How the innate and adaptive immune systems cooperate in the natural history of allergic diseases has been largely unknown. Plant-derived allergen, papain, and mite allergens, Der f 1 and Der p 1, belong to the same family of cysteine proteases. We examined the role of protease allergens in the induction of Ab production and airway inflammation after repeated intranasal administration without adjuvants and that in basophil/mast cell stimulation in vitro. Papain induced papain-specific IgE/IgG1 and lung eosinophilia. Der f 1 induced Der f 1-specific IgG1 and eosinophilia. Although papain-, Der f 1-, and Der p 1-stimulated basophils expressed allergy-inducing cytokines, including IL-4 in vitro, basophil-depleting Ab and mast cell deficiency did not suppress the papain-induced in vivo responses. Protease inhibitor-treated allergens and a catalytic site mutant did not induce the responses. These results indicate that protease activity is essential to Ab production and eosinophilia in vivo and basophil activation in vitro. IL-33-deficient mice lacked eosinophilia and had reduced papain-specific IgE/IgG1. Coadministration of OVA with papain induced OVA-specific IgE/IgG1, which was reduced in IL-33-deficient mice. We demonstrated IL-33 release, subsequent IL-33-dependent IL-5/IL-13 release, and activation of T1/ST2-expressing lineage(-)CD25(+)CD44(+) innate lymphoid cells in the lung after papain inhalation, suggesting the contribution of the IL-33-type 2 innate lymphoid cell-IL-5/IL-13 axis to the papain-induced airway eosinophilia. Rag2-deficient mice, which lack adaptive immune cells, showed significant, but less severe, eosinophilia. Collectively, these results suggest cooperation of adaptive immune cells and IL-33-responsive innate cells in protease-dependent allergic airway inflammation.
Mammalian eukaryotic translation initiation factor 3 (eIF3) is the largest complex of the translation initiation factors. The eIF3 complex is comprised of thirteen subunits, which are named eIF3a to eIF3 m in most multicellular organisms. The eIF3e gene locus is one of the most frequent integration sites of mouse mammary tumor virus (MMTV), which induces mammary tumors in mice. MMTV-integration events result in the expression of C-terminal-truncated eIF3e proteins, leading to mammary tumor formation. We have shown that tumor formation can be partly caused by activation of hypoxia-inducible factor 2α. To investigate the function of eIF3e in mammals, we generated eIF3e-deficient mice. These eIF3e-/- mice are embryonically lethal, while eIF3e+/- mice are much smaller than wild-type mice. In addition, eIF3e+/- mouse embryonic fibroblasts (MEFs) contained reduced levels of eIF3a and eIF3c subunits and exhibited reduced cellular proliferation. These results suggest that eIF3e is essential for embryonic development in mice and plays a role in maintaining eIF3 integrity.
The high-affinity IgE receptor, FcεRI, which is composed of α-, β-, and γ-chains, plays an important role in IgE-mediated allergic responses. In the current study, involvement of the transcription factors, PU.1, GATA1, and GATA2, in the expression of FcεRI on human mast cells was investigated. Transfection of small interfering RNAs (siRNAs) against PU.1, GATA1, and GATA2 into the human mast cell line, LAD2, caused significant downregulation of cell surface expression of FcεRI. Quantification of the mRNA levels revealed that PU.1, GATA1, and GATA2 siRNAs suppressed the α transcript, whereas the amount of β mRNA was reduced in only GATA2 siRNA transfectants. In contrast, γ mRNA levels were not affected by any of the knockdowns. Chromatin immunoprecipitation assay showed that significant amounts of PU.1, GATA1, and GATA2 bind to the promoter region of FCER1A (encoding FcεRIα) and that GATA2 binds to the promoter of MS4A2 (encoding FcεRIβ). Luciferase assay and EMSA showed that GATA2 transactivates the MS4A2 promoter via direct binding. These knockdowns of transcription factors also suppressed the IgE-mediated degranulation activity of LAD2. Similarly, all three knockdowns suppressed FcεRI expression in primary mast cells, especially PU.1 siRNA and GATA2 siRNA, which target FcεRIα and FcεRIβ, respectively. From these results, we conclude that PU.1 and GATA1 are involved in FcεRIα transcription through recruitment to its promoter, whereas GATA2 positively regulates FcεRIβ transcription. Suppression of these transcription factors leads to downregulation of FcεRI expression and IgE-mediated degranulation activity. Our findings will contribute to the development of new therapeutic approaches for FcεRI-mediated allergic diseases.
Interleukin-33 (IL-33) is a member of the IL-1 cytokine family, which includes IL-1 and IL-18. IL-33 is considered to be crucial for induction of Th2-type cytokine-associated immune responses such as host defense against nematodes and allergic diseases by inducing production of such Th2-type cytokines as IL-5 and IL-13 by Th2 cells, mast cells, basophils and eosinophils. In addition, IL-33 is involved in the induction of non-Th2-type acute and chronic inflammation as a proinflammatory cytokine, similar to IL-1 and IL-18. In this review, we summarize and discuss the current knowledge regarding the roles of IL-33 and IL-33 receptors in host defense and disease development.
