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• W2024543722 abstract "The stem cell factor (SCF) plays a central role in the regulation of mast cell function and growth. However, roles of transcription factors involved in these processes remain incompletely defined. The early growth response factor-1 (Egr-1) is a member of the zinc finger transcription factor family. A role for Egr-1 in SCF-induced mast cell activation and growth was investigated in mouse bone marrow-derived mast cells (BMMC). The stimulation of BMMC with SCF induced a strong expression of Egr-1 mRNA. SCF-induced Egr-1 nuclear translocation and DNA binding were demonstrated by electrophoretic mobility shift assay (EMSA) and immunofluorescence assay. SCF-induced IL-13 expression was significantly reduced at both mRNA and protein levels in Egr-1-deficient BMMC. In addition, the synergy between IgE and SCF on IL-13 and IL-4 production was reduced in Egr-1-deficient mast cells. Interestingly, Egr-1 deficiency had little effect on SCF-induced mast cell growth. SCF-induced Egr activation likely requires tyrosine phosphorylation because a tyrosine kinase inhibitor PP2 blocked SCF-induced nuclear protein binding to Egr probe as determined by EMSA. Thus, Egr-1 is required for SCF-induced IL-13 expression, but not mast cell growth. Egr-1 represents a novel mechanism for SCF-induced mast cell activation. The stem cell factor (SCF) plays a central role in the regulation of mast cell function and growth. However, roles of transcription factors involved in these processes remain incompletely defined. The early growth response factor-1 (Egr-1) is a member of the zinc finger transcription factor family. A role for Egr-1 in SCF-induced mast cell activation and growth was investigated in mouse bone marrow-derived mast cells (BMMC). The stimulation of BMMC with SCF induced a strong expression of Egr-1 mRNA. SCF-induced Egr-1 nuclear translocation and DNA binding were demonstrated by electrophoretic mobility shift assay (EMSA) and immunofluorescence assay. SCF-induced IL-13 expression was significantly reduced at both mRNA and protein levels in Egr-1-deficient BMMC. In addition, the synergy between IgE and SCF on IL-13 and IL-4 production was reduced in Egr-1-deficient mast cells. Interestingly, Egr-1 deficiency had little effect on SCF-induced mast cell growth. SCF-induced Egr activation likely requires tyrosine phosphorylation because a tyrosine kinase inhibitor PP2 blocked SCF-induced nuclear protein binding to Egr probe as determined by EMSA. Thus, Egr-1 is required for SCF-induced IL-13 expression, but not mast cell growth. Egr-1 represents a novel mechanism for SCF-induced mast cell activation. Mast cells play a central role in allergic reactions. They originate from pluripotential progenitor cells in bone marrow and acquire mature phenotype in tissues through migration and differentiation (1Metcalfe D.D. Baram D. Mekori Y.A. Physiol. Rev. 1997; 77: 1033-1079Crossref PubMed Scopus (1789) Google Scholar). Mast cells are characterized by expression of high affinity IgE receptor (FcɛRI) and c-Kit (CD117), the stem cell factor (SCF) 3The abbreviations used are: SCF, stem cell factor; BMMC, bone marrow-derived mast cells; PI, phosphatidylinositol; PKC, protein kinase C; MAP, mitogen-activated protein; IL, interleukin; FACS, fluorescent-activated cell sorting; FITC, fluorescein isothiocyanate; Egr-1, early growth response factor-1; EMSA, electrophoretic mobility shift assay; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TNP, trinitrophenyl; BSA, bovine serum albumin; DAPI, 4′,6-diamidino-2-phenylindole. receptor (1Metcalfe D.D. Baram D. Mekori Y.A. Physiol. Rev. 1997; 77: 1033-1079Crossref PubMed Scopus (1789) Google Scholar). The c-Kit ligand, SCF, is critical for the development, survival, and proliferation of mast cells (reviewed in Ref. 2Galli S.J. Zsebo K.M. Geissler E.N. Adv. Immunol. 