IgE-sensitized wt and fyn−/− BMMCs activated with varying concentrations of Ag. B, RT-PCR analysis was used to detect IL-2, IL-3, and IL-4 mRNA levels in fyn−/− BMMCs. Panels show a representative experiment of three individual experiments.

Close modal
FIGURE 7.

FcεRI-stimulated cytokine secretion in fyn−/− BMMCs. Secretion of TNF (A), IL-6 (B), CCL4 (C), CCL2 (D), and IL-13 (E) was determined by ELISA. IgE-sensitized BMMCs were activated with the indicated concentrations of Ag for 3 h, and the amount of lymphokine released into the cell-free culture medium was quantitated. The data are representative of at least three or more individual experiments conducted with different BMMC cultures. Data are the mean ± SEM.

FIGURE 7.

FcεRI-stimulated cytokine secretion in fyn−/− BMMCs. Secretion of TNF (A), IL-6 (B), CCL4 (C), CCL2 (D), and IL-13 (E) was determined by ELISA. IgE-sensitized BMMCs were activated with the indicated concentrations of Ag for 3 h, and the amount of lymphokine released into the cell-free culture medium was quantitated. The data are representative of at least three or more individual experiments conducted with different BMMC cultures. Data are the mean ± SEM.

Close modal

The late phase of the mast cell response is largely based on the de novo synthesis and secretion of inflammatory mediators (1). The link between gene expression and Src family kinases has long been recognized (43); however, this varies with cell lineage. Fyn has been demonstrated to contribute to TCR and CD43-mediated production of regulatory cytokines, like IL-2 (44, 45). Fyn also appears to contribute in the generation of survival signals for naive T cells, which is likely to be dependent on cytokine production (46). The role of Fyn in BCR-mediated gene expression seems to be minor relative to its role in regulating IL-mediated gene expression (like IL-4 and IL-5) (47, 48). BCR stimulation of Fyn-deficient B cells showed a modest impairment of downstream signals, whereas IL-5 signaling was completely blocked. B cell stimulation with IL-4 selectively activated Fyn kinase but not Lyn kinase, and its increased activity was associated with the induction of Ig Cε gene expression (48). There is also increasing evidence that differential compartmentalization of Fyn, in a given cell type, is important for its particular function in that cell (49). These observed differences in the cellular role of Fyn, as well as its differential use by particular receptors, emphasizes the importance of exploring the role of Fyn in mast cell FcεRI-induced gene expression.

It was important to assess the role of FcεRIγ because of its essential nature in transduction of signals that initiate the late-phase events (24, 50). We observed no significant alterations in the amount of FcεRIγ phosphorylated in the absence of Fyn compared with wt cells. In contrast, only a small fraction of FcεRIγ is phosphorylated in Lyn-deficient mast cells, consistent with the previous report of residual FcεRIβ phosphorylation in these cells (51). Previously (52), we failed to detect phosphorylated FcεRI in Lyn-deficient mast cells. Two technical differences may account for the apparent discrepancy: 1) in this study, only phosphorylated receptor was immunoprecipitated. This contrasts to the previous study where immunoprecipitation of total (both nonphosphorylated and phosphorylated) receptors was performed. 2) We now used a mixture of two Abs to phosphotyrosine for immunoprecipitation (4G10 and PY20), whereas, in the past experiments, only one Ab was used for detection (PY20). Together, these modifications likely increased the sensitivity of phosphotyrosine detection. However, importantly, no correlation was established, in either this or the previous study (52), between the extent of FcεRI phosphorylation and the ability of the mast cells to degranulate or induce cytokine gene expression and secretion. An apparent incongruity arises from the loss of JNK activation in Fyn-deficient BMMC under circumstances where LAT and Vav1 phosphorylation is normal. Our previous findings (14, 27, 28) demonstrated that LAT- or Vav1-deficiencies caused a loss in JNK activation and that Vav1 contributed to IL-6 production through Rac/JNK-mediated signals. This suggests that, in mast cells, there is cross-talk between LAT or Vav1 and Fyn kinase. The view of Fyn-Vav1 cross-talk is supported by multiple studies in lymphocytes demonstrating the importance of Fyn activity in Vav1 phosphorylation (53, 54), the presence of both Vav1 and Fyn in macromolecular signaling complexes (55), and the role of both Fyn and Vav1 in JNK activation (56). This defines a possible point where Fyn kinase and Lyn-dependent LAT and Vav1 signals may intersect to influence gene expression, as LAT and Vav1-deficiencies also caused loss of cytokine gene expression (14, 27).