Abstract The IL-1-related molecules, IL-1 and IL-18, can promote Th2 cytokine production by IgE/antigen-FcεRI-stimulated mouse mast cells. Another IL-1-related molecule, IL-33, was identified recently as a ligand for T1/ST2. Although mouse mast cells constitutively express ST2, the effects of IL-33 on mast cell function are poorly understood. We found that IL-33, but not IL-1β or IL-18, induced IL-13 and IL-6 production by mouse bone marrow-derived, cultured mast cells (BMCMCs) independently of IgE. In BMCMCs incubated with the potently cytokinergic SPE-7 IgE without specific antigen, IL-33, IL-1β, and IL-18 each promoted IL-13 and IL-6 production, but the effects of IL-33 were more potent than those of IL-1β or IL-18. IL-33 promoted cytokine production via a MyD88-dependent but Toll/IL-1R domain-containing adaptor-inducing IFN-β-independent pathway. By contrast, IL-33 neither induced nor enhanced mast cell degranulation. At 200 ng/ml, IL-33 prolonged mast cell survival in the absence of IgE and impaired survival in the presence of SPE-7 IgE, whereas at 100 ng/ml, IL-33 had no effect on mast cell survival in the absence of IgE and reduced mast cell survival in the presence of IgE. These observations suggest potential roles for IL-33 in mast cell- and Th2 cytokine-associated immune responses and disorders.
IL-33, a member of the IL-1-related cytokines, is considered to be a proallergic cytokine that is especially involved in Th2-type immune responses. Moreover, like IL-1α, IL-33 has been suggested to act as an “alarmin” that amplifies immune responses during tissue injury. In contrast to IL-1, however, the precise roles of IL-33 in those settings are poorly understood. Using IL-1- and IL-33-deficient mice, we found that IL-1, but not IL-33, played a substantial role in induction of T cell-mediated type IV hypersensitivity such as contact and delayed-type hypersensitivity and autoimmune diseases such as experimental autoimmune encephalomyelitis. Most notably, however, IL-33 was important for innate-type mucosal immunity in the lungs and gut. That is, IL-33 was essential for manifestation of T cell-independent protease allergen-induced airway inflammation as well as OVA-induced allergic topical airway inflammation, without affecting acquisition of antigen-specific memory T cells. IL-33 was significantly involved in the development of dextran-induced colitis accompanied by T cell-independent epithelial cell damage, but not in streptozocin-induced diabetes or Con A-induced hepatitis characterized by T cell-mediated apoptotic tissue destruction. In addition, IL-33-deficient mice showed a substantially diminished LPS-induced systemic inflammatory response. These observations indicate that IL-33 is a crucial amplifier of mucosal and systemic innate, rather than acquired, immune responses.
The mi transcription factor (MITF) is a basic-helix-loop-helix leucine zipper (bHLH-Zip) transcription factor that is important for the normal phenotypic expression of mast cells. Most transcription factors function in cooperation with other factors by protein-protein interactions. To search proteins interacting with MITF, we carried out a yeast two-hybrid screen and isolated Myc-associated zinc-finger protein related factor (MAZR) as a partner of MITF. When expressed with MITF in NIH/3T3 cells, MAZR was colocalized with MITF. The association of MAZR with MITF was further confirmed by a co-immunoprecipitation study and in vitrobinding assay. The zinc-finger domain of MAZR and the Zip domain of MITF were essential for the interaction. MAZR was expressed in cultured mast cells and MST mastocytoma cells containing mouse mast cell protease (mMCP)-6 transcript abundantly. The overexpression of dominant negative MAZR in MST mastocytoma cells reduced the amount of mMCP-6 mRNA. The simultaneous transfection of MAZR and MITF significantly increased the promoter activity of the mMCP-6 gene, indicating that the MAZR and MITF synergistically transactivated the mMCP-6 gene. MAZR appeared to play important roles in the normal phenotypic expression of mast cells in association with MITF. The mi transcription factor (MITF) is a basic-helix-loop-helix leucine zipper (bHLH-Zip) transcription factor that is important for the normal phenotypic expression of mast cells. Most transcription factors function in cooperation with other factors by protein-protein interactions. To search proteins interacting with MITF, we carried out a yeast two-hybrid screen and isolated Myc-associated zinc-finger protein related factor (MAZR) as a partner of MITF. When expressed with MITF in NIH/3T3 cells, MAZR was colocalized with MITF. The association of MAZR with MITF was further confirmed by a co-immunoprecipitation study and in vitrobinding assay. The zinc-finger domain of MAZR and the Zip domain of MITF were essential for the interaction. MAZR was expressed in cultured mast cells and MST mastocytoma cells containing mouse mast cell protease (mMCP)-6 transcript