1994; 55: 1-96Crossref PubMed Scopus (561) Google Scholar). SCF initiates its effects by binding to c-Kit, which possesses intrinsic tyrosine kinase activity (2Galli S.J. Zsebo K.M. Geissler E.N. Adv. Immunol. 1994; 55: 1-96Crossref PubMed Scopus (561) Google Scholar). Binding of SCF to c-Kit results in receptor dimerization and activation of protein kinase activity. Activation of c-Kit initiates multiple signaling pathways as a result of interaction with several enzymes and adaptor proteins (3Ronnstrand L. Cell. Mol. Life Sci. 2004; 61: 2535-2548Crossref PubMed Scopus (336) Google Scholar, 4Roskoski Jr., R. Biochem. Biophys. Res. Commun. 2005; 337: 1-13Crossref PubMed Scopus (225) Google Scholar). These include Src kinase, phosphatidylinositol-3 (PI 3)-kinase, phospholipase C (PLC)-γ, mitogen-activated protein (MAP) kinase pathways, and others (3Ronnstrand L. Cell. Mol. Life Sci. 2004; 61: 2535-2548Crossref PubMed Scopus (336) Google Scholar, 4Roskoski Jr., R. Biochem. Biophys. Res. Commun. 2005; 337: 1-13Crossref PubMed Scopus (225) Google Scholar). SCF-induced activation of the Src family of tyrosine kinases has been implicated in gene transcription (5Lennartsson J. Blume-Jensen P. Hermanson M. Ponten E. Carlberg M. Ronnstrand L. Oncogene. 1999; 18: 5546-5553Crossref PubMed Scopus (173) Google Scholar, 6Bondzi C. Litz J. Dent P. Krystal G.W. Cell Growth Differ. 2000; 11: 305-314PubMed Google Scholar) and mast cell chemotaxis (3Ronnstrand L. Cell. Mol. Life Sci. 2004; 61: 2535-2548Crossref PubMed Scopus (336) Google Scholar, 4Roskoski Jr., R. Biochem. Biophys. Res. Commun. 2005; 337: 1-13Crossref PubMed Scopus (225) Google Scholar, 7O'Laughlin-Bunner B. Radosevic N. Taylor M.L. Shivakrupa DeBerry C. Metcalfe D.D. Zhou M. Lowell C. Linnekin D. Blood. 2001; 98: 343-350Crossref PubMed Scopus (81) Google Scholar). SCF-induced PI 3-kinase is essential for mast cell development (3Ronnstrand L. Cell. Mol. Life Sci. 2004; 61: 2535-2548Crossref PubMed Scopus (336) Google Scholar, 4Roskoski Jr., R. Biochem. Biophys. Res. Commun. 2005; 337: 1-13Crossref PubMed Scopus (225) Google Scholar, 8Fukao T. Yamada T. Tanabe M. Terauchi Y. Ota T. Takayama T. Asano T. Takeuchi T. Kadowaki T. Hata Ji J. Koyasu S. Nat. Immunol. 2002; 3: 295-304Crossref PubMed Scopus (184) Google Scholar). Thus, each SCF-induced signaling pathway is associated with specific biological functions. Upon activation, mast cells secrete three major categories of mediators including preformed mediators such as histamine, lipid mediators such as leukotrienes and various cytokines and chemokines, such as IL-13 (1Metcalfe D.D. Baram D. Mekori Y.A. Physiol. Rev. 1997; 77: 1033-1079Crossref PubMed Scopus (1789) Google Scholar, 9Kinet J.P. Annu. Rev. Immunol. 1999; 17: 931-972Crossref PubMed Scopus (855) Google Scholar). Although mast cells are able to produce all three categories of mediators, the kinetic, amount, and type of particular mediators secreted are dependent upon the nature of individual stimuli (10Galli S.J. Kalesnikoff J. Grimbaldeston M.A. Piliponsky A.M. Williams C.M. Tsai M. Annu. Rev. Immunol. 2005; 23: 749-786Crossref PubMed Scopus (1065) Google Scholar). Studies of the regulation of release of these mediators from mast cells have largely focused on FcɛRI-mediated signaling pathways. However, FcɛRI functions physiologically in the context of c-Kit. FcɛRI and c-Kit serve distinct as well as overlapping functions in mast cells (11Hundley T.R. Gilfillan A.M. Tkaczyk C. Andrade M.V. Metcalfe D.D. Beaven M.A. Blood. 