Gene array analysis revealed that Fyn deficiency in mast cells resulted in selectively impaired FcεRI-induced gene expression. We verified only a small number of the identified genes, focusing primarily on cytokines. Among the nonverified down-regulated genes, the T cell death-associated protein (TDAG51), which was demonstrated to link TCR signaling to Fas (CD95) expression and whose expression is regulated by Akt (18, 19), was viewed as an internal control because FcεRI-dependent Akt activation is defective in Fyn-deficient mast cells (Ref.4 and Fig. 2 A) and thus the expression of Akt-regulated genes should be impaired. In the absence of Fyn, its FcεRI-dependent expression was reduced by 5.3-fold. The early response genes ATF3 and AP-1 are transcription factors whose expression is dependent on JNK activity (15, 57). Thus, the loss of their FcεRI-mediated expression is consistent with the observed defect in JNK activation in Fyn-deficient BMMC. AP-1 expression is also regulated by PKC, with the β and ε isoforms contributing to induction of c-fos and c-jun in mast cells (58). The finding that the PKCβII-dependent phosphorylation of Akt (Ser473) was defective is consistent with the view that activation of this isoform PKC is impaired and likely affects AP-1 expression. This is also consistent with the reduced PDK1 activity in these cells and the requirement for this enzyme in PKC activation (59).

Multiple signaling pathways converge in the metabolic/catabolic regulation of lipid metabolism. Among them, a role for MAP kinases has been demonstrated in generation of arachidonate (34, 39, 60, 61). Our findings demonstrate that FcεRI-dependent JNK activation is significantly impaired and that p38MAPK activation is partly impaired in the absence of Fyn. Depending on the cell type, one or both of these pathways have been implicated in the activation of cPLA2, which is the essential provider of arachidonic acid to the 5-lipoxygenase and cyclooxgenase pathways and is required for activation-dependent induction of prostaglandin endoperoxidase synthetase 2 in mast cells (35, 62). Given the defect in JNK and p38MAPK activation in the absence of Fyn, our finding that cPLA2 S505 phosphorylation was normal argues against this site being a target of JNK or p38MAPK activity in these cells. Nonetheless, the JNK inhibitor SP600125 inhibited arachidonic acid release in wt BMMCs, whereas the p38MAPK inhibitor SB203580 did not, demonstrating a dominant role for JNK in arachidonic acid metabolism. JNK activity has been linked both upstream and downstream of arachidonic acid production, suggesting a key relationship between the activity of this kinase and generation of this lipid metabolite. The failure of fyn−/− BMMC to release arachidonic acid and to generate both LTB4 and LTC4, along with the normal phosphorylation of cPLA2 in these cells, points to a defect that is likely to be upstream of cPLA2 activation. Based on requirement of calcium mobilization and S505 phosphorylation (39) for cPLA2 activation and the ability of Fyn-deficient BMMC to initiate these responses, our findings suggest that either a substrate for cPLA2 is limiting or that cPLA2 is mislocalized. Interestingly, the observed phenotype of mast cell cPLA2 deficiency mirrors that of Fyn deficiency with respect to arachidonic acid release and eicosanoid secretion (35, 36).