abundantly. The overexpression of dominant negative MAZR in MST mastocytoma cells reduced the amount of mMCP-6 mRNA. The simultaneous transfection of MAZR and MITF significantly increased the promoter activity of the mMCP-6 gene, indicating that the MAZR and MITF synergistically transactivated the mMCP-6 gene. MAZR appeared to play important roles in the normal phenotypic expression of mast cells in association with MITF. basic-helix-loop-helix leucine zipper mitranscription factor cultured mast cells mastocytoma cell line polyomavirus enhancer-binding protein Myc-associated zinc-finger protein-related factor broad-complex-tramtrack-bric-a-brac glutathioneS-transferase green fluorescent protein antibody(ies) nucleotide(s) amino acid(s) The mi locus of mice encodes a member of the basic-helix-loop-helix leucine zipper (bHLH-Zip)1 protein family of transcription factors (hereafter called MITF) (1Hodgkinson C.A. Moore K.J. Nakayama A. Steingrimsson E. Copeland N.G. Jenkins N.A. Arnheiter H. Cell. 1993; 74: 395-404Abstract Full Text PDF PubMed Scopus (942) Google Scholar, 2Hughes J.J. Lingrel J.B. Krakowsky J.M. Anderson K.P. J. Biol. Chem. 1993; 268: 20687-20690Abstract Full Text PDF PubMed Google Scholar). The MITF encoded by the mutant mi allele (mi-MITF) deletes one of four consecutive arginines in the basic domain (1Hodgkinson C.A. Moore K.J. Nakayama A. Steingrimsson E. Copeland N.G. Jenkins N.A. Arnheiter H. Cell. 1993; 74: 395-404Abstract Full Text PDF PubMed Scopus (942) Google Scholar, 3Steingrimsson E. Moore K.J. Lamoreux M.L. Ferre-D'Amare A.R. Burley S.K. Zimring D.C. Skow L.C. Hodgkinson C.A. Arnheiter H. Copeland N.G. Jenkins N.A. Nat. Genet. 1994; 8: 256-263Crossref PubMed Scopus (441) Google Scholar, 4Hemesath T.J. Streingrimsson E. McGill G. Hansen M.J. Vaught J. Hodgkinson C.A. Arnhheiter H. Copeland N.G. Jenkins N.A. Fisher D.E. Gene. Dev. 1994; 8: 2770-2780Crossref PubMed Scopus (546) Google Scholar). Themi/mi mutant mice show microphthalmia, depletion of pigmentation in both hair and eyes, osteopetrosis, and deficient natural killer activity (3Steingrimsson E. Moore K.J. Lamoreux M.L. Ferre-D'Amare A.R. Burley S.K. Zimring D.C. Skow L.C. Hodgkinson C.A. Arnheiter H. 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Kanakura Y. Jippo-Kanemoto T. Tsujimura T. Furitsu T. Ikeda H. Adachi S. Kasugai T. Nomura S. Kanayama Y. Kitamura Y. Blood. 1992; 80: 1454-1462Crossref PubMed Google Scholar, 12Kasugai T. Oguri K. Jippo-Kanemoto T. Morimoto M. Yamatodani A. Yoshida K. Ebi Y. Isozaki K. Tei H. Tsujimura T. Nomura S. Okayama M. Kitamura Y. Am. J. Pathol. 1993; 143: 1337-1347PubMed Google Scholar, 13Isozaki K. Tsujimura T. Nomura S. Morii E. Koshimizu U. Nishimune Y. Kitamura Y. Am. J. Pathol. 1994; 145: 827-836PubMed Google Scholar, 14Jippo T. Ushio H. Hirota S. Mizuno H. Yamatodani A. Nomura S. Matsuda H. Kitamura Y. Blood. 1994; 84: 2977-2983Crossref PubMed Google Scholar, 15Kim D.K. Morii E. Ogihara H. Lee Y.M. Jippo T. Adachi S. Maeyama K. Kim H.M. Kitamura Y. Blood. 1999; 93: 4179-4186Crossref PubMed Google Scholar). Cultured mast cells (CMCs) derived from the spleen of mi/mimice are deficient in the expression of various genes, such as mouse mast cell protease (mMCP)-4 (16Jippo T. Lee Y.M. Katsu Y. Tsujino K. Morii E. Kim D.K. Kitamura Y. Blood. 1999; 93: 1942-1950Crossref PubMed Google Scholar), mMCP-5 (17Morii E. Jippo T. Tsujimura T. Hashimoto K. Kim D.K. Lee Y.M. Ogihara H. Tsujino K. Kim H.M. Kitamura Y. Blood. 1997; 90: 3057-3066Crossref PubMed Google Scholar), mMCP-6 (18Morii E. Tsujimura T. Jippo T. Hashimoto K. Takebayashi K. Tsujino K. Nomura S. Yamamoto M. Kitamura Y. Blood. 1996; 88: 2488-2494Crossref PubMed Google Scholar), mMCP-7 (19Ogihara H. Morii E. Kim D.K. Oboki K. Kitamura Y. Blood. 2001; 97: 645-651Crossref PubMed Scopus (38) Google Scholar), c-kit (20Tsujimura T. Morii E. Nozaki M. Hashimoto K. Moriyama Y. Takebayashi K. Kondo T. Kanakura Y. Kitamura Y. Blood. 1996; 88: 1225-1233Crossref PubMed Google Scholar), p75 nerve growth factor receptor (21Jippo T. Morii E. Tsujino K. Tsujimura T. Lee Y.M. Kim D.K. Matsuda H. Kim H.M. Kitamura Y. Blood. 1997; 90: 2601-2608Crossref PubMed Google Scholar), granzyme B (22Ito A. Morii E. Maeyama K. Jippo T. Kim D.K. Lee Y.M. Ogihara H. Hashimoto K. Kitamura Y. Nojima H. Blood. 1998; 91: 3210-3221Crossref PubMed Google Scholar), tryptophan hydroxylase (22Ito A. Morii E. Maeyama K. Jippo T. Kim D.K. Lee Y.M. Ogihara H. Hashimoto K. Kitamura Y. Nojima H. Blood. 1998; 91: 3210-3221Crossref PubMed Google Scholar), integrin α4 subunit (23Kim D.K. Morii E. Ogihara H. Hashimoto K. Oritani K. Lee Y.M. Jippo T. Adachi S. Kanakura Y. Kitamura Y. Blood. 1998; 92: 1973-1980Crossref PubMed Google Scholar) and α−melanocyte-stimulating hormone receptor genes (24Adachi S. Morii E. Kim D. Ogihara H. Jippo T. Ito A. Lee Y.M. Kitamura Y. J. Immunol. 2000; 164: 855-860Crossref PubMed Scopus (52) Google Scholar). Among the genes whose expression was deficient in mi/miCMCs, the transactivation mechanism of the mMCP-6 gene has been studied most intensively (18Morii E. Tsujimura T. Jippo T. Hashimoto K. Takebayashi K. Tsujino K. Nomura S. Yamamoto M. Kitamura Y. Blood. 