2004; 104: 2410-2417Crossref PubMed Scopus (134) Google Scholar). This is because FcɛRI and c-Kit mediate unique and convergent signals for release of inflammatory mediators from mast cells (11Hundley T.R. Gilfillan A.M. Tkaczyk C. Andrade M.V. Metcalfe D.D. Beaven M.A. Blood. 2004; 104: 2410-2417Crossref PubMed Scopus (134) Google Scholar). Recently we demonstrated that a transcription factor, early growth response-1 (Egr-1) is required for the FcɛRI-induced cytokine TNF and IL-13 production by mast cells (12Li B. Power M.R. Lin T.J. Blood. 2006; 107: 2814-2820Crossref PubMed Scopus (37) Google Scholar). It is unclear whether Egr-1 regulates SCF-induced mast cell cytokine production or SCF-dependent mast cell growth. Egr-1 is an 80–82-kDa zinc finger transcription factor. It is the prototype of the Egr family that includes Egr-1, Egr-2, Egr-3, Egr-4, and the Wilm’s tumor product (13Gashler A. Sukhatme V.P. Prog. Nucleic Acids Res. Mol. Biol. 1995; 50: 191-224Crossref PubMed Scopus (557) Google Scholar, 14Beckmann A.M. Wilce P.A. Neurochem Int. 1997; 31 (; discussion 517-476): 477-510Crossref PubMed Scopus (277) Google Scholar). Members of the Egr family have been associated in a large number of biological effects including cell growth and gene expression (13Gashler A. Sukhatme V.P. Prog. Nucleic Acids Res. Mol. Biol. 1995; 50: 191-224Crossref PubMed Scopus (557) Google Scholar, 14Beckmann A.M. Wilce P.A. Neurochem Int. 1997; 31 (; discussion 517-476): 477-510Crossref PubMed Scopus (277) Google Scholar). Importantly, the biological effects of Egr-1 appear to be cell type-specific (13Gashler A. Sukhatme V.P. Prog. Nucleic Acids Res. Mol. Biol. 1995; 50: 191-224Crossref PubMed Scopus (557) Google Scholar, 14Beckmann A.M. Wilce P.A. Neurochem Int. 1997; 31 (; discussion 517-476): 477-510Crossref PubMed Scopus (277) Google Scholar, 15Nguyen H.Q. Hoffman-Liebermann B. Liebermann D.A. Cell. 1993; 72: 197-209Abstract Full Text PDF PubMed Scopus (352) Google Scholar, 16Faour W.H. Alaaeddine N. Mancini A. He Q.W. Jovanovic D. Di Battista J.A. J. Biol. Chem. 2005; 280: 9536-9546Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Through interaction with other transcription factors or tissue-specific factors, Egr-1 acts to activate or inhibit gene expression or cell differentiation (13Gashler A. Sukhatme V.P. Prog. Nucleic Acids Res. Mol. Biol. 1995; 50: 191-224Crossref PubMed Scopus (557) Google Scholar, 14Beckmann A.M. Wilce P.A. Neurochem Int. 1997; 31 (; discussion 517-476): 477-510Crossref PubMed Scopus (277) Google Scholar, 15Nguyen H.Q. Hoffman-Liebermann B. Liebermann D.A. Cell. 1993; 72: 197-209Abstract Full Text PDF PubMed Scopus (352) Google Scholar, 16Faour W.H. Alaaeddine N. Mancini A. He Q.W. Jovanovic D. Di Battista J.A. J. Biol. Chem. 2005; 280: 9536-9546Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). In this study, we examined SCF-induced expression and function of Egr-1 in mast cells. SCF stimulated a rapid expression of Egr-1 mRNA and protein. By using Egr-1-deficient mast cells, we showed that SCF-induced IL-13 production requires Egr-1. Inhibition of Src family kinases, but not PI 3-kinase, PKC, or MAP kinase blocked SCF-induced Egr-1 activation, suggesting a role for Src kinases in SCF-induced Egr-1 activation. Surprisingly, SCF induced mast cell growth was not affected in the absence of Egr-1. Taken together, Egr-1 represents a novel mechanism in SCF-induced cytokine IL-13 production by mast cells. Animals—Egr-1-deficient mice and control C57BL/6NTac mice were purchased from Taconic Farms (Germantown, NY). The protocols were approved by the University Committee on Laboratory Animals, Dalhousie University, in accordance with the guidelines of the Canadian Council on Animal Care. Antibodies and Reagents—Antibodies to Egr-1 (sc-189 for immunofluorescence