Fyn kinase is also important for de novo production of various cytokines. IL-2, IL-3, IL-4, IL-6, TNF, CCL2, and CCL4 mRNAs were considerably reduced and the secretion of several of these cytokines was dramatically affected confirming the gene array analysis. A key regulatory signal for cytokines, downstream of Fyn, is the transcription factor NF-κB, a well-characterized regulator of inflammation in health and disease (63). NF-κB is an essential regulator of IL-6 and TNF production in mast cells (41, 42). Fyn controls the activation of NF-κΒ through signals leading to the phosphorylation of IκB and its degradation. The link between Fyn kinase and these events is not yet clear. In SrcPTK triple-deficient B cells (Blk, Fyn, and Lyn), IKKαβ activation and NFκB activity were also defective (64). The impaired NF-κB induction could be overcome by expression of PKCλ, suggesting that a defect in its activation linked the SrcPTKS to IKKαβ activation. The role for PKCs in NF-κB activation is also addressed in a study analyzing the function of a Fyn binding protein called signaling lymphocyte activation molecule-associated protein (SAP), whose mutation causes X-linked lymphoproliferative disease (65). SAP-deficient T cells have impaired NF-κB activation as well as impaired recruitment of PKCθ to the immunological synapse (65). Indeed, this phenotype was reproduced in Fyn-deficient T cells demonstrating that Fyn-SAP interactions may play an important role in NF-κB activation in these cells. These findings may extrapolate to mast cells, as we previously demonstrated (4), and presently expand the evidence for decreased PKC activity in Fyn-deficient mast cells. Given that both PKCλ and PKCθ have been implicated in different cell types, it is of considerable interest to determine which isoform(s) is/are responsible in mast cells. Regardless, the defective NF-κB activation in Fyn-deficient mast cells makes a strong case for the reduced inflammatory competence of these cells.

The key findings of this study argue that Fyn kinase exerts a major influence on the delayed inflammatory responses of mast cells. To a large extent, this is mediated through its role in arachidonic acid production and NF-κB-mediated induction of proinflammatory cytokines. This is seemingly independent of the Lyn-Syk-LAT signaling axis and thus underscores the importance of Fyn-mediated signals in mast cell-mediated inflammation. We do not exclude the contribution of Fyn-independent signals to mast cell inflammatory responses as we could still detect considerable production (mRNA) and some release of both proinflammatory and immunoregulatory cytokines. The enhanced levels of IL-13 mRNA and protein are consistent with a negative regulatory role of Fyn on expression of this gene. These results also serve to dismiss the possible generalized inactivity of Fyn-deficient BMMC to FcεRI stimulation. The dominant role of transcription factors, like NFAT1 and GATA, in regulating the expression of IL-13 in mast cells has been demonstrated (66) suggesting possible candidates for the negative regulatory effects of Fyn. Interestingly, the gene expression analysis also revealed up-regulation of mast cell protease 4, granzyme B, and cathepsin D (data not shown), suggesting other possible candidate genes in which Fyn may play a negative role. While still early in our understanding, the findings promote the general conclusion that Fyn kinase is important for mast cells inflammatory responses. The revelation that Fyn kinase negatively regulates selected mast cell responses suggests an intriguing complexity that warrants further exploration.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by the Department of Health and Human Services and the National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health and the United States-Israel Binational Science Foundation Grant Number 2000016 (to J.R.). C.G.-E. was supported by Grant Number 39726-Q from Consejo Nacíonal de Ciencia y Tecnologia. J.J.R. was supported by National Institutes of Health Grants 1RO1AI43433 and 1R01CA91839.

3

Abbreviations used in this paper: LT, leukotriene; SAP, signaling lymphocyte activation molecule (SLAM)-associated protein; LAT, linker for activation of T cells; BMMC, bone marrow-derived mast cell; WCL, whole cell lysate; cPLA2, cytosolic phospholipase A2; wt, wild type; IKK, IκB kinase; RPA, RNase protection assay; SrcPTK, Src family protein tyrosine kinase; PLD, phospholipase D; PDK1, PI3K-dependent kinase 1; PKC, protein kinase C; SCF, stem cell factor; ATF, activated transcription factor.