1996; 88: 2488-2494Crossref PubMed Google Scholar, 25Morii E. Takebayashi K. Motohashi H. Yamamoto M. Nomura S. Kitamura Y. Biochem. Biophys. Res. Commun. 1994; 205: 1299-1304Crossref PubMed Scopus (52) Google Scholar, 26Ogihara H. Kanno T. Morii E. Kim D.K. Lee Y.M. Sato M. Kim W.Y. Nomura S. Ito Y. Kitamura Y. Oncogene. 1999; 18: 4632-4639Crossref PubMed Scopus (31) Google Scholar). The expression of mMCP-6 gene was deficient not only in mi/mi CMCs but also in CMCs derived from other mutants at the mi locus. The tg/tgCMCs that lack MITF did not express the mMCP-6 gene (27Ito A. Morii E. Kim D.K. Kataoka T.R. Jippo T. Maeyama K. Nojima H. Kitamura Y. Blood. 1999; 93: 1189-1196Crossref PubMed Google Scholar). Themiew/miew andmice/mice CMCs, in which the basic domain and the Zip domain of MITF were deleted, respectively, also did not express the mMCP-6 gene (28Morii E. Ogihara H. Kim D.K. Ito A. Oboki K. Lee Y.M. Jippo T. Maeyama K. Nomura S. Lamoreux M.L. Kitamura Y. Blood. 2001; 97: 2038-2044Crossref PubMed Scopus (29) Google Scholar, 29Morii E. Ogihara H. Oboki K. Kataoka T.R. Maeyama K. Fisher D.E. Lamoreux M.L. Kitamura Y. Blood. 2001; 98: 2577-2579Crossref PubMed Scopus (20) Google Scholar). These findings indicated that the normal (+) MITF was essential for the expression of the mMCP-6 gene. The +-MITF bound the three motifs in the promoter region of the mMCP-6 gene (18Morii E. Tsujimura T. Jippo T. Hashimoto K. Takebayashi K. Tsujino K. Nomura S. Yamamoto M. Kitamura Y. Blood. 1996; 88: 2488-2494Crossref PubMed Google Scholar). The mutation of each of these three motifs reduced the magnitude of transactivation by +-MITF. Among them, the GACCTG motif appeared to play the most important role since the magnitude of reduction was greatest after the mutation of the GACCTG motif (18Morii E. Tsujimura T. Jippo T. Hashimoto K. Takebayashi K. Tsujino K. Nomura S. Yamamoto M. Kitamura Y. Blood. 1996; 88: 2488-2494Crossref PubMed Google Scholar). We found that the GACCTG motif was partly overlapped by the binding motif recognized by another transcription factor, polyomavirus enhancer-binding protein (PEBP2). PEBP2 interacted with +-MITF and showed functional synergy in the transcription of mMCP-6 gene (26Ogihara H. Kanno T. Morii E. Kim D.K. Lee Y.M. Sato M. Kim W.Y. Nomura S. Ito Y. Kitamura Y. Oncogene. 1999; 18: 4632-4639Crossref PubMed Scopus (31) Google Scholar). In the present study, we searched the factor that cooperated with +-MITF using the yeast two-hybrid screen and found theMyc-associated zinc-finger protein-related factor (MAZR) as a protein that interacted with +-MITF. MAZR possesses the broad-complex-tramtrack-bric-a-brac (BTB) domain and the zinc-finger domain (30Kobayashi A. Yamagiwa H. Hoshino H. Muto A. Sato K. Morita M. Hayashi N. Yamamoto M. Igarashi K. Mol. Cell. Biol. 2000; 20: 1733-1746Crossref PubMed Scopus (95) Google Scholar). The overexpression of dominant negative MAZR reduced the expression of the mMCP-6 gene in mastocytoma cells. MAZR showed functional synergy with +-MITF for transcription of the mMCP-6 gene. MAZR appeared to play roles in the normal phenotype expression of mast cells in association with +-MITF. The bait plasmid pGBKT7-MITF was constructed by inserting a portion of MITF cDNA, which was deleted in the N-terminal 161 residues, into pGBKT7 vector (CLONTECH, Palo Alto, CA). The yeast two-hybrid screen was performed according to the instructions for the MATCHMAKER two-hybrid system 3 (CLONTECH) using pGBKT7-MITF as bait and a mouse lymphoma cell cDNA library (CLONTECH). Approximately 2 × 108Hf7c yeast transformants were screened for His autotrophy. β-galactosidase assay was used for isolating positive clones. The isolated clones were sequenced in both directions with ABI 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA). CMCs were obtained by culturing spleen cells of +/+ mice as described previously (17Morii E. Jippo T. Tsujimura T. Hashimoto K. Kim D.K. Lee Y.M. Ogihara H. Tsujino K. Kim H.M. Kitamura Y. Blood. 1997; 90: 3057-3066Crossref PubMed Google Scholar). MST cells were kindly provided by Dr. J. D. Esko of University of California, San Diego (31Montgomery R.I. Lidholt K. Flay N.W. Liang J. Vertel B. Lindahl U Esko J.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11327-11331Crossref PubMed Scopus (28) Google Scholar), and Jurkat cells were maintained in RPMI 1640 medium (Sigma) supplemented with 10% fetal calf serum (Nippon Bio Supply Center, Tokyo, Japan). NIH/3T3 cells and 293T cells were maintained in Dulbecco's modification of Eagle's medium (Flow Laboratories, Irvine, UK) supplemented with 10% fetal calf serum. The DNA fragment encoding the entire open reading frame of MAZR was obtained by PCR from the reverse-transcribed product of CMCs. The used primers were 5′-ATGGAGCGGGTCAACGACGCTTCTTGCGGT and 5′-TCACTTCCCTTCAGGCCCCATGGGCTGCTG. The amplified DNA fragment was subcloned into pBluescript (Stratagene, La Jolla, CA). The amplified fragment was also subcloned into pGEX-3X glutathioneS-transferase (GST)-expressing vector (Amersham