study and sc-189X for the blockade of DNA-protein complex formation), Egr-2 (sc-20690), Egr-3 (sc-22801X), and Egr-4 (sc-19868X) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Alexa 594-conjugated goat anti-rabbit IgG F(ab′)2 fragment was purchased from Molecular Probes (Eugene, OR). FITC anti-mouse CD117 mAb (CL8936F), FITC rat IgG2a (CLCR2A01) were purchased from Cedarlane Laboratories Limited (Ontario, Canada). Recombinant murine stem cell factor (SCF, catalog no. 250-03) was purchased from PeproTech Incorporation (Rocky Hill, NJ). Mast Cell Culture and Stimulation—Murine primary cultured bone marrow-derived mast cells (BMMC) were cultured as previously described (12Li B. Power M.R. Lin T.J. Blood. 2006; 107: 2814-2820Crossref PubMed Scopus (37) Google Scholar). Following 4–5 weeks of culture, mast cell purity of greater than 98% was achieved as assessed by toluidine blue staining (pH 1.0) of fixed cytocentrifuged preparations. BMMC (1 × 106 cells/ml) were activated by addition of various concentrations of SCF (0.5–100 ng/ml) for various times (5 min to 24 h). In the case of IgE+antigen stimulation, BMMC were sensitized with IgE directed against trinitrophenyl (TNP) as previously described (12Li B. Power M.R. Lin T.J. Blood. 2006; 107: 2814-2820Crossref PubMed Scopus (37) Google Scholar). TNP-bovine serum albumin (BSA; Biosearch Technologies, Inc., Novato, CA) was used as an antigen. Nuclear Extract Preparation and Electrophoretic Mobility Shift Assay (EMSA)—Nuclear protein extracts were obtained using a nuclear extract kit (catalog no. 40010; Active Motif, Carlsbad, CA), according to the manufacturer’s protocol. All preparation procedures were carried out at 4 °C. Total protein concentration was determined using the Bio-Rad protein assay (catalog no. 500-0006; Bio-Rad Laboratories). EMSA was carried out as previously described (12Li B. Power M.R. Lin T.J. Blood. 2006; 107: 2814-2820Crossref PubMed Scopus (37) Google Scholar). The following double-stranded oligonucleotides were used: wild-type Egr-1 probe, 5′-GGA TCC AGC GGG GGC GAG CGG GGG CGA ACG-3′ (catalog no. 1200011; Geneka Biotechnology, Montreal, QC, Canada. Note, this probe recognizes all 4 Egr family members, although it is indicated as an Egr-1 probe by the manufacturer); mutant Egr-1 probe, 5′-GGA TCC AGC GGG GTA GAG CGG GTA CGA ACG-3′ (catalog no. 1400011; Geneka Biotechnology); Sp-1 probe, 5′-ATT CGA TCG GGG CGG GGC GAG C-3′ (catalog no. E323B; Promega, Madison, WI), and Ap-1 probe, 5′-CGC TTG ATG AGT CAG CCG GAA-3′ (catalog no. E320B; Promega, Madison, WI). Probe-labeling was accomplished by treatment with T4 poly kinase (catalog no. M410A; Promega) in the presence of adenosine 5′-[32P]triphosphate (catalog no. PB10218; Amersham Biosciences). Labeled oligonucleotides were purified using a MicroSpin G-25 column (catalog no. 275325; Amersham Biosciences). Nuclear protein (8 μg) was added to a total volume of 10 μl of the binding reaction with 1 μl of (0.75 μg/μl) poly(dI-dC) (catalog no. 27-7880-02; Amersham Biosciences) and incubated at room temperature for 15 min. Labeled oligonucleotides were added to each reaction mixture and incubated at room temperature for 30 min. Samples were then separated by electrophoresis on a 6% polyacrylamide gel in 0.5× Tris boric acid-EDTA buffer at room temperature for 1 h at 300 V. Gels were pre-run for 30 min at 100 V before samples were loaded. Gels were vacuum-dried and subjected to autoradiography overnight at -80 °C. For competition assays, 1 μl of nonradiolabeled wild-type Egr-1, mutant Egr-1, Sp1, or Ap-1 oligonucleotides (50-fold excess of radiolabeled probe) were added and incubated for 15 min before the addition of the radiolabeled probe. For assays using antibodies to block the DNA-protein