1
Galli, S., C. Lantz.
1998
. Allergy. W. Paul, ed.
Fundamental Immunology
1127
-1174. Raven Press, New York.
2
Pribluda, V. S., C. Pribluda, H. Metzger.
1994
. Transphosphorylation as the mechanism by which the high-affinity receptor for IgE is phosphorylated upon aggregation.
Proc. Natl. Acad. Sci. USA
91
:
11246
-11250.
3
Rivera, J..
2002
. Molecular adapters in FcεRI signaling and the allergic response.
Curr. Opin. Immunol.
14
:
688
-693.
4
Parravicini, V., M. Gadina, M. Kovarova, S. Odom, C. Gonzalez-Espinosa, Y. Furumoto, S. Saitoh, L. E. Samelson, J. J. O’Shea, J. Rivera.
2002
. Fyn kinase initiates complementary signals required for IgE-dependent mast cell degranulation.
Nat. Immunol.
3
:
741
-748.
5
Ahn, B.-H., S. Y. Kim, E. H. Kim, K. S. Choi, T. K. Kwon, Y. H. Lee, J.-S. Chang, M.-S. Kim, Y.-H. Jo, D. S. Min.
2003
. Transmodulation between phospholipase D and c-Src enhances cell proliferation.
Mol. Cell. Biol.
23
:
3103
-3115.
6
Choi, W. S., T. Hiragun, J. H. Lee, Y. M. Kim, H. P. Kim, A. Chahdi, E. Her, J. W. Han, M. A. Beaven.
2004
. Activation of RBL-2H3 mast cells is dependent on tyrosine phosphorylation of phospholipase D by Fyn and Fgr.
Mol. Cell. Biol.
24
:
6980
-6992.
7
Choi, W. S., Y. M. Kim, C. Combs, M. A. Frohman, M. A. Beaven.
2002
. Phospholipases D1 and D2 regulate different phases of exocytosis in mast cells.
J. Immunol.
168
:
5682
-5689.
8
Lin, P., G. A. Wiggan, A. M. Gilfillan.
1991
. Activation of phospholipase D in a rat mast (RBL 2H3) cell line: a possible unifying mechanism for IgE-dependent degranulation and arachidonic acid metabolite release.
J. Immunol.
146
:
1609
-1616.
9
Gu, H., K. Saito, L. D. Klaman, J. Shen, T. Fleming, Y. Wang, J. C. Pratt, G. Lin, B. Lim, J.-P. Kinet, B. G. Neel.
2001
. Essential role for Gab2 in the allergic response.
Nature
412
:
186
-190.
10
Odom, S., G. Gomez, M. Kovarova, Y. Furumoto, J. J. Ryan, H. V. Wright, C. Gonzalez-Espinosa, M. L. Hibbs, K. W. Harder, J. Rivera.
2004
. Negative regulation of IgE-dependent allergic responses by Lyn kinase.
J. Exp. Med.
199
:
1491
-1502.
11
Wedemeyer, J., M. Tsai, S. J. Galli.
2000
. Role of mast cells and basophils in innate and acquired immunity.
Curr. Opin. Immunol.
12
:
624
-631.
12
Liu, F. T., J. W. Bohn, E. L. Ferry, H. Yamamoto, C. A. Molinaro, L. A. Sherman, N. R. Klinman, D. H. Katz.
1980
. Monoclonal dinitrophenyl-specific murine IgE antibody: preparation, isolation, and characterization.
J. Immunol.
124
:
2728
-2737.
13
Gonzalez-Espinosa, C., S. Odom, A. Olivera, J. P. Hobson, M. E. Martinez, A. Oliveira-Dos-Santos, L. Barra, S. Spiegel, J. M. Penninger, J. Rivera.
