Introduction

Mast cells are centrally important in inflammatory and immediate allergic reactions (Metcalfe et al., 1997). Ligation of the high-affinity IgE receptor (FcRI) triggers the release of pre-formed inflammatory mediators including histamine and the synthesis of specific proinflammatory cytokines. Mast cells also express the tyrosine kinase-encoded receptor c-Kit and are responsive to its ligand, c-Kit ligand (KL, also referred to as stem cell factor, SCF). In mast cell lines or bone marrow-derived mast cells, mitogen-activated protein kinase (MAPK) pathways including extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK) and p38 MAPK are activated by antigen–IgE ligation of FcRI and KL ligation of c-Kit (Tsai et al., 1993; Ishizuka et al., 1996, 1998, 1999). An important role for these MAPK pathways in mast cell function is predicted based on the ability of ERK1/2, JNK and p38 to regulate the transcriptional activity of genes important in mast cell function, such as cytokine genes (Rooney et al., 1995; Faris et al., 1996; Ishizuka et al., 1997; Nishina et al., 1997).

MAPKs are regulated by a family of proteins known as MAPK kinases (MKKs), which are in turn regulated by a family of MKK kinases (MKKKs or MAP3Ks) (Widmann et al., 1999). Transfection studies in mast cells and Jurkat T cells hint at a role for the MEKK group of MAP3Ks in antigen regulation of cytokine production (Faris et al., 1996; Ishizuka et al., 1997). We used kinase-inactive mutants of different MAP3Ks in initial transfection screens to define the role of specific MAPK pathways in mast cell functions responsive to FcRI- and c-Kit-mediated stimulation. In MC/9 mouse mast cells, these studies identified MEKK2 as having a role in the regulation of JNK activation and tumor necrosis factor- (TNF-) production following FcRI ligation. MEKK2 is a 70 kDa member of the MEKK group of MAP3Ks that has been shown to regulate the JNK and ERK5 pathways (Blank et al., 1996; Sun et al., 2000). We have shown recently that MEKK2 is recruited to the T-cell receptor (TCR) signaling complex upon presentation of antigen to T cells (Schaefer et al., 1999). MEKK2 was activated in response to antigen presentation and was required for stabilization of conjugates of T cells and antigen-presenting cells. MEKK1 and MEKK3 were not recruited to the TCR signaling complex in response to antigen, showing the selectivity of this response to MEKK2. Also, MEKK2^-/- embryonic stem (ES) cells were used for Rag2^-/- blastocyst complementation to define the role of MEKK2 in lymphocyte development (B.C. Schaefer, T.P.Garrington, S.Webb, D.M.Russell, E.W.Gelfand, J.W.Kappler, G.L.Johnson and P.Marrack, submitted). It was found that MEKK2 was required for B- and T⁺-cell development beyond the pre-BCR and pre-TCR signaling checkpoints, respectively.

To define further the involvement of MEKK2 in antigen-responsive receptor signaling, the technique of in vitro differentiation of ES cells to ES cell-derived mast cells (ESMC) was employed. ESMC provided a powerful system to define the role of MEKK2 in signaling by the mast cell high-affinity IgE receptor, FcRI. Our results demonstrate that MEKK2 is a critical MAP3K in mast cell receptor signaling and in the control of cytokine production in mast cells.

Results

MEKK2 is stimulated by FcRI ligation and can stimulate TNF- promoter activity

Activation of FcRI in MC/9 mouse mast cells by sensitization with anti-ovalbumin IgE followed by cross-linking with ovalbumin (IgE-ova) stimulated TNF- promoter-regulated expression of luciferase (Figure 1A). IgE-ova activation of FcRI activated MEKK2 in MC/9 cells as measured by immunoprecipitation of MEKK2 from control and stimulated cells followed by an in vitro kinase assay (Figure 1B). MEKK2 expression in MC/9 cells also activated TNF- promoter-regulated expression of luciferase (Figure 1C). Cumulatively, the findings in MC/9 cells indicate that the mast cell high-affinity IgE receptor, FcRI, activates MEKK2 in a manner similar to that observed with TCR ligation in D10 T cells (Schaefer et al., 1999). Furthermore, MEKK2 expression is able to stimulate TNF- promoter activity similarly to IgE-ova activation of FcRI. These results provide evidence for pathways utilizing MEKK2 to link FcRI ligation with stimulation of TNF- promoter activity. In order to define unequivocally the role of MEKK2 in mast cell receptor signaling, the MEKK2 gene was inactivated by targeted gene disruption.