Biosciences, Inc.) and into pEF-BOS expression vector kindly provided by Dr. S. Nagata of Osaka University (32Mizushima S. Nagata S. Nucleic Acids Res. 1990; 185322Crossref PubMed Scopus (1499) Google Scholar). Various fragments of MAZR were amplified by PCR and subcloned into pBluescript or pEF-BOS. The expression plasmid containing MAZR fused with green fluorescent protein (GFP) and the expression plasmid containing MAZR fused with FLAG epitope tag were described before (30Kobayashi A. Yamagiwa H. Hoshino H. Muto A. Sato K. Morita M. Hayashi N. Yamamoto M. Igarashi K. Mol. Cell. Biol. 2000; 20: 1733-1746Crossref PubMed Scopus (95) Google Scholar). The expression plasmid and the pBluescript containing normal or mutant MITFs, the expression plasmid containing Myc-tagged normal or mutant MITFs, and the GST-expressing plasmid containing +-MITF were also described previously (19Ogihara H. Morii E. Kim D.K. Oboki K. Kitamura Y. Blood. 2001; 97: 645-651Crossref PubMed Scopus (38) Google Scholar, 33Morii E. Ogihara H. Kanno T. Kim D.K. Nomura S. Ito Y. Kitamura Y. Biochem. Biophys. Res. Comm. 1999; 261: 53-57Crossref PubMed Scopus (7) Google Scholar, 34Morii E. Ogihara H. Oboki K. Sawa C. Sakuma T. Nomura S. Esko J.D. Handa H. Kitamura Y. Blood. 2001; 97: 3032-3039Crossref PubMed Scopus (31) Google Scholar). All of the PCR fragments were verified by sequencing. Each RNA sample was prepared from 1.0 × 107 CMCs, MST cells, and Jurkat cells by the lithium chloride-urea method (35Auffray C. Rougenon F. Eur. J. Biochem. 1980; 107: 303-314Crossref PubMed Scopus (2085) Google Scholar). Northern blot analysis was performed using the full-length MAZR (30Kobayashi A. Yamagiwa H. Hoshino H. Muto A. Sato K. Morita M. Hayashi N. Yamamoto M. Igarashi K. Mol. Cell. Biol. 2000; 20: 1733-1746Crossref PubMed Scopus (95) Google Scholar), mMCP-6 (36Reynolds D.S. Gurley D.S. Austen K.F. Serafin W.E. J. Biol. Chem. 1991; 266: 3847-3853Abstract Full Text PDF PubMed Google Scholar), and glyceraldehyde-3-phosphate dehydrogenase (37Sabath D.E. Broome H.E. Prystowsky M.B. Gene. 1990; 91: 185-191Crossref PubMed Scopus (380) Google Scholar) cDNAs labeled with [α-32P]dCTP (PerkinElmer Life Sciences; 10 mCi/ml) by random oligonucleotide priming. After hybridization at 42 °C, blots were washed to a final stringency of 0.2 × SSC (1 × SSC is 150 mmol/liter NaCl and 15 mmol/liter trisodium citrate, pH 7.4) and subjected to autoradiography. The expression plasmid containing +-MITF and the expression plasmid containing MAZR fused with GFP were cotransfected to NIH/3T3 cells using TransFast Transfection Reagent (Promega, Madison, WI) according to the manufacturer's instructions. The cells were fixed with 100% methanol and permiated by treatment with 0.2% Triton X-100 in phosphate-buffered saline. The cells were incubated with polyclonal rabbit anti-MITF antibody (Ab). Immunoreacted cells were detected with anti-rabbit IgG Ab conjugated with rhodamine (MBL, Nagoya, Japan). The cells expressing +-MITF were detected with the red filter, and the cells expressing MAZR fused with GFP were detected with the green filter. The specimens were observed with a confocal laser scanning microscope (LSM 510; Carl Zeiss, Jena, Germany). The Myc-tagged normal or mutant MITF was coexpressed with FLAG-tagged MAZR in 293T cells using TransFast Transfection Reagent (Promega). The nuclear or whole cell extract was obtained by the method described previously (38Takebayashi K. Chida K. Tsukamoto I. Morii E. Munakata H. Arnheiter H. Kuroki T. Kitamura Y. Nomura S. Mol. Cell. Biol. 1996; 16: 1203-1211Crossref PubMed Google Scholar). The nuclear or whole cell extract was incubated with LIP buffer (10 mm HEPES, 250 mm NaCl, 0.1% Nonidet P-40, 5 mm EDTA, 1 mm phenylmethylsulfonyl fluoride) and protein G-Sepharose (Amersham Biosciences, Inc.) for 1 h with gentle rocking and centrifuged at 3,000 rpm for 3 min. The supernatants were transferred into new tubes and incubated with protein G-Sepharose and anti-Myc monoclonal Ab (9E10, PharMingen, San Diego, CA) or anti-FLAG Ab (Sigma) for 1 h in LIP buffer. Immunocomplexes were washed four times with LIP buffer, resuspended in loading buffer, boiled, and analyzed by immunoblot with anti-Myc monoclonal Ab. The 35S-labeled normal or mutant MITF protein was synthesized using the reticulocyte lysate system (TNT system, Promega). The 35S-labeled normal or mutant MAZR protein was also synthesized. For the binding assays, the35S-labeled normal or mutant MITF protein was incubated for 1 h at room temperature with GST-MAZR or GST alone immobilized on glutathione-agarose beads. The 35S-labeled normal or mutant MAZR protein was also incubated with GST-+-MITF or GST alone immobilized on beads. The beads were washed four times. Proteins retained on the beads were subsequently analyzed by SDS-PAGE and autoradiography. The transfection to MST cells and to Jurkat cells were performed by electroporation. The reporter plasmid that contained the promoter region of the mMCP-6 gene starting from nucleotide (nt) −171 or nt −151 and the reporter plasmid with the minimal mMCP-6 promoter starting from nt −61 were described previously (26Ogihara H. Kanno T. Morii E. Kim D.K. Lee Y.M. Sato M. Kim W.Y. Nomura S. Ito Y. Kitamura Y. Oncogene. 