complex formation, nuclear extracts were incubated in a total volume of 20 μl with 6 μl of appropriate polyclonal antibodies to Egr-1 (sc-189X), Egr-2 (sc-190X), Egr-3 (sc-22 801X), or Egr-4 (sc-19868X), respectively, for 2 h on ice before the addition of the radiolabeled probe. Real-time Quantitative PCR—Total RNA was isolated from BMMCs using TRIzol Reagent (catalog no. 15596-026; Invitrogen) and reverse-transcribed using SuperScript II RNase H-Reverse Transcriptase (catalog no. 18064-014; Invitrogen) according to the manufacturer’s instruction. Real-time quantitative polymerase chain reaction (PCR) was performed using a 7000 Sequence detector (PE Applied Biosystems, Foster City, CA). Specific quantitative assays for Egr-1, 2, and IL-13 were performed using Assays-on-Demand reagents containing 6-FAM dye-labeled TaqMan MGB probes (Applied Biosystems). GAPDH was used as an endogenous reference. Data were analyzed using relative standard curve method according to the manufacturer’s protocol. An average value of each gene after GAPDH normalization at the time point showing highest expression was used as a calibrator to determine the relative levels of Egr-1, 2, or IL-13 at different conditions as previously described (12Li B. Power M.R. Lin T.J. Blood. 2006; 107: 2814-2820Crossref PubMed Scopus (37) Google Scholar). In addition, PCR products of Egr-1, 2, IL-13, and GAPDH were separated on 2% agarose gel and stained with ethidium bromide. Immunofluorescence Study—BMMCs (3 × 106 cells/sample) were stimulated with SCF (100 ng/ml) for 2 h, or pretreated with SCF (100 ng/ml) for 1 h, then stimulated with various concentrations of TNP-BSA for an additional 1 h. After stimulation, BMMCs were fixed and permeabilized using Cytofix/Cytoperm solution (catalog no. 554714; BD Biosciences Pharmingen, San Diego, CA) for 20 min at 4 °C. Cells were then blocked with 5% normal goat serum (catalog no. CL1200, Cedarlane Laboratories) for 1 h at 4 °C. Then, cells were incubated with 4 μg/ml rabbit polyclonal anti-Egr-1 antibody (catalog no. sc-189; Santa Cruz Biotechnology) for 3 h at 4 °C. Cells were then stained for 2 h with Alexa Fluor-594-conjugated goat anti-rabbit IgG (F(ab)2) (Molecular Probes). Fluorescence-labeled mast cells were cytocentrifuged (Cytospin 3, Shandon, United Kingdom) onto slides at 4.5 × g (200 rpm) for 5 min. To visualize cell nuclei, slides were mounted with DAPI, a fluorescent groove-binding probe for DNA, before coverslip attachment. Cells were examined using a fluorescence microscope (Nikon Eclipse E600; Nikon, Tokyo, Japan). Fluorescence-activated Cell Sorting (FACS) Analysis—For analysis of c-Kit expression, BMMCs were stained with a FITC-conjugated rat anti-mouse CD117 (c-kit) monoclonal Ab (mAb) (IgG2a) for 30 min at 4 °C. FITC-rat IgG2a was used as an isotypic control. Cells were analyzed by a FACScaliber flow cytometer (BD Biosciences). Fluorescence-labeled mast cells also were cytocentrifuged (Cytospin 3, Shandon, UK) onto slides at 4.5g (200 rpm) for 5 min. To visualize cell nuclei, slides were mounted with DAPI, a fluorescent groove-binding probe for DNA, before coverslip attachment. Cells were examined using a fluorescence microscope (Nikon Eclipse E600; Nikon, Tokyo, Japan). For analysis of FcɛRI expression, BMMC were sensitized with IgE and then stained with FITC-conjugated rat anti-mouse IgE mAb (BD Biosciences) for 1 h at 4 °C. FITC-rat IgG1 was used as a control. For cell proliferation assay, BMMCs (1 × 106) were incubated with 1 μm 5 (6Bondzi C. Litz J. Dent P. Krystal G.W. Cell Growth Differ. 