Aggregation of the FcϵRI on Mast Cells Stimulates c-Jun Amino-terminal Kinase Activity

Aggregation of the high-affinity Fc receptors for immunoglobulin E (IgE) (FcεRI) on the surface of mast cells initiates intracellular signal transduction pathways including the tyrosine phosphorylation of cellular proteins, phosphoinositide hydrolysis, an increase in intracellular calcium, and protein kinase C activation. These signals are believed to be involved in the exocytic release of inflammatory mediators such as vasoactive amines, cytokines, and lipid metabolites. However, the downstream consequences of these early activation events are not well defined. One exception is the activation of the extracellular signal-regulated kinases/mitogen-activated protein kinases. One member of the mitogen-activated protein kinase superfamily, designated c-Jun amino-terminal kinase (JNK), has been recently identified. JNK is activated following dual phosphorylation at a Thr-Pro-Tyr motif in response to diverse stimuli including tumor necrosis factor-α, heat shock, or ultraviolet irradiation. We found that JNK was strongly activated by antigen cross-linking in a mouse mast cell line passively sensitized with ovalbumin-specific IgE. Anti-mouse IgE antibody also activated JNK. MEK kinase 1 (MEKK1) which activates the JNK activator, JNK kinase (JNKK), was similarly activated by antigen stimulation. JNK but not p42erk2 activation induced by antigen was significantly inhibited in the presence of wortmannin, a known inhibitor of phosphatidylinositol 3-kinase. These results indicate that in response to the aggregation of FcεRI on mast cells, phosphatidylinositol 3-kinase activation is involved in the stimulation of the MEKK1, JNKK, JNK pathway.


Aggregation of the high-affinity Fc receptors for immunoglobulin E (IgE) (Fc⑀RI) on the surface of mast cells initiates intracellular signal transduction path-
ways including the tyrosine phosphorylation of cellular proteins, phosphoinositide hydrolysis, an increase in intracellular calcium, and protein kinase C activation. These signals are believed to be involved in the exocytic release of inflammatory mediators such as vasoactive amines, cytokines, and lipid metabolites. However, the downstream consequences of these early activation events are not well defined. One exception is the activation of the extracellular signal-regulated kinases/mitogen-activated protein kinases. One member of the mitogen-activated protein kinase superfamily, designated c-Jun amino-terminal kinase (JNK), has been recently identified. JNK is activated following dual phosphorylation at a Thr-Pro-Tyr motif in response to diverse stimuli including tumor necrosis factor-␣, heat shock, or ultraviolet irradiation. We found that JNK was strongly activated by antigen cross-linking in a mouse mast cell line passively sensitized with ovalbumin-specific IgE. Anti-mouse IgE antibody also activated JNK. MEK kinase 1 (MEKK1) which activates the JNK activator, JNK kinase (JNKK), was similarly activated by antigen stimulation. JNK but not p42 erk2 activation induced by antigen was significantly inhibited in the presence of wortmannin, a known inhibitor of phosphatidylinositol 3-kinase. These results indicate that in response to the aggregation of Fc⑀RI on mast cells, phosphatidylinositol 3-kinase activation is involved in the stimulation of the MEKK1, JNKK, JNK pathway.
Mast cells play a central role in inflammatory and immediate allergic reactions. The multivalent binding of antigen to receptor-bound IgE and the subsequent aggregation of the highaffinity Fc receptors for IgE (Fc⑀RI) provide the trigger for activation of mast cells. Fc⑀RI aggregation results in the release of inflammatory mediators from secretory granules, which contain preformed mediators such as histamine, and the generation of leukotriene C4, prostaglandin D2, and a variety of cytokines (1)(2)(3)(4). These mast cell responses are regulated by intracellular signal transduction pathways initiated by Fc⑀RI aggregation. The first demonstrable response to Fc⑀RI aggregation is tyrosine phosphorylation and activation of phospholipase C␥, which catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate resulting in the liberation of inositol 1,4,5trisphosphate and diacylglycerol (5). The elevation of diacylglycerol and the mobilization of Ca 2ϩ from intracellular and extracellular sources result in the activation of protein kinase C (6). In addition to the activation of phospholipase C␥ and protein kinase C, which appears to be essential for the Fc⑀RI-mediated release of preformed mediators (7), it was recently shown that the aggregation of Fc⑀RI on rat basophilic leukemia 2H3 (RBL-2H3) cells induces histamine and leukotriene release following activation of the phosphatidylinositol 3-kinase (PI3-kinase) 1 (8). Changes such as calcium mobilization and the activation of tyrosine kinases, protein kinase C, and PI3-kinase can affect more downstream events, including the increased transcription of certain activation-related genes and cell proliferation.