2003
. Preferential signaling and induction of allergy-promoting lymphokines upon weak stimulation of the high affinity IgE receptor on mast cells.
J. Exp. Med.
197
:
1453
-1465.
14
Manetz, T. S., G. Gonzalez-Espinosa, R. Arudchandran, S. Xirasagar, V. Tybulewicz, J. Rivera.
2001
. Vav1 regulates phospholipase Cγ activation and calcium responses in mast cells.
Mol. Cell. Biol.
21
:
3763
-3774.
15
Inoue, K., T. Zama, T. Kamimoto, R. Aoki, Y. Ikeda, H. Kimura, M. Hagiwara.
2004
. TNFα-induced ATF3 expression is bidirectionally regulated by the JNK and ERK pathways in vascular endothelial cells.
Genes Cells
9
:
59
-70.
16
Luo, K..
2004
. Ski and SnoN: negative regulators of TGF-β signaling.
Curr. Opin. Gen. Dev.
14
:
65
-70.
17
Chiao, P. J., S. Miyamoto, I. M. Verma.
1994
. Autoregulation of IκBα activity.
Proc. Natl. Acad. Sci. USA
91
:
28
-32.
18
Park, C. G., S. Y. Lee, G. Kandala, S. Y. Lee, Y. Choi.
1996
. A novel gene product that couples TCR signaling to Fas (CD95) expression in activation-induced death.
Immunity
4
:
583
-591.
19
Kuhn, I., M. F. Bartholdi, H. Salamon, R. I. Feldman, R. A. Roth, P. H. Johnson.
2001
. Indentification of AKT-regulated genes in inducible MERAkt cells.
Physiol. Genomics
7
:
105
-114.
20
Jakobsson, P.-J., R. Morgenstern, J. Mancini, A. Ford-Hutchinson, B. Persson.
2000
. Membrane-associated proteins in eicosanoid and glutathione metabolism (MAPEG).
Am. J. Respir. Crit. Care. Med.
161
:
S20
-S24.
21
Yoshida, K., S. Furuya, S. Osuka, J. Mitoma, Y. Shinoda, M. Watanabe, N. Azuma, H. Tanaka, T. Hashikawa, S. Itohara, Y. Hirabayashi.
2004
. Targeted disruption of the mouse 3-phosphoglycerate dehydrogenase gene causes severe neural developmental defects and results in embryonic lethality.
J. Biol. Chem.
279
:
3573
-3577.
22
Lin, S., C. Cicala, A. M. Scharenberg, J. P. Kinet.
1996
. The FcεRIβ subunit functions as an amplifier of FcεRIγ-mediated cell activation signals.
Cell
85
:
985
-995.
23
Dombrowicz, D., S. Q. Lin, V. Flamand, A. T. Brini, B. H. Koller, J. P. Kinet.
1998
. Allergy-associated FcRβ is a molecular amplifier of IgE- and lgG-mediated in vivo responses.
Immunity
8
:
517
-529.
24
Takai, T., M. Li, D. Sylvestre, R. Clynes, J. V. Ravetch.
1994
. FcRγ chain deletion results in pleiotrophic effector cell defects.
Cell
76
:
519
-529.
25
Van den Herik-Oudijk, I. E., M. W. H. Ter Bekke, M. J. Tempelman, P. J. A. Capel, J. G. J. Van de Winkel.
1995
. Functional differences between two Fc receptor ITAM signaling motifs.
Blood
86
:
3302
-3307.
26
Saitoh, S., S. Odom, G. Gomez, C. L. Sommers, H. A. Young, J. Rivera, L. Samelson.
2003
. The four distal tyrosines are required for LAT-dependent signaling in FcεRI-mediated mast cell activation.
J. Exp. Med.
198
:
831
-843.
27
Saitoh, S., R. Arudchandran, T. S. Manetz, W. Zhang, C. L. Sommers, P. E. Love, J. Rivera, L. E. Samelson.