2000; 11: 305-314PubMed Google Scholar)-carboxyfluorescein diacetate N-succinimidyl ester (CFSE, catalog no. 21888, Sigma-Aldrich) in phosphate buffer for 8 min, and were then washed with 2% FBS RPMI 1640 medium for three times. CFSE-labeled BMMCs were cultured at 37 °C, 5% CO2 incubator for 5 days with 0, 0.5, 5, 25, 50, or 100 ng/ml of SCF. Cells were counted using hemocytometer and also analyzed by flow cytometer. Cytokine Assays—The levels of IL-13, IL-4, IL-5, IL-10, MCP-1 (CCL-2), and RANTES (CCL-5) were measured by enzyme-linked immunosorbent assay (ELISA) using Quantikine Mouse IL-13 Immunoassay (for IL-13) and Duoset antibody pairs (for all other cytokines and chemokines) from R&D Systems (Minneapolis, MN) according to the manufacturer’s protocol. SCF-induced Expression of Egr Products in Mouse BMMC—To determine if SCF stimulation induces expression of Egr family members in mast cells, mouse BMMC were treated with SCF at the concentration of 50 ng/ml for 15, 30, 60, or 180 min. Expression of Egr products were examined by quantitative real-time PCR. Egr levels were normalized to GAPDH in each sample. An average value of Egr products after GAPDH normalization at the time point of 15 min (the highest Egr expression level) was used as a calibrator to determine the relative levels of Egr expression at various conditions (Fig. 1A). SCF treatment induced a rapid and transient expression of Egr-1 (Fig. 1, A and B) and Egr-2 (supplemental Fig. S1). We also examined expression of Egr-3, Egr-4, and the Wilm’s tumor product. Little or no Egr-3, Egr-4, or the Wilm’s tumor product can be detected in these samples (data not shown). PCR products were also separated in agarose gels and visualized by ethidium bromide staining. Representative gels were presented in Fig. 1B and supplemental Fig. S1. A rapid and strong Egr-1 and Egr-2 expression can be seen at 15 and 30 min after SCF treatment. Egr-1 and Egr-2 levels in the unstimulated mast cells were undetectable. SCF-induced expression of Egr-1 protein was determined by immunofluorescence assay. BMMC after treatment with SCF (100 ng/ml) for 2 h were fixed, permeabilized and stained with anti-Egr-1 Ab. Alexa-594-conjugated secondary Ab was used to visualize Egr-1 expression. Nuclei were stained by DAPI. SCF-induced Egr-1 protein localized in the nucleus of the BMMC (Fig. 2). SCF-induced Egr Activation in Mouse BMMC—To determine if SCF stimulation induces Egr activation, nucleus proteins from SCF-treated BMMC were examined by EMSA. An Egr DNA probe, which can bind Egr products (Egr-1, -2, -3, and -4) was used. Significant Egr DNA binding can be seen after treatment with SCF (50 ng/ml) for 40, 60, and 120 min. Egr DNA binding activity declined after 3–6 h (Fig. 3A). Dose-dependent experiment showed that mast cells responded to SCF even at the very low concentration (0.5 ng/ml). A strong Egr binding was observed when BMMC were stimulated with 5 ng/ml of SCF (Fig. 3B), a concentration close to the physiological SCF level of 3.3 ng/ml in human serum (17Langley K.E. Bennett L.G. Wypych J. Yancik S.A. Liu X.D. Westcott K.R. Chang D.G. Smith K.A. Zsebo K.M. Blood. 1993; 81: 656-660Crossref PubMed Google Scholar). Significant Egr binding was found when BMMC were stimulated with SCF at the concentrations of 25–100 ng/ml (Fig. 3B). Egr DNA binding specificity was confirmed by competitive binding with unlabeled Egr probe, but not mutant Egr probe or Sp-1 probe (Fig. 3C). Because Sp-1 has been shown to be closely associated with Egr-1 function because of the similarity in their DNA binding sequences, we examined whether SCF induced Sp-1 DNA binding. No Sp-1 activation can be observed in SCF-stimulated mast cells (Fig. 3C). As a control, the specific binding of Sp-1 was shown using nuclear extracts from Hela cells (Fig. 3D). To further confirm Egr binding specificity, antibodies to Egr-1, -2, -3, and -4 were used in an EMSA supershift assay. Anti-Egr-1 