Production of homozygous MEKK2^-/- ES cells

Targeted disruption of the MEKK2 gene in mouse ES cells was accomplished using the gene targeting strategy shown in Figure 2A. The targeting vector was constructed by replacing a 5.7 kb fragment of the MEKK2 gene containing the start site exon, the downstream exon and the intervening intron with a neomycin resistance gene. Both exons were interrupted, the start site was lost and a frameshift mutation was introduced. NotI, BamHI and XhoI restriction sites were inserted by PCR to facilitate placement of the gene fragments into the targeting vector. Outside of the region of homology there was a thymidine kinase gene to allow positive–negative selection. The linearized construct was introduced into R1 ES cells by electroporation. The cells were grown in G418 (400 g/ml) and ganciclovir (2 M) for positive–negative selection. G418- and ganciclovir-resistant clones were screened for homologous recombination by Southern (DNA) blotting. The frequency of homologous recombination was 14%. For the generation of MEKK2^-/- ES cells, the MEKK2^+/- clones were grown in high concentration (10 mg/ml) G418 to select for clones in which replacement of the remaining wild-type MEKK2 gene by the mutant gene had occurred. A Southern blot analysis of wild-type (MEKK2^+/+), heterozygous (MEKK2^+/-) and homozygous (MEKK2^-/-) ES cells is shown in Figure 2B. Two feeder-independent R1 MEKK2^-/- clones were developed for further studies, and the clones were screened for MEKK2 protein expression by western blotting. Figure 2C shows that the MEKK2 protein is absent in homozygous -/- ES cell clones.

In vitro differentiation of MEKK2^-/- ES cells into mast cells is normal

Mast cells were differentiated in vitro from ES cells (see Materials and methods). Wild-type and MEKK2^-/- ES cells were first differentiated to embryoid bodies (EBs) for 6 days in culture. The EBs were dissociated with trypsin and the cells were cultured for 4–12 weeks in media containing interleukin-3 (IL-3) and KL. Light microscopy after May Grünwald/Giemsa staining showed similar morphologies of wild-type and MEKK2^-/- ESMC (not shown). Electron microscopy showed that wild-type and MEKK2^-/- ESMC had microvilli and granules characteristic of mast cells (Figure 3A). These findings indicated that the loss of MEKK2 expression did not alter morphological differentiation of ES cells to mast cells. Granules with the MEKK2^-/- and wild-type ESMC contained heparin and chymase (Figure 3B and C). Flow cytometric analysis of cell surface expression of c-Kit and FcRI also indicated similar receptor expression in MEKK2^-/- and wild-type ESMC (Figure 3D and E) (Valent and Bettelheim, 1992). Finally, the growth rate, differentiation potential and degranulation capability of MEKK2^-/- and wild-type ESMC were indistinguishable (not shown). Thus, loss of MEKK2 expression had no measurable effect on the growth and differentiation characteristics, morphology, granule content, degranulation or surface receptor expression of ESMC.

Cytokine mRNA biosynthesis is reduced markedly in MEKK2^-/- ESMC responding to stimulation through c-Kit and FcRI