The extracellular signal-regulated kinases (ERKs), ERK1 and ERK2, are serine/threonine protein kinases that are themselves activated through concomitant phosphorylation of tyrosine and threonine residues (9 -12). It is thought that ERKs are one of the intermediates in the transduction pathway leading to increases in gene transcription and proliferation. ERKs phosphorylate specific transcription factors including members of the Ets family such as Elk-1 (13). It has been reported that ERKs are activated via Fc⑀RI on mast cells (14,15).
A distant member of the mitogen-activated protein kinase (MAPK) superfamily, designated c-Jun amino-terminal kinase (JNK), has been recently identified (16,17). JNK is activated by dual phosphorylation at a Thr-Pro-Tyr motif during response to cellular stresses including heat shock and ultraviolet (UV) irradiation (16 -18). Costimulation of T cells with antibodies to the T cell receptor and CD28 or the stimulation of B cells with anti-CD40 antibody also induces the activation of JNK (19,20). JNK functions to phosphorylate c-Jun at the amino-terminal regulatory sites, serine 63 and serine 73, mapping within its transactivation domain (16 -18). Phosphorylation of these sites in response to UV irradiation also results in the transcriptional activation of c-Jun (16 -18). Here we show that JNK is activated following aggregation of Fc⑀RI in a * This work was supported in part by Grants AI HL-36577 (to E. W. G.) and DK-37871 (to G. L. J.) from the National Institutes of Health. 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.

MATERIALS AND METHODS
Cell Line and Agents-The MC/9 murine mast cell clone was obtained from the American Type Culture Collection (Rockville, MD) and maintained in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 5 ϫ 10 Ϫ5 M 2-mercaptoethanol (Life Technologies, Inc.), 10% fetal bovine serum (Summit Biotechnology, Ft Collins, CO), and 5% conditioned medium (rat growth factor obtained from Collaborative Biomedical (Bedford, MA)). Affinity-purified rabbit polyclonal anti-mouse MEK kinase 1 (MEKK1) antibody was prepared by immunizing rabbits with a recombinant fragment of the amino-terminal domain of MEKK1 (21). Purified rat anti-mouse IgE monoclonal antibody (R35-72) was purchased from Pharmingen (San Diego, CA). The mouse monoclonal anti-mouse ERK2 antibody and bovine myelin basic protein were obtained from Upstate Biotechnology (Lake Placid, NY). Goat affinity-purified polyclonal anti-ERK2 (C-14, amino acids 345-358) antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Ovalbumin (OVA, grade V) was obtained from Sigma. Recombinant protein G-Sepharose 4B was purchased from Zymed Laboratories (San Francisco, CA). Wortmannin was obtained from Calbiochem and stored as a 10 mM stock in dimethyl sulfoxide (Me 2 SO).
Passive Sensitization and Stimulation of MC/9 Cells-MC/9 cells (5 ϫ 10 6 /ml) were cultured with 500 ng/ml anti-OVA IgE for 2 h. The cells were washed with medium three times and cultured with fresh medium for an additional 2 h. OVA dissolved in PBS or anti-IgE was added for the stimulation, and PBS was used as a control vehicle.
anti-ERK 2 antibody (200 g/69 l, Upstate Biotechnology) was added to the blocking buffer (1:1000), and blots were incubated for an additional 1 h at room temperature. The blots were washed in TBST (25 mM Tris (pH 8.0), 125 mM NaCl, 0.025% Tween 20), and specific reactive proteins were detected by an enhanced chemiluminescence method, employing a sheep anti-mouse Ig antibody linked to horseradish peroxidase (Amersham Corp.).
Kinase Assay of ERK2-In vitro kinase assay of ERK2 was carried out as described previously (24).
Statistical Analysis-Student's t test, Welch's t test, or a paired t test was used for the statistical analysis.