or anti-Egr-2 antibodies diminished Egr DNA binding activity, whereas anti-Egr-3 or anti-Egr-4 antibodies had little effect. These results were consistent with that shown in Fig. 1 that Egr-1 and -2 but not Egr-3 or -4 were induced by SCF stimulation. SCF-induced Mast Cell Growth in the Absence of Egr-1—Because SCF is a major mast cell growth factor, we determined if SCF-induced Egr-1 expression is required for mast cell growth. Bone marrow cells from Egr-1-deficient mice and wild-type control mice were used to culture for BMMC. We used conventional method to culture BMMC by using WEHI-3B conditioned medium as a source for IL-3. Egr-1-deficient BMMC showed similar level of c-kit and IgE receptor expression when compared with wild-type BMMC (Fig. 4A and supplemental Fig. S2) (12Li B. Power M.R. Lin T.J. Blood. 2006; 107: 2814-2820Crossref PubMed Scopus (37) Google Scholar). Egr-1-deficient BMMC and wild-type BMMC were labeled with CFSE and treated with various concentrations of SCF (0.5, 5, 25, 50, or 100 ng/ml) for 5 days. Cells were then examined by flow cytometry. Treatment with SCF induced a concentration dependent reduction of CFSE fluorescence intensity, suggesting cell proliferation. Egr-1 deficiency did not affect SCF-induced decrease of CFSE fluorescence intensity (mast cell proliferation) (Fig. 4B). Moreover, a similar trend of SCF-induced increase of mast cell numbers was observed in both Egr-1-deficient BMMC and wild-type BMMC (Fig. 4C). In addition, no morphological difference was observed between Egr-1-deficient and wild-type BMMC by toluidine blue staining or electron microscopic analysis (data not shown). These results suggest that Egr-1 is not required for SCF-induced mast cell growth in vitro. To examine if Egr-1 is needed for mast cell development in vivo, tissues from the ear and back skin of Egr-1-deficient and wild-type mice were used for alcian blue staining for mast cells. Consistent with the in vitro results, no difference of mast cell number in these tissues was observed (supplemental Fig. S3, A and B). To determine if Egr-1 has a role in maintaining mast cell viability, WEHI-3B supernatant (a source for IL-3) was removed from the culture media to induce cell death. Mast cell viability was examined by trypan blue exclusion. The withdrawal of WEHI-3B component from the culture media induced similar levels of cell death in Egr-1-deficient and wild-type mast cells (supplemental Fig. S4A). To further examine if Egr-1 affects mast cell apoptosis, BMMC were treated with camptothecin (5 μm, 24 h). Cell lysates were used to examine the cleavage of D4-GDI, an endogenous caspase 3 substrate. Similar level of D4-GDI cleavage in Egr-1-deficient and wild-type BMMC was observed (supplemental Fig. S4B). Decreased IL-13 Production in Egr-1-deficient Mast Cells Following SCF Stimulation—In addition to promotion of mast cell growth, SCF modulates mast cell function by regulating expression of various inflammatory mediators. SCF has been shown to directly induce IL-13 expression by human mast cells (11Hundley T.R. Gilfillan A.M. Tkaczyk C. Andrade M.V. Metcalfe D.D. Beaven M.A. Blood. 2004; 104: 2410-2417Crossref PubMed Scopus (134) Google Scholar). To determine if Egr-1 is required for SCF-dependent IL-13 production, Egr-1-deficient mast cells were treated with SCF (50 ng/ml) for various times. Expression of IL-13 mRNA and protein was determined. Quantitative real-time PCR analysis showed that SCF-induced IL-13 mRNA expression was reduced in Egr-1-deficient BMMC when compared with wild-type BMMC (Fig. 5A). Reduced IL-13 expression by Egr-1-deficient BMMC was also confirmed by analysis of IL-13 PCR product using agarose gel (Fig. 5B). Similarly, SCF-induced production of IL-13 at the protein