Transfection studies in MC/9 mast cells indicated that MEKK2 can regulate TNF- promoter activity (Figure 1C). Several cytokines, including IL-1, IL-3, IL-4, IL-5, IL-6, IL-8, IL-13, granulocyte–macrophage colony-stimulating factor (GM-CSF) and TNF-, are synthesized and released by mature mast cells and are believed to be important in the responses initiated by mast cells and in their recruitment of other immune cells to sites of inflammation (Plaut et al., 1989; Paul et al., 1993; Jaffe et al., 1995, 1996; Okayama et al., 1995a, b; Gagari et al., 1997; Moller et al., 1998). The targeted disruption of MEKK2 expression was exploited to test directly the role of MEKK2 in regulating cytokine gene expression in response to receptor activation of mast cells. RNase protection assays (RPAs) were performed to measure the expression of specific cytokine mRNAs in ESMC. Wild-type and MEKK2^-/- ESMC were challenged with KL to stimulate c-Kit, IgE-ova to stimulate FcRI, or the combination of KL and IgE-ova, and then analyzed for cytokine mRNA expression. A panel of cytokines including IL-2, IL-4, IL-5, IL-6, IL-9, IL-15, GM-CSF, TNF- and interferon- (IFN-) was screened in control and stimulated ESMC. Figure 4A shows a time course for fold increase in IL-4 mRNA production in response to KL stimulation in wild-type and MEKK2^-/- ESMC. IL-4 mRNA expression peaked at 30–60 min following stimulation, and mRNA levels were markedly reduced in the MEKK2^-/- ESMC compared with wild-type ESMC. Figure 4B shows RPA results for five different cytokines 1 h after stimulation with KL, IgE-ova or the combination of KL and IgE-ova. The induction of cytokine mRNA expression was altered significantly in stimulated MEKK2^-/- ESMC compared with MEKK2^+/+ ESMC. In several independent experiments, the ability of FcRI or c-Kit ligation, alone or in combination, to stimulate the expression of the mRNAs for IL-4, IL-6, GM-CSF and TNF- was diminished markedly in MEKK2^-/- ESMC compared with wild-type ESMC. Quantitation of cytokine mRNA expression relative to control GAPDH and L32 mRNA expression is shown in Figure 4C. Interestingly, stimulation of IL-9 expression was not affected, indicating that the regulation of cytokine expression by MEKK2 is selective for specific cytokine genes. When FcRI and c-Kit were co-stimulated, there was synergy in the ability to stimulate cytokine mRNA expression. The mRNA levels for IL-4, IL-6, TNF- and GM-CSF were nonetheless reduced significantly in MEKK2^-/- relative to wild-type ESMC co-stimulated with IgE-ova and KL. The results clearly show that MEKK2 expression is required for normal receptor-stimulated induction of specific cytokine gene expression in mast cells.

Receptor stimulation of cytokine mRNA biosynthesis is normal in MEKK1^-/-ESMC

To demonstrate the specificity of MEKK2 in mast cell receptor regulation of cytokine production, MEKK1^-/- ESMC were analyzed for their response to ligation of c-Kit by KL. We have shown previously that MEKK1 is required for JNK activation in response to perturbation of the microtubule cytoskeleton (Yujiri et al., 1998). As with MEKK2^-/- ES cells, MEKK1^-/- ES cells differentiate normally to mast cells (not shown). Figure 5 shows expression of mRNA for IL-4 and TNF- in MEKK1^-/- ESMC compared with wild-type ESMC 1 h after stimulation with KL. IL-4 and TNF- mRNA expression is similar in wild-type and MEKK1^-/- ESMC, indicating that MEKK2, but not MEKK1, is required for c-Kit receptor-mediated regulation of mast cell cytokine production.

JNK activation is lost in response to c-Kit and FcRI stimulation of ESMC

Targeted disruption of different MAP3Ks results in the selective loss of specific signaling functions in response to different upstream stimuli, demonstrating a unique role for each of the different MAP3Ks in the regulation of MAPK signaling pathways. Figure 6 defines the regulation of MAPK signaling pathways in wild-type and MEKK2^-/- ESMC. We have shown previously that JNK activation in response to stress stimuli, including UV irradiation, heat shock, cold shock, hyperosmolarity, anisomycin, cytochalasin D and nocodazole, is normal in MEKK2^-/- ES cells (Yujiri et al., 1999; unpublished observations). MEKK2^-/- ESMC also have a normal JNK activation in response to exposure to UV irradiation (Figure 6A). Thus, the signal response for stress activation is intact in MEKK2^-/- ESMC. Dramatically, the ability of both FcRI and c-Kit to activate the JNK pathway is lost in MEKK2^-/- ESMC (Figure 6B). This result was observed in two independently generated MEKK2^-/- clones differentiated to ESMC. In contrast, ligation of FcRI (Figure 6C) or c-Kit (not shown) in MEKK1^-/- ESMC produces a JNK response similar to that in wild-type ESMC. Thus, loss of JNK activation in response to FcRI ligation is specific to the loss of MEKK2 expression. In contrast to the loss of JNK activation in MEKK2^-/- cells following stimulation of FcRI or c-Kit, the activation of ERK1/2 and p38 is normal (Figure 6D and E). Therefore, MEKK2 is required for FcRI and c-Kit activation of the JNK pathway but not for ERK1/2 or p38 activation. Importantly, the findings show that the knockout of MEKK1 or MEKK2 expression does not disrupt the overall integrity of MAPK pathway regulation. Rather, selectivity in loss of signaling to a specific stimulus is observed.