1999; 18: 4632-4639Crossref PubMed Scopus (31) Google Scholar). The reporter plasmid mutated at the MITF-binding GACCTG motif in the mMCP-6 promoter and the reporter plasmid possessing the tetrameric fragment between nt −171 and −151 upstream from the minimal mMCP-6 promoter were also described previously (26Ogihara H. Kanno T. Morii E. Kim D.K. Lee Y.M. Sato M. Kim W.Y. Nomura S. Ito Y. Kitamura Y. Oncogene. 1999; 18: 4632-4639Crossref PubMed Scopus (31) Google Scholar). The pEF-BOS expression plasmid containing +-MITF, mutant MITFs, MAZR, or mutant MAZRs was used as the effector plasmid. In luciferase assays, 5 μg of a reporter, 2 μg of effector plasmids, and 1 μg of an expression vector containing the β-galactosidase gene were cotransfected to Jurkat cells. The expression vector containing the β-galactosidase gene was used as an internal control. When two kinds of effector plasmids were used, equal amounts of both plasmids were transfected. In some experiments, 5 μg of a reporter, 2 μg of the expression plasmid containing +-MITF, 2 or 4 μg of the expression plasmid containing the dominant negative form of MAZR and 1 μg of an expression vector containing the β-galactosidase gene were cotransfected to MST cells. The cells were harvested 48 h after the transfection and lysed with 0.1 mol/liter potassium phosphate buffer (pH 7.4) containing 1% Triton X-100. Soluble extracts were then assayed for luciferase activity with a luminometer LB96P (Berthold GmbH, Wildbad, Germany) and for β-galactosidase activity. The luciferase activity was normalized by the β-galactosidase activity and the total protein concentration as described previously (18Morii E. Tsujimura T. Jippo T. Hashimoto K. Takebayashi K. Tsujino K. Nomura S. Yamamoto M. Kitamura Y. Blood. 1996; 88: 2488-2494Crossref PubMed Google Scholar). The normalized value was divided by the value obtained without effector plasmids, and the divided value was expressed as the relative luciferase activity. We searched for proteins that were associated with +-MITF by yeast two-hybrid screening. The full-length +-MITF was not suitable as bait, since the full-length +-MITF fused to the Gal4 DNA binding domain was a strong transactivator of reporter genes. We deleted the transactivation domain and obtained the new construct, truncating the N-terminal 161 amino acids (aa) of MITF (MITF-(162–419). MITF-(162–419) contained the bHLH-Zip domain. MITF-(162–419) fused with the Gal4 DNA binding domain was used as bait. Positive transformants were selected for His autotrophy and β-galactosidase assay. We isolated eighteen positive cDNA clones. Four of the 18 clones encoded the ubiquitin-conjugating enzyme UBC9, and one of the 18 clones encoded protein kinase C-interacting protein. UBC9 and protein kinase C-interacting protein were previously shown to associate with MITF (39Xu U. Gong L. Haddad M.M. Biscof O. Campisi J. Yeh E.T.H. Medrano E.E. Exp. Cell Res. 2000; 255: 135-143Crossref PubMed Scopus (209) Google Scholar,40Razin E. Zhang Z.C. Nechushtan H. Frenkel S. Lee Y.N. Arudchandran R. Rivela J. J. Biol. Chem. 1999; 274: 34272-34276Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). One of the other clones was found to encode a part of cDNA of a transcription factor, MAZR. MAZR consisted of 641 aa and contains a BTB domain in the N terminus (aa 1–145) and seven zinc-finger domains in the C terminus (aa 288–641) (30Kobayashi A. Yamagiwa H. Hoshino H. Muto A. Sato K. Morita M. Hayashi N. Yamamoto M. Igarashi K. Mol. Cell. Biol. 2000; 20: 1733-1746Crossref PubMed Scopus (95) Google Scholar). By Northern blotting, the expression of the MAZR gene was detected in CMCs and MST mastocytoma cells, but was hardly detectable in Jurkat T cells (Fig.1). The interaction of MAZR to +-MITF was confirmed by two experiments. First, MAZR fused with GFP was coexpressed with +-MITF in NIH/3T3 cells, and their subcellular localization was examined. The +-MITF and MAZR were colocalized in the nucleus of NIH/3T3 cells (Fig.2 A). Next, the co-immunoprecipitation studies were performed. Myc-tagged +-MITF and FLAG-tagged MAZR were coexpressed in 293T cells, and their nuclear extract was analyzed. The immunoprecipitated product with anti-FLAG Ab contained Myc-tagged +-MITF (Fig. 2 B). To identify the region of +-MITF that was required for the interaction with MAZR, we carried out in vitro binding experiments. Various mutants of MITF were constructed (Fig.3 A). The mi-MITF deletes one arginine in the basic domain. MITF-(1–298) possesses the Zip domain, but deletes the C-terminal region downstream of the Zip domain. MITF-(1–260) deletes the Zip domain. The35S-labeled normal or mutant MITF was subjected to coprecipitation with GST or GST-MAZR fusion protein that was immobilized on glutathione-agarose beads. Protein complexes were analyzed by SDS-PAGE. The complex of +-MITF and GST-MAZR was detected, but that of +-MITF and GST was not (Fig. 3 B). Themi-MITF and MITF-(1–298) bound GST-MAZR-coated beads but not GST-coated beads (Fig. 3 B). In