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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Morii, E. Articles by Kitamura, Y. Search for Related Content PubMed PubMed Citation Articles by Morii, E. Articles by Kitamura, Y. Related Collections Hematopoiesis and Stem Cells Gene Expression Brief Reports Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 15 October 2001, Vol. 98, No. 8, pp. 2577-2579 BRIEF REPORT Effect of a large deletion of the basic domain of mi transcription factor on differentiation of mast cells Eiichi Morii, Hideki Ogihara, Keisuke Oboki, Tatsuki R. Kataoka, Kazutaka Maeyama, David E. Fisher, M. Lynn Lamoreux, and Yukihiko Kitamura From the Department of Pathology, Osaka University Medical School, Suita, Japan; Department of Pharmacology, Ehime University Medical School, Japan; Division of Pediatric Hematology/Oncology, Dana Farber Cancer Institute and Children's Hospital, Harvard Medical School, Boston, MA; and Department of Veterinary Pathobiology, Texas A&M University, College Station, TX.     Abstract Top Abstract Introduction Study design Results and discussion References The mi transcription factor (MITF) is a basic-helix-loop-helix-leucine zipper transcription factor that is important for the development of mast cells. Cultured mast cells (CMCs) of mi/mi genotype express abnormal MITF (mi-MITF), but CMCs of tg/tg genotype do not express any MITFs. It was previously reported that mi/mi CMCs showed more severe abnormalities than tg/tg CMCs, indicating that mi-MITF had inhibitory function. Whereas mi-MITF contains a single amino acid deletion in the basic domain, MITF encoded by miew allele (ew-MITF) deletes 16 of 21 amino acids of the basic domain. Here the effect of a large deletion of the basic domain was examined. In miew/miew CMCs, the expression pattern of genes whose transcription was affected by MITF was comparable to that of tg/tg CMCs rather than to that of mi/mi CMCs. This suggested that ew-MITF lacked any functions. The part of the basic domain deleted in ew-MITF appeared necessary for either transactivation or inhibition of transactivation. (Blood. 2001;98:2577-2579) © 2001 by The American Society of Hematology.     Introduction Top Abstract Introduction Study design Results and discussion References The mi locus of mice encodes a member of the basic-helix-loop-helix-leucine zipper protein family of transcription factors (hereafter called MITF).1 MITF plays an important role in the development of mast cells.2-9 Mast cells of mi/mi genotype express normal amounts of abnormal MITF (mi-MITF), whereas mast cells of tg/tg genotype do not express any MITFs.10-12 Because tg is considered to be a null mutant allele, the tg/tg mice are useful as a standard for evaluating the function of other mutant MITFs.13-15 We previously compared the phenotype of mast cells of mi/mi mice with that of tg/tg mice.13 The transcription of mouse mast cell protease (mMCP)-4, mMCP-5, and mMCP-6 genes in cultured mast cells (CMCs) derived from mi/mi mice is reduced to the level comparable to that of