Our finding of loss of JNK activation in response to receptor stimulation in MEKK2^-/- ESMC prompted us to investigate whether or not the JNK pathway was acting as the primary mediator of IgE-ova- and KL-stimulated cytokine production in mast cells. We were unsuccessful in transfecting ESMC using a variety of methods, including lipofectamine, calcium phosphate and electroporation. For this reason, we used MC/9 mast cells to express a constitutively active MKK7–JNK fusion protein along with the same TNF- promoter-regulated luciferase reporter plasmid as used in Figure 1. Activated JNK failed to stimulate luciferase expression (data not shown), indicating that activated JNK alone is insufficient to stimulate cytokine gene expression in mast cells. We have shown recently that MEKK2 activates the BMK1/ERK5 pathway as well as the protein kinase C-related kinase, PRK2 (Sun et al., 2000; W.Sun, K.Kesavan, B.Schaefer, N.L.Johnson, M.Ware, E.W.Gelfand and G.L.Johnson, submitted). Figure 6F shows that in MC/9 mast cells, IgE-ova and KL are capable of activating BMK1/ERK5. Thus, it is likely that in addition to JNK, IgE-ova and KL are activating the BMK1/ERK5 pathway in ESMC. The expression of BMK1/ERK5 in ESMC is extremely low, and, although we can measure BMK1/ERK5 activation in MC/9 cells, we have been unable to measure BMK1/ERK5 activity in ESMC. Nonetheless, our cumulative work with MEKK2 indicates that it regulates more than one MAPK pathway and that the sum of its downstream effects, not limited to JNK activation and possibly including BMK1/ERK5 activation, is involved with controlling cytokine gene expression. While further work is required to determine the specific downstream pathways affected in addition to loss of JNK activation in MEKK2^-/- ESMC, our targeted gene disruption studies demonstrate that MEKK2 is the pivotal MAP3K that regulates the receptor stimulation of cytokine gene expression in mast cells.

Discussion

Our findings provide direct evidence that MEKK2-dependent signaling pathways regulate production of several cytokines in mast cells. The regulation of IL-4, IL-6, TNF- and GM-CSF is highly dependent on MEKK2, while the regulation of IL-9 shows no dependence on MEKK2 signaling. The promoters for these cytokine genes contain binding sites for several transcription factors, including AP-1, NF-AT, NF-IL6, STAT family members and GATA family members (Luo et al., 1996; Zhu et al., 1996; Shannon et al., 1997; Szabo et al., 1997; Grassl et al., 1999). TNF-, IL-6, IL-9 and GM-CSF gene transcription is also regulated in part by NF-B (Zhu et al., 1996; Blackwell and Christman, 1997). We have been unable to transfect ESMC successfully. For this reason, we have been unable to dissect promoter elements responsible for the differential regulation of IL-9 mRNA expression, which is MEKK2 independent, versus other cytokines such as IL-4 and TNF-, whose mRNA expression in response to receptor activation is dependent on MEKK2 expression.

More recently, we have shown that MEKK2 is critical for both B-cell and T⁺-cell development (B.C.Schaefer, T.P.Garrington, S.Webb, D.M.Russell, E.W.Gelfand, J.W.Kappler, G.L.Johnson and P.Marrack, submitted), a response that is dependent, respectively, on pre-BCR and pre-TCR antigen receptor signaling. Thus, MEKK2 is involved in the signaling of antigen receptors in both lymphocytes and mast cells. Interestingly, MEKK3, the MAP3K that is most closely related in homology to MEKK2, is not required for these responses. We have shown recently that MEKK2, but not MEKK3, binds to adaptor proteins that are recruited to activated protein tyrosine kinases such as Lck that associate with antigen receptors (W.Sun, K.Kesavan, B.Schaefer, N.L.Johnson, M.Ware, E.W.Gelfand and G.L.Johnson, submitted). The specificity for MEKK2 in antigen receptor signaling relative to MEKK1 or MEKK3 is predicted to be a function of MEKK2's interaction with these adaptor proteins.