contrast, MITF-(1–260), which lacked the Zip domain, did not bind GST-MAZR-coated beads (Fig.3 B). The region required for the interaction with MAZR was also examined by a co-immunoprecipitation study. Myc-tagged +-MITF, Myc-taggedmi-MITF, and Myc-tagged MITF-(1–260) were coexpressed with FLAG-tagged MAZR in 293T cells, and their whole cell lysate was analyzed. The immunoprecipitated product with anti-FLAG Ab contained Myc-tagged +-MITF or Myc-tagged mi-MITF, but did not contain Myc-tagged MITF-(1–260) that lacked the Zip domain (Fig.3 C). Next, the region of MAZR necessary for the interaction with MITF was examined by in vitro binding experiments. MAZR deleting the zinc-finger domain (MAZR-(1–288)), MAZR deleting the BTB domain (MAZR-(145–641)), and MAZR containing the zinc-finger domain alone (MAZR-(288–641)) were constructed (Fig.4 A). MAZR, MAZR-(145–641), and MAZR-(288–641) bound GST-+-MITF-coated beads but not GST-coated beads (Fig. 4 B). In contrast, MAZR-(1–288) that lacked the zinc-finger domain did not bind GST-+-MITF-coated beads (Fig.4 B). Since the +-MITF strongly transactivated the mMCP-6 promoter in mast cells (18Morii E. Tsujimura T. Jippo T. Hashimoto K. Takebayashi K. Tsujino K. Nomura S. Yamamoto M. Kitamura Y. Blood. 1996; 88: 2488-2494Crossref PubMed Google Scholar), the effect of MAZR on the function of +-MITF was examined. To examine whether the endogenous MAZR increased the amount of mMCP-6 mRNA, we used a dominant negative MAZR. The dominant negative MAZR is a mutant that possesses only a part of the zinc-finger domain (aa 409–496). We overexpressed the dominant negative MAZR in MST cells that expressed both mMCP-6 and MAZR mRNAs. After 6 days of culture, the expression of mMCP-6 gene was examined by Northern blotting. The overexpression of dominant negative MAZR significantly reduced the amount of mMCP-6 mRNA (Fig.5 A). Next, we cotransfected the dominant negative MAZR to MST cells with the reporter plasmid containing the promoter region of the mMCP-6 gene. This reporter plasmid possessed the GACCTG motif to which +-MITF bound. When various amounts of dominant negative MAZR were cotransfected, the luciferase activity of the reporter plasmid decreased in a dose-dependent manner (Fig. 5 B). The functional synergy between +-MITF and MAZR was examined. To remove the possible involvement of endogenous MITF and MAZR, we used Jurkat cells that expressed neither MITF (19Ogihara H. Morii E. Kim D.K. Oboki K. Kitamura Y. Blood. 2001; 97: 645-651Crossref PubMed Scopus (38) Google Scholar) nor MAZR. The expression plasmid containing +-MITF cDNA and that containing MAZR cDNA were cotransfected to Jurkat cells with the reporter plasmid used in Fig.5 B. The transfection of the plasmid containing +-MITF cDNA alone and the transfection of the plasmid containing MAZR cDNA alone did not increase the luciferase activity. In contrast, the simultaneous transfection of these plasmids increased the luciferase activity ∼40-fold (Fig.6). Various mutants of MITF were cotransfected with MAZR. The synergy with MAZR was not observed when mi-MITF or MITF-(1–260) deleting the Zip domain was cotransfected (Fig.7). Next, various mutants of MAZR were cotransfected with +-MITF. MAZR-(145–641) and MAZR-(288–641) showed the synergy with +-MITF, but MAZR-(1–288) deleting the zinc-finger domain did not (Fig. 7). Then, we examined the DNA element that was necessary for the synergy between MAZR and +-MITF. The +-MITF bound the GACCTG motif in the mMCP-6 promoter but not the mutated GTCCAG motif (Fig. 8 A) (18Morii E. Tsujimura T. Jippo T. Hashimoto K. Takebayashi K. Tsujino K. Nomura S. Yamamoto M. Kitamura Y. Blood. 1996; 88: 2488-2494Crossref PubMed Google Scholar). The mutation or deletion of the GACCTG motif abolished the synergy (Fig.8 B). The tetrameric fragments between nt −171 and −151 were cloned into the plasmid with the minimal mMCP-6 promoter starting from nt −61. A significant synergy between +-MITF and MAZR was observed in the reporter plasmid containing the tetrameric fragments but not in the reporter plasmid containing the minimal mMCP-6 promoter alone (Fig. 8 C). No synergy was observed in the reporter plasmid that contained the tetrameric fragments mutated at the GACCTG motif (Fig. 8 C). MAZR was isolated as a protein that interacted with +-MITF by yeast two-hybrid screening. MAZR was first cloned as the protein interacting with Bach2, which is a transcription factor possessing both the BTB domain and the b-Zip domain (30Kobayashi A. Yamagiwa H. Hoshino H. Muto A. Sato K. Morita M. Hayashi N. Yamamoto M. Igarashi K. Mol. Cell. Biol. 2000; 20: 1733-1746Crossref PubMed Scopus (95) Google Scholar). MAZR transactivates the c-myc gene in B cells and is important for the development of B cells in association with Bach2 (30Kobayashi A. Yamagiwa H. Hoshino H. Muto A. Sato K. Morita M. Hayashi N. Yamamoto M. Igarashi K. Mol. Cell. Biol. 2000; 20: 1733-1746Crossref PubMed Scopus (95) Google Scholar). In mast cells, MAZR transactivated the mMCP-6 gene in cooperation with +-MITF. MAZR may show synergy with +-MITF in the transcription of other genes in mast cells. MAZR appeared to play some roles in the normal phenotype expression of mast cells in association with +-MITF. The deletion of the zinc-finger domain of MAZR abolished the physical interaction and functional synergy with +-MITF. This suggested that the zinc-finger domain of MAZR was essential for the interaction with +-MITF. The Zip domain of MITF mediated the physical interaction and functional synergy with MAZR. The Zip domain of cAMP-response element-binding protein interacted with the zinc-finger domain of YY1 (41Zhou Q. Gedrich R.W. Engel D.A. J. Virol. 