JNK Is Activated by Antigen or Anti-IgE in MC/9 Cells-
Following addition of OVA to MC/9 cells sensitized with OVAspecific IgE (OVA-IgE), JNK was significantly activated in a dose-dependent manner. 10 g/ml OVA induced maximal activation of JNK (Fig. 1A). JNK was significantly activated within 5 min, and its activation was maximal at 15-20 min after the addition of OVA (Fig. 1, B and C). JNK activation by OVA was not induced in MC/9 cells sensitized with TNP-specific IgE (TNP-IgE) and BSA did not activate JNK in MC/9 cells sensitized with OVA-IgE. Anti-mouse IgE antibody activated JNK in both TNP-IgE and OVA-IgE-sensitized cells (Fig. 1D).
MEKK1 Is Activated by Antigen in MC/9 Cells-Addition of OVA (10 g/ml) induced MEKK1 activation in MC/9 cells sensitized with OVA-IgE. As a positive control in the kinase assay for MEKK1, cell lysates from Cos cells that transiently overexpressed full-length MEKK1 were used. MEKK1 activation in MC/9 cells was observed 30 s after the addition of OVA to IgE-sensitized cells. MEKK1 activity reached maximal levels 3 min after OVA addition. MEKK1 activity was increased to 2.5-3-fold over basal activity. Kinase activities decreased gradually by 10 min after addition of OVA (Fig. 2, A and B). The same membrane in the kinase assay was probed with the anti-MEKK1 antibody used for immunoprecipitation, and reactivity was visualized by the alkaline phosphatase system to ensure that the same amounts of MEKK1 were present in each sample. Immunoblotting showed a 98-kDa band of MEKK1, and the density in each sample was comparable (data not shown).
ERK2 Is Phosphorylated and Activated by Antigen Ligation in MC/9 Cells-ERK2 activation induced by tyrosine-threonine phosphorylation was observed in immunoblots using anti-ERK2 antibody. ERK2 was phosphorylated in the presence of 1-100 g/ml OVA (Fig. 3A). This phosphorylation was elicited within 30 s, and a clear shift in mobility was observed at 1-20 min after 10 g/ml OVA stimulation. Phosphorylated ERK2 protein was decreased 30 -40 min after OVA addition (Fig. 3B). Kinase activity of ERK2 was measured as 32 P incorporation into myelin basic protein. ERK2 was significantly activated at 1 min after the addition of OVA, and its activation was maximal at 5-20 min. Significant activation was still observed at 90 min after OVA stimulation (Fig. 3C).
Effect of Wortmannin on JNK Activation and ERK2 Activation-In many systems wortmannin has been shown to inhibit PI3-kinase when used at concentrations below 1 M. We examined the effect of wortmannin (3 nM to 1 M) on JNK and ERK2 activation induced by 10 g/ml OVA stimulation in OVA-IgE- sensitized MC/9 cells. Wortmannin was added 15 min before addition of OVA. Equivalent Me 2 SO (0.01%) amounts were used as a control vehicle. Wortmannin inhibited JNK activity in a dose-dependent manner. The kinase activity in cells treated with 100 nM wortmannin was decreased to 8% of that observed in the absence of treatment (Fig. 4, A and B). In contrast, 100 -300 nM wortmannin did not significantly inhibit ERK2 activation induced by OVA (Fig. 4C). DISCUSSION Mast cells are responsible for initiation of immediate hypersensitivity responses in allergic diseases because they have Fc⑀RI and release preformed inflammatory mediators such as histamine. Furthermore, they produce LTC4, PGD2, and cyto-kines in response to allergen cross-linking and may be involved in airway hyperreactivity and late asthmatic responses in patients with bronchial asthma. A number of mast cell lines have been utilized to investigate intracellular signaling and function. MC/9, a mouse mast cell line, was originally derived from fetal liver cells cultured in concanavalin A-conditioned medium followed by culture with irradiated syngeneic bone marrow cells. The MC/9 mast cell line was identified as a source of mast cells by light microscopy and the appearance of metachromatic granules. Further characterization includes the findings on electron microscopy and the ability to release histamine upon stimulation with A23187 or with antigen following passive sensitization with IgE (25). MC/9 cells express Fc⑀RI on the cell surface (26).