Our results also demonstrate that specific MEKKs selectively control specific MAPK pathways. MEKK2 is required for JNK activation in response to FcRI and c-Kit ligation in mast cells. In MEKK1^-/- ES cells, JNK activation is diminished in response to cold shock, hyperosmolarity and treatment of the ES cells with lysophosphatidic acid, taxol or nocodazole. MEKK2^-/- cells have a normal JNK response to these stimuli (Yujiri et al., 1999). Thus, targeted disruption of MEKK1 or MEKK2 expression results in the selective loss of JNK activation in response to different stimuli. MEKK2 gene disruption has little if any effect on receptor-mediated or stress stimulation of ERK or p38 activity in mast cells. In activated T cells, however, MEKK2 has been found to regulate the activity of the ERK and p38 pathways (Schaefer et al., 1999), suggesting that protein scaffolding and subcellular localization processes unique to different cell types may be playing a role in determining MEKK2's downstream targets (Garrington and Johnson, 1999).

In addition to the JNK pathway, MEKK2 regulates BMK1/ERK5 and PRK2, a protein kinase C-related kinase (Sun et al., 2000; W.Sun, K.Kesavan, B.Schaefer, N.L.Johnson, M.Ware, E.W.Gelfand and G.L.Johnson, submitted). Thus, MEKK2 regulates several kinase relay pathways that are capable of regulating specific gene transcription. Regarding cytokine production, the phenotype of MEKK2^-/- ESMC appears different from that of the JNK1/JNK2 double knockout in T cells (Dong et al., 2000). MEKK2^-/- ESMC lose cytokine production in response to receptor activation, whereas the JNK1/JNK2 double knockouts are hyper-responsive with regard to TCR stimulation of IL-2 production. This difference is probably related in part to the fact that JNK is downstream of MEKK2 and represents only one arm of MEKK2-regulated signaling pathways. An important concept that emerges from these studies is that the phenotype of a MAP3K knockout, such as that for MEKK2, can include the very specific loss of a cellular function, such as receptor-stimulated cytokine production, even though the downstream MAPKs are responsive to alternative stimuli. Thus, while several MAP3Ks capable of stimulating a downstream target such as JNK may be present in a given cell, activation of a given pathway in response to a specific stimulus is dependent on the presence of a specific MAP3K member, giving greater selectivity and specificity of response to the system. The targeted disruption of MAP3K expression will define the selective role of different MAP3Ks in regulating kinase relay pathways in response to different cellular stimuli.

Materials and methods

Luciferase assay

For luciferase assays, MC/9 mast cells were first transfected by electroporation with 2.5 g of a pGL3 Basic vector (Promega) containing a 5' TNF- promoter sequence, along with 7.5 g of other experimental plasmids, including wild-type MEKK2, kinase-inactive MEKK2 (K385M) and pCMV5 empty vector. For electroporation, 1 10⁷ MC/9 cells were suspended in 0.5 ml of RPMI with 10% fetal calf serum (FCS) and kept at room temperature for 10 min. Cells were electroporated using a Gene Pulser II® (Bio-Rad) with pulse conditions of 800 F and 320 V, kept an additional 10 min at room temperature and then placed overnight in mast cell medium. For FcRI stimulation, cells were then incubated overnight at 37°C with 0.8 g/ml anti-ova IgE, washed and treated with 10 g/ml ovalbumin for 6 h. The MC/9 mast cells were then washed twice with phosphate-buffered saline (PBS) and lysed using Reporter Lysis Buffer (Promega). Luciferase assays were done using a Luciferase Assay System Kit (Promega).