2000
. LAT is essential for FcεRI-mediated mast cell activation.
Immunity
12
:
525
-535.
28
Song, J. S., J. Gomez, L. F. Stancato, J. Rivera.
1996
. Association of a p95 Vav-containing signaling complex with the FcεRI γ chain in the RBL-2H3 mast cell line: evidence for a constitutive in vivo association of Vav with Grb2, Raf-1, and ERK2 in an active complex.
J. Biol. Chem.
271
:
26962
-26970.
29
Kawakami, Y., H. Nishimoto, J. Kitaura, M. Maeda-Yamamoto, R. M. Kato, D. R. Littman, M. Leitges, D. J. Rawlings, T. Kawakami.
2004
. Protein kinase CβII regulates Akt phosphorylation on Ser-473 in a cell type-and stimulus-specific fashion.
J. Biol. Chem.
279
:
47720
-47725.
30
Azzolina, A., A. Bongiovanni, N. Lampiasi.
2003
. Substance P induces TNF-α and IL-6 production through NF κB in peritoneal mast cells.
Biochim. Biophys. Acta
1643
:
75
-83.
31
Jijon, H., B. Allard, C. Jobin.
2004
. NF-κB inducing kinase activates NF-κB transcriptional activity independently of IκB kinase γ through a p38 MAPK-dependent RelA phosphorylation pathway.
Cell Signal
16
:
1023
-1032.
32
Darieva, Z., E. B. Lasunskaia, M. N. Campos, T. L. Kipnis, W. D. Da Silva.
2004
. Activation of phosphatidylinositol 3-kinase and c-Jun-N-terminal kinase cascades enhances NF-κB-dependent gene transcription in BCG-stimulated macrophages through promotion of p65/p300 binding.
J. Leukocyte Biol.
75
:
689
-697.
33
Borsch-Haubold, A. G., F. Ghomashchi, S. Pasquet, M. Goedert, P. Cohen, M. H. Gelb, S. P. Watson.
1999
. Phosphorylation of cytosolic phospholipase A2 in platelets is mediated by multiple stress-activated protein kinase pathways.
Eur. J. Biochem.
265
:
195
-203.
34
Hirasawa, N., F. Santini, M. A. Beaven.
1995
. Activation of the mitogen-activated protein kinase/cytosolic phospholipase A2 pathway in a rat mast cell line: indications of different pathways for release of arachidonic acid and secretory granules.
J. Immunol.
154
:
5391
-5402.
35
Fujishima, H., R. O. Sanchez Mejia, C. O. Bingham, B. K. Lam, B. A. Sapirstein, J. V. Bonventre, K. F. Austen, J. P. Arm.
1999
. Cytosolic phospholipase A2 is essential for both the immediate and the delayed phases of eicosanoid generation in mouse bone marrow-derived mast cells.
Proc. Natl. Acad. Sci. USA
96
:
4803
-4807.
36
Nakatani, N., N. Uozumi, K. Kume, M. Murakami, I. Kudo, T. Shimizu.
2000
. Role of cytosolic phospholipase A2 in the production of lipid mediators and histamine release in mouse bone-marrow-derived mast cells.
Biochem. J.
352
:
311
-317.
37
Subbaramaiah, K., W. J. Chung, A. J. Dannenberg.
1998
. Ceramide regulates the transcription of cyclooxygenase-2: evidence for involvement of extracellular signal-regulated kinase/c-Jun N-terminal kinase and p38 mitogen-activated protein kinase pathways.
J. Biol. Chem.
273
:
32943
-32949.
38
Hundley, T. R., A. R. Prasad, M. A. Beaven.
2001
. Elevated levels of cyclooxygenase-2 in antigen-stimulated mast cells is associated with minimal activation of p38 mitogen-activated protein kinase.
J. Immunol.
167
:
1629
-1636.