The aggregation of Fc⑀RI initiates diverse signal transduction pathways. In addition to the release of mast cell granule contents, these pathways lead to late responses such as the increase in c-fos and c-jun expression and modulation of cytokine gene expression. The Fc⑀RI is composed of three subunits, single ␣ and ␤ chains, and a homodimer of disulfide-linked ␥ chains (27,28). The intracellular tails of the ␤ and ␥ chains contain a motif that is important for signal transduction. This motif has been called the antigen recognition activation motif or tyrosine activation motif, which is thought to couple the Fc⑀RI to protein-tyrosine kinases (29 -31). Activation of protein-tyrosine kinases is one of the earliest signaling events induced by aggregation of the Fc⑀RI on mast cells (32). Aggregation of Fc⑀RI in the RBL-2H3 cell line results in the rapid tyrosine phosphorylation of its ␤ and ␥ chains (33). Proteintyrosine kinase activation is thought to be proximal to the activation of phospholipase C␥ and protein kinase C, which appears essential for mast cell activation (7) since proteintyrosine kinase inhibitors prevent the production of inositol 1,4,5-trisphosphate and histamine release (34). In the RBL-2H3 cells, Lyn and Syk are associated with Fc⑀RI, and their kinase activity is stimulated by aggregation of the Fc⑀RI (35)(36)(37). In addition to these protein-tyrosine kinases, Bruton tyrosine kinase is involved in mast cell activation (38). It is not associated with Fc⑀RI, but its activation takes place prior to protein kinase C activation. In mast cells, activation of ERKs is apparent from increases in ERK activity and the tyrosine phosphorylation and shift in the electrophoretic migration of ERK2 and, much less so, of ERK1 (14,15). In our investigation, electrophoretic migration and activation of ERK2 was observed in antigen-stimulated MC/9 cells. One role for ERKs in mast cells may be the activation of cytosolic phospholipase A2 (39), which would result in the production of arachidonic acid derivatives such as LTC4 and PGD2.
Aggregation of Fc⑀RI results in the rapid activation of both JNK and ERK2 in MC/9 cells. As predicted, MEKK1 activation precedes JNK activation in response to ligation of the Fc⑀RI. Strikingly, wortmannin, at concentrations that inhibit PI3kinase activity (40 -45), also inhibited JNK activation but not ERK activation. This finding is the first demonstration of a role for PI3-kinase in regulating a JNK pathway by an Src family tyrosine kinase-associated receptor. Thus, in mast cells the regulation of the MEKK1, JNKK, JNK pathway is dependent on the activation of PI3-kinase. Mechanistically, these results indicate that there is a very early separation in the signal pathways activated by the Fc⑀RI to differentially regulate JNK and ERK sequential protein kinase pathways. How PI3-kinase activity is involved in activating the MEKK1, JNKK, and JNK pathway in mast cells is unclear except that it must be downstream of tyrosine kinases and upstream of MEKK1. The regulation of Rac/Cdc 42 GTP-binding proteins in a PI3-kinase-dependent manner has been described (46); Rac and Cdc 42 have FIG. 4. Effect of wortmannin on JNK and ERK2 activation by antigen in mast cells. A and B, MC/9 cells sensitized with OVA-IgE were incubated with 0.01% Me 2 SO (control and 0 nM) or 3-1000 nM wortmannin for 15 min. The cells were then incubated with 10 g/ml OVA or PBS (control) for 10 min. A representative autoradiography from four independent experiments (A) and the inhibition of JNK activity (mean Ϯ S.D., n ϭ 4) (B) are shown. GST, glutathione S-transferase. The data are expressed as the percentage of JNK activity detected in the presence of 10 g/ml OVA and 0.01% Me 2 SO. (*, p Ͻ 0.05; **, p Ͻ 0.01.) C, MC/9 cells sensitized with OVA-IgE were incubated with 0.01% Me 2 SO (0 nM) or 3-1000 nM wortmannin for 15 min. The cells were then incubated together with 10 g/ml OVA for 5 min. ERK2 activity was measured as described under "Materials and Methods." The data (mean Ϯ S.D. of five independent experiments) are expressed as the percentage of ERK2 activity stimulated by 10 g/ml OVA in the presence of 0.01% Me 2 SO. been shown to activate JNK (47,48). Functionally, antigenstimulated JNK activity in mast cells may function in the regulation of AP-1 activity and cytokine gene expression (2, 3, 49 -52). Our studies provide the first evidence for the regulation of the JNK pathway in mast cells and will allow genetic analysis of the role of this pathway in mast cell biology.