MEKK2 kinase assay

MEKK2 kinase activity was assayed using a previously described method (Fanger et al., 1997). MC/9 cells were lysed in buffer containing 20 mM Tris–HCl pH 7.6, 0.5% NP-40, 0.25 M NaCl, 3 mM EDTA, 3 mM EGTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), 2 mM sodium vanadate, 10 g/ml aprotinin, 5 g/ml leupeptin and 1 mM dithiothreitol (DTT). Endogenously expressed MEKK2 was immunoprecipitated with rabbit anti-C-terminal MEKK2 antibody (1:100). The immunoprecipitate was incubated at 30°C for 20 min in kinase buffer (20 mM HEPES pH 7.5, 10 mM MgCl₂, 10 mM -glycerophosphate, 10 mM p-nitrophenyl phosphate, 1 mM DTT, 50 M sodium vanadate) containing wild-type JNK (polyhistidine-human JNK1), wild-type SEK1 (GST–mouse SEK1) and 50 M ATP. This reaction was then incubated with GST–c-Jun substrate conjugated to Sepharose beads for 20 min at 4°C, before being washed and incubated for 20 min at 30°C in kinase buffer containing 10 Ci of [-³²P]ATP. The kinase reaction was stopped by addition of SDS sample buffer, separated by SDS–PAGE and visualized with autoradiography. Phosphorylation of GST–c-Jun was quantified by PhosphorImager analysis (Molecular Dynamics).

Screening of a genomic DNA library and MEKK2 knockout vector construction

Targeted gene disruption of MEKK1 has been described previously (Yujiri et al., 1998). For targeted disruption of MEKK2, an MEKK2 genomic clone was isolated by screening a FixII phage library prepared from mouse strain 129/sv (Stratagene) using an MEKK2 cDNA fragment that included bp 593–1032 of the MEKK2 gene. The targeting vector was constructed by inserting a 4 kb fragment from the 5' end of the genomic clone in which NotI and BamHI restriction sites were engineered by PCR for ligation into the vector, called Osdupdel (a gift from Dr O.Smithies). A 2.5 kb fragment from the 3' end of the genomic clone was inserted, using an NheI site and an XhoI site inserted by PCR. This construct deleted 40 codons, including the ATG start site of the MEKK2 gene, and inserted the neomycin resistance gene with a polyadenylation signal in the antisense orientation. Outside of the region of homology, there was a thymidine kinase gene for negative selection of randomly inserted vectors.

ES cell electroporation, and selection of +/- and -/- clones

The targeting vector was linearized using NotI and electroporated into an R1 ES cell line using a Bio-Rad Gene Pulser™. A total of 1 10⁷ cells suspended in 0.8 ml of PBS were electroporated using pulse conditions of 250 V and 500 F. Cells were then placed on ice for 5 min and plated onto 100 mm dishes with feeder cells. The cells were then grown in medium containing G418 (400 g/ml) and ganciclovir (2 mM) for 8 days. Surviving clones were picked and grown in medium lacking G418 and ganciclovir. A portion of the cells was collected for DNA analysis by Southern blotting, and the remaining cells were frozen in liquid N₂. For selection of MEKK2^-/- clones, MEKK2^+/- clones were grown in medium containing G418 (10 mg/ml). After 7 days, surviving clones were picked, grown and used for DNA analysis or for freezing.

Mast cell differentiation

For differentiation of ES cells into mast cells, feeder cell-independent ES cells were first maintained on gelatinized dishes in medium containing Dulbecco's modified Eagle's medium (DMEM) (Gibco) with 15% FCS, 1.5 10^-4 M monothioglycerol (MTG) (Sigma) and10 ng/ml leukemia inhibitory factor (LIF) (R&D Systems). At 24–48 h prior to differentiation, the cells were placed in medium containing Iscove's modified Dulbecco's medium (IMDM) (Gibco) with15% FCS, 1.5 10^-4 M MTG and 10 ng/ml LIF. The ES cells were then trypsinized and washed in IMDM with 15% FCS and suspended at a concentration of 7.5 10³–12.5 10³ cells/ml in differentiation medium [15% FCS, 2 mM L-glutamine (Gibco), 300 g/ml transferrin (Boehringer Mannheim), 4 10^-4 M MTG, 50 g/ml ascorbic acid (Sigma), 5% protein-free hybridoma medium (PFHM-II) (Gibco) and IMDM (to 100%)] to a volume of 5 ml in 60 15 mm Petri grade dishes (Fisher).