1995; 69: 4323-4330Crossref PubMed Google Scholar). The present result may be another example of such interactions. The MITF encoded by the mutant mice allele deletes the Zip domain (3Steingrimsson E. Moore K.J. Lamoreux M.L. Ferre-D'Amare A.R. Burley S.K. Zimring D.C. Skow L.C. Hodgkinson C.A. Arnheiter H. Copeland N.G. Jenkins N.A. Nat. Genet. 1994; 8: 256-263Crossref PubMed Scopus (441) Google Scholar). The mMCP-6 gene was not expressed in mast cells of mice/mice genotype (28Morii E. Ogihara H. Kim D.K. Ito A. Oboki K. Lee Y.M. Jippo T. Maeyama K. Nomura S. Lamoreux M.L. Kitamura Y. Blood. 2001; 97: 2038-2044Crossref PubMed Scopus (29) Google Scholar). Since the Zip domain of MITF mediated the interaction with MAZR, the loss of expression of the mMCP-6 gene might be attributable to the abolishment of synergy between MITF and MAZR inmice/mice mast cells. The MITF encoded by the mutant miew allele (miew -MITF) deletes most of the portion of the basic domain (3Steingrimsson E. Moore K.J. Lamoreux M.L. Ferre-D'Amare A.R. Burley S.K. Zimring D.C. Skow L.C. Hodgkinson C.A. Arnheiter H. Copeland N.G. Jenkins N.A. Nat. Genet. 1994; 8: 256-263Crossref PubMed Scopus (441) Google Scholar). The mMCP-6 gene was also not expressed in mast cells of the miew/miew genotype (29Morii E. Ogihara H. Oboki K. Kataoka T.R. Maeyama K. Fisher D.E. Lamoreux M.L. Kitamura Y. Blood. 2001; 98: 2577-2579Crossref PubMed Scopus (20) Google Scholar). Since the basic domain of MITF mediates the DNA binding (4Hemesath T.J. Streingrimsson E. McGill G. Hansen M.J. Vaught J. Hodgkinson C.A. Arnhheiter H. Copeland N.G. Jenkins N.A. Fisher D.E. Gene. Dev. 1994; 8: 2770-2780Crossref PubMed Scopus (546) Google Scholar), the loss of expression of the mMCP-6 gene might be attributable to the deficient DNA binding of the complex ofmiew -MITF and MAZR inmiew/miew mast cells. MAZR recognizes the G-rich motif of DNA (30Kobayashi A. Yamagiwa H. Hoshino H. Muto A. Sato K. Morita M. Hayashi N. Yamamoto M. Igarashi K. Mol. Cell. Biol. 2000; 20: 1733-1746Crossref PubMed Scopus (95) Google Scholar). The consensus binding sequence of MAZR is (G/C)GGGGGGGG(A/C)C (30Kobayashi A. Yamagiwa H. Hoshino H. Muto A. Sato K. Morita M. Hayashi N. Yamamoto M. Igarashi K. Mol. Cell. Biol. 2000; 20: 1733-1746Crossref PubMed Scopus (95) Google Scholar). In the promoter region of the mMCP-6 gene, there was a sequence GTGGTGGGGAC between nt −138 and −128, in which nine of 11 nucleotides were matched to the MAZR-consensus sequence. However, this sequence was not essential for the synergy between +-MITF and MAZR, since the synergy was observed in the reporter plasmid containing the fragment between nt −171 and −151 alone upstream of the minimal mMCP-6 promoter. No G-rich motif was found in the fragment between nt −171 and −151 or in the minimal mMCP-6 promoter. The G-rich motif appeared dispensable for the synergy between MAZR and +-MITF. Instead, the GACCTG motif between nt −166 and −161, to which +-MITF bound, was required for the synergy, since the mutation at the GACCTG motif abolished it. The binding of +-MITF to the GACCTG motif activated the mMCP-6 promoter, and binding of MAZR to +-MITF might further enhance the promoter activity. PEBP2 showed synergy with +-MITF for the transcription of the mMCP-6 gene (26Ogihara H. Kanno T. Morii E. Kim D.K. Lee Y.M. Sato M. Kim W.Y. Nomura S. Ito Y. Kitamura Y. Oncogene. 1999; 18: 4632-4639Crossref PubMed Scopus (31) Google Scholar). PEBP2 recognized the TGTGGTC motif, partly overlapped the MITF-binding GACCTG motif (the overlapped nucleotides were underlined) (26Ogihara H. Kanno T. Morii E. Kim D.K. Lee Y.M. Sato M. Kim W.Y. Nomura S. Ito Y. Kitamura Y. Oncogene. 1999; 18: 4632-4639Crossref PubMed Scopus (31) Google Scholar). The +-MITF interacted with PEBP2 through the region upstream of the basic domain (33Morii E. Ogihara H. Kanno T. Kim D.K. Nomura S. Ito Y. Kitamura Y. Biochem. Biophys. Res. Comm. 1999; 261: 53-57Crossref PubMed Scopus (7) Google Scholar), whereas the +-MITF interacted with MAZR through the Zip domain. The formation of the triple complex consisting of +-MITF, PEBP2, and MAZR might be possible. The triple complex might efficiently enhance the transcription of the mMCP-6 gene in mast cells. Taken together, MAZR interacted with +-MITF and synergistically transactivated the mMCP-6 promoter. MAZR appeared important for the normal phenotypic expression of mast cells. We thank Dr. S. Nagata of Osaka University for pEF-BOS, Dr. Jeffrey D. Esko of University of California, San Diego for MST cells, and C. Murakami for technical assistance.