level by Egr-1-deficient BMMC was significantly reduced comparing to that by wild-type BMMC (Fig. 5C). SCF not only directly stimulates mast cell mediator secretion, it also modulates IgE-dependent cytokine production. SCF synergistically potentiated antigen (TNP)/IgE-induced IL-13 (Fig. 6A) and IL-4 (Fig. 6B) production. Comparing to wild-type BMMC, Egr-1-deficient BMMC showed 69% reduction for IL-13 (Fig. 6A) and 53% reduction for IL-4 (Fig. 6B) in response to SCF + TNP/IgE stimulation. This result suggests that Egr-1 is required for the full scale production of IL-13 and IL-4 during SCF and TNP/IgE co-stimulation. Interestingly, Egr-1 deficiency had little or no effects on SCF + IgE-induced MCP-1 production (supplemental Fig. S5). In addition, RAN-TES, IL-10, and IL-5 were undetectable in any BMMC samples (data not shown). To examine if SCF and TNP/IgE co-stimulation has synergistic effects on Egr-1 expression and activation, BMMC were treated with various concentrations of TNP-BSA (0.5, 2, 10 ng/ml) alone or in the presence of SCF (50 ng/ml). Egr-1 expression at mRNA and protein levels was examined by real-time PCR and immunofluorescence, respectively. DNA binding activity was examined by EMSA. No synergistic or limited additive effect between SCF and TNP/IgE on Egr DNA binding activity was observed when analyzed by EMSA (Fig. 6, C and D). Similarly, little additive effects between SCF and TNP/IgE on Egr-1 mRNA and protein expression were observed (supplemental Fig. S6, A and B). It is likely that the synergistic effect between SCF and IgE on IL-13 and IL-4 production involves additional mechanisms. Tyrosine Kinase Activity in SCF-induced Egr Activation—To examine possible mechanisms involved in SCF-induced Egr activation, BMMC were pretreated for 1 h with various inhibitors for different protein kinases and phosphatases. These inhibitors include PP2 (tyrosine kinases), SB 203580 (p38 mitogen-activated protein kinase, p38 MAPK), PD 98059 (extracellular signal-regulated kinase, ERK), wortmannin (phosphatidylinositol 3-kinase, PI 3-kinase), Ro 31-8220 (protein kinase C), rapamycin (mammalian target of rapamycin, mTOR), and okadaic acid (protein phosphatase 2A, PP2A). BMMC were then stimulated with SCF (50 ng/ml) for an additional 2 h. Nuclear proteins were then extracted and subjected to EMSA. Tyrosine kinase inhibitor PP2 and protein phosphatase 2A inhibitor okadaic acid blocked SCF-induced Egr activation (Fig. 7A). However, other kinase inhibitors including SB 203580, PD 98059, wortmannin, Ro 31-8220, or rapamycin had no effect (Fig. 7A). A concentration-dependent inhibition of PP2 on SCF-induced Egr activation was demonstrated in Fig. 7B. Increased mast cell numbers and increased mast cell functional activities are major features of allergic disorders. SCF is a critical factor for both mast cell growth and functional activation (2Galli S.J. Zsebo K.M. Geissler E.N. Adv. Immunol. 1994; 55: 1-96Crossref PubMed Scopus (561) Google Scholar). Mast cell precursors as well as mature mast cells express the SCF receptor, c-Kit, on their surface (2Galli S.J. Zsebo K.M. Geissler E.N. Adv. Immunol. 1994; 55: 1-96Crossref PubMed Scopus (561) Google Scholar). In mice, mutations on the c-Kit or the SCF (steel) loci lead to mast cell deficiency (18Geissler E.N. Ryan M.A. Housman D.E. Cell. 1988; 55: 185-192Abstract Full Text PDF PubMed Scopus (1037) Google Scholar, 19Zsebo K.M. Williams D.A. Geissler E.N. 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• W2024543722 title "The Early Growth Response Factor-1 Is Involved in Stem Cell Factor (SCF)-induced Interleukin 13 Production by Mast Cells, but Is Dispensable for SCF-dependent Mast Cell Growth" @default.
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