The ES cells were then maintained in the differentiation medium for 5–6 days, forming embryoid bodies (EBs). The EBs were transferred to 50 ml conical tubes and allowed to settle by gravity for 10 min. The supernatant was removed and the EBs were dissociated by addition of 3 ml of trypsin-EDTA, incubation for 5 min at 37°C, and addition of 1 ml of FCS, followed by passage twice through a syringe with a 20 gauge needle. The dissociated cells were then centrifuged at 1200 r.p.m. for 5 min, and the pellet was resuspended in mast cell medium [10% FCS, 1.5 10^-4 M MTG, 1 ng/ml IL-3 (R&D Systems), 100 ng/ml c-Kit ligand (R&D Systems) and IMDM (to 100%)] and placed in 6-well dishes at a concentration of 1.0 10⁶–2.0 10⁶ cells/ml in a final volume of 3–4 ml. Non-adherent cells were transferred to new plates with fresh mast cell medium 24 h later, and again transferred 48–72 h later to remove any remaining adherent cells. Half of the medium was then replaced every 2–3 days, and the mast cells were grown and expanded over the following 4–12 weeks.

DNA extracts and Southern blotting

For DNA analysis, cells were harvested, washed twice in PBS and lysed in buffer containing 0.35 M NaCl, 15 mM sodium citrate pH 7.0, 0.5% SDS, 1 mM DTT and 80 g/ml proteinase K, and incubated overnight at 37°C. DNA was then extracted twice in 1:1 phenol:chloroform and once in chloroform, followed by ethanol precipitation. The DNA was then digested overnight with XbaI, resolved on a 0.8% agarose gel, and transferred by Southern blot to Hybond™-N nylon membrane (Amersham). The blots were hybridized with [-³²P]CTP-labeled DNA probe overnight, washed and visualized by autoradiography.

Protein extracts and western blotting

Cells were harvested, washed twice in PBS and lysed in buffer containing 20 mM Tris–HCl pH 7.5, 0.5% NP-40, 0.25 M NaCl, 3 mM EDTA, 3 mM EGTA, 1 mM PMSF, 2 mM sodium vanadate, 0.2 mg/ml aprotinin, 1 mg/ml leupeptin and 1 mM DTT. Nuclei were removed by centrifugation at 15 000 g for 10 min. A total of 200 g of protein per sample was resolved on SDS–10% polyacrylamide gels. Protein was then electroblotted to nitrocellulose membrane. MEKK2 protein was detected using rabbit antiserum (1:500 dilution) against the N-terminus of MEKK2, followed by protein A conjugated to horseradish peroxidase (protein A–HRP) (Zymed). Western blots were then visualized by chemiluminescence using ECL (Amersham). Western blots were also done using rabbit antiserum (1:500 dilution) against the C-terminus of MEKK2. Western blots for ERK1/2 activity used a phospho-p44/42 MAPK (T202/Y204) rabbit polyclonal antibody (New England Biolabs) followed by an HRP-linked donkey anti-rabbit IgG antibody (Jackson Immunoresearch Laboratories). Western blots for p38 activity used a phospho-p38 MAPK (Thr180/Tyr182) rabbit polyclonal antibody (Cell Signaling Technology) followed by an HRP-linked donkey anti-rabbit IgG antibody.

For BMK1/ERK5 western blots, cells were lysed in buffer containing 25 mM Tris–HCl pH 7.4, 50 mM NaCl, 0.5% sodium deoxycholate, 2% NP-40, 0.2% SDS, 1 mM PMSF, 50 g/ml aprotinin and 50 M leupeptin at 4°C for 15 min. Samples were boiled and resolved on SDS–7% polyacrylamide, and the protein was electroblotted to nitrocellulose membrane. ERK5 protein was detected using a rabbit polyclonal antibody (1:2000 dilution) to a synthetic peptide corresponding to the C-terminus of ERK5 (Sigma), followed by a protein G–HRP conjugate (Bio-Rad) at a 1:2000 dilution.