39
Clark, J. D., A. R. Schievella, E. A. Nalefski, L.-L. Lin.
1995
. Cytosolic phospholipase A2.
J. Lipid Mediat. Cell Signal
12
:
83
-117.
40
Hirasawa, N., A. Scharenberg, H. Yamamura, M. A. Beaven, J.-P. Kinet.
1995
. A requirement for Syk in the activation of the microtubule-associated protein kinase/phospholipase A2 pathway by FcεRI is not shared by a G protein-coupled receptor.
J. Biol. Chem.
270
:
10960
-10967.
41
Marquardt, D. L., L. L. Walker.
2000
. Dependence of mast cell IgE-mediated cytokine production on nuclear factor-κB activity.
J. Allergy Clin. Immunol.
105
:
500
-505.
42
Pelletier, C., N. Varin-Blank, J. Rivera, B. Iannascoli, F. Marchand, B. David, A. Weyer, U. Blank.
1998
. FcεRI-mediated induction of TNF-α gene expression in the RBL-2H3 mast cell line: regulation by a novel NF-κB-like nuclear binding complex.
J. Immunol.
161
:
4768
-4776.
43
Parsons, S. J., J. T. Parsons.
2004
. Src family kinases, key regulators of signal transduction.
Oncogene
23
:
7906
-7909.
44
Palacios, E. H., A. Weiss.
2004
. Function of the Src-family kinases, Lck and Fyn, in T-cell development and activation.
Oncogene
23
:
7990
-8000.
45
Pedraza-Alva, G., L. B. Merida, S. J. Burakoff, Y. Rosenstein.
1996
. CD43-specific activation of T cells induces association of CD43 to Fyn kinase.
J. Biol. Chem.
271
:
27564
-27568.
46
Zamoyska, R., A. Basson, A. Filby, G. Legname, M. Lovatt, B. Seddon.
2003
. The influence of the src-family kinases, Lck, Fyn, on T cell differentiation, survival and activation.
Immunol. Rev.
191
:
107
-118.
47
Appleby, M. W., J. D. Kerner, S. Chien, C. R. Maliszewski, S. Bondada, R. M. Perlmutter.
1995
. Involvement of p59fynT in interleukin-5 receptor signaling.
J. Exp. Med.
182
:
811
-820.
48
Ikizawa, K., K. Kajiwara, T. Koshio, Y. Yanagihara.
1994
. Possible role of tyrosine kinase activity in interleukin 4-induced expression of germ-line Cε transcripts in a human Burkitt lymphoma B-cell line, DND39.
J. Allergy Clin. Immunol.
94
:
620
-624.
49
Filipp, D., M. Julius.
2004
. Lipid rafts: resolution of the “fyn problem”?.
Mol. Immunol.
41
:
645
-656.
50
Kinet, J. P..
1999
. The high-affinity IgE receptor (FcεRI): from physiology to pathology.
Annu. Rev. Immunol.
17
:
931
-972.
51
Hernandez-Hansen, V., A. J. Smith, Z. Surviladze, A. Chigaev, T. Mazel, J. Kalesnikoff, C. A. Lowell, G. Krystal, L. A. Sklar, B. S. Wilson, J. M. Oliver.
2004
. Dysregulated FcεRI signaling and altered Fyn and SHIP activities in Lyn-deficient mast cells.
J. Immunol.
173
:
100
-112.
52
Kovarova, M., P. Tolar, R. Arudchandran, L. Draberova, J. Rivera, P. Draber.
2001
. Structure-function analysis of Lyn kinase association with lipid rafts and initiation of early signaling events after Fcε receptor I aggregation.
Mol. Cell. Biol.
21
:
8318
-8328.
53
Michel, F., L. Grimaud, L. Tuosto, O. Acuto.
1998
. Fyn and ZAP-70 are required for Vav phosphorylation in T cells stimulated by antigen-presenting cells.
J. Biol. Chem.
273
:
31932
-31938.
54
Pedraza-Alva, G., L. B. Merida, S. J. Burakoff, Y. Rosenstein.
1998
. T cell activation through the CD43 molecule leads to Vav tyrosine phosphorylation and mitogen-activated protein kinase pathway activation.
J. Biol. Chem.
273
:
14218
-14224.
55
Foucault, I., S. Le Bras, C. Charvet, C. Moon, A. Altman, M. Deckert.
2005
. The adaptor protein 3BP2 associates with VAV guanine nucleotide exchange factors to regulate NFAT activation by the B-cell antigen receptor.
Blood
105
:
1106
-1113.
56
Hehner, S. P., R. Breitkreutz, G. Shubinsky, H. Unsoeld, K. Schulze-Osthoff, M. L. Schmitz, W. Droge.
2000
. Enhancement of T cell receptor signaling by a mild oxidative shift in the intracellular thiol pool.
J. Immunol.
165
:
4319
-4328.
57
Minden, A., A. Lin, T. Smeal, B. Dérijard, M. Cobb, R. Davis, M. Karin.
1994
. c-Jun N-terminal phosphorylation correlates with activation of the JNK subgroup but not the ERK subgroup of mitogen-activated protein kinases.
Mol. Cell. Biol.
14
:
6683
-6688.
58
Razin, E., Z. Szallasi, M. G. Kazanietz, P. M. Blumberg, J. Rivera.
1994
. Protein kinases C-β and C-ε link the mast cell high-affinity receptor for IgE to the expression of c-fos and c-jun.
Proc. Natl. Acad. Sci. USA
91
:
7722
-7726.
59
Le Good, J. A., W. H. Ziegler, D. B. Parekh, D. R. Alessi, P. Cohen, P. J. Parker.
1998
. Protein kinase C isotypes controlled by phosphoinositide 3-kinase through the protein kinase PDK1.
Science
281
:
2042
-2045.
60
Nakatani, Y., M. Murakami, I. Kudo, K. Inoue.
1994
. Dual regulation of cytosolic phospholipase A2 in mast cells after cross-linking of Fcε-receptor.
J. Immunol.
153
:
796
-803.
61
Kramer, R. M., E. F. Roberts, S. L. Um, A. G. Börsch-Haubold, S. P. Watson, M. J. Fisher, J. A. Jakubowski.
1996
. p38 Mitogen-activated protein kinase phosphorylates cytosolic phospholipase A2 (cPLA2) in thrombin-stimulated platelets: evidence that proline-directed phosphorylation is not required for mobilization of arachidonic acid by cPLA2.
J. Biol. Chem.
271
:
27723
-27729.
62
Bingham, C. O., K. F. Austen.
1999
. Phospholipase A2 enzymes in eicosanoid generation.
Proc. Assoc. Am. Physicians
111
:
516
-524.
63
Li, X., G. R. Stark.
2002
. NFκB-dependent signaling pathways.
Exper. Hematol.
30
:
285
-296.
64
Saijo, K., C. Schmedt, I. Su, H. Karasuyama, C. A. Lowell, M. Reth, T. Adachi, A. Patke, A. Santana, A. Tarakhovsky.
2003
. Essential role of Src-family protein tyrosine kinases in NF-κB activation during B cell development.
Nat. Immunol.
4
:
274
-279.
65
Cannons, J. L., L. J. Yu, B. Hill, L. A. Mijares, D. Dombroski, K. E. Nichols, A. Antonellis, G. A. Koretzky, K. Gardner, P. L. Schwartzberg.
2004
. SAP regulates Th2 differentiation and PKC-θ-mediated activation of NF-κB1.
Immunity
21
:
693
-706.
66
Monticelli, S., D. C. Solymar, A. Rao.
2004
. Role of NFAT proteins in IL13 gene transcription in mast cells.
J. Biol. Chem.
279
:
36210
-36218.