Flow cytometry

For analysis of c-Kit expression, mast cells were treated with 1 g/ml fluorescein isothiocyanate (FITC)-conjugated anti-c-Kit antibody (PharMingen) for 30 min at 4°C, washed with PBS and analyzed by flow cytometry. Negative controls were untreated. For analysis of FcRI expression, mast cells were treated with 1 g/ml anti-DNP IgE for 30 min at 4°C, washed with culture medium then treated with 1 g/ml FITC–anti-IgE (PharMingen) for 30 min at 4°C and analyzed. Negative controls were treated with FITC–anti-IgE alone.

Assay of JNK activity

JNK activity was measured using GST–c-Jun_(1–79) coupled to glutathione–Sepharose 4B (Hibi et al., 1993). Cells were lysed in buffer containing 20 mM Tris–HCl pH 7.6, 0.5% NP-40, 0.25 M NaCl, 3 mM EDTA, 3 mM EGTA, 1 mM PMSF, 2 mM sodium vanadate, 0.20 g/ml aprotinin, 5 g/ml leupeptin and 1 mM DTT. Nuclei were removed by centrifugation at 15 000 g for 10 min, and the supernatants (100 g of protein) were mixed with 10 l of a slurry of GST–c-Jun_(1–79)–Sepharose [3–5 g of GST–c-Jun_(1–79)]. The mixture was rotated at 4°C for 1 h, and washed twice in lysis buffer and once in kinase buffer (20 mM HEPES pH 7.5, 10 mM MgCl₂, 20 mM -glycerophosphate, 10 mM p-nitrophenyl phosphate, 1 mM DTT, 50 M sodium vanadate). Beads were suspended in 40 l of kinase assay buffer containing 10 Ci of [-³²P]ATP and incubated at 30°C for 20 min. The kinase reaction was stopped by addition of SDS sample buffer, and phosphorylated proteins were resolved on SDS–10% polyacrylamide gels. Results were visualized by autoradiography and quantified by PhosphorImaging (Molecular Dynamics).

Assay of p38 kinase activity

p38 activity was measured using an in vitro kinase assay (Abdel-Hafiz et al., 1992; Han et al., 1994). Cells were lysed in buffer containing 20 mM Tris–HCl pH 7.6, 0.5% NP-40, 1% Triton X-100, 150 mM NaCl, 20 mM NaF, 1 mM EDTA, 1 mM EGTA, 5 mM PMSF and 0.2 mM sodium vanadate. p38 was immunoprecipitated from cell lysates (500 g of protein) by mixture with rabbit antiserum (1:100 dilution) raised against the C-terminal peptide sequence of p38 to a final volume of 500 l and rotation at 4°C for 1 h. Immune complexes were captured by adding 10 l of a 1:1 slurry of protein A–Sepharose (Sigma). The mixture was rotated at 4°C for an additional hour and washed twice in lysis buffer and once in kinase buffer (25 mM HEPES, 25 mM -glycerophosphate, 25 mM MgCl₂, 2 mM DTT, 0.1 mM sodium vanadate). Beads were suspended in 40 l of kinase assay buffer containing 3 Ci of [-³²P]ATP and 2 g of ATF-2 as a substrate, and incubated at 30°C for 30 min. Reaction mixtures were added to Laemmli sample buffer, boiled, and phosphorylated proteins were resolved on SDS–10% polyacrylamide gels. Results were visualized by autoradiography.

RPA analysis

For RPA analysis, a RiboQuant® Multi-Probe RNase Protection Assay System (PharMingen) was used. An mCK-1 custom probe set (PharMingen) was designed, containing DNA templates for IL-2, IL-4, IL-5, IL-6, IL-9, IL-15, GM-CSF, TNF-, IFN-, L32 and GAPDH. The DNA template set was used for T7 RNA-polymerase-directed synthesis of [-³²P]UTP-labeled antisense RNA probes. The probes were hybridized with 20 ng of RNA isolated from mast cells using RNAzol™B (Tel-Test, Inc). Samples were then digested with RNases to remove single-stranded (non-hybridized) RNA. The remaining probes were resolved on denaturing polyacrylamide gels. Quantitation was done by PhosphorImaging (Molecular Dynamics).

Acknowledgements

We thank Dr Oliver Smithies for the Osdupdel gene targeting vector. Supported in part by the Leukemia Research Foundation, the Cancer League of Colorado and NIH grants CA85276, DK37871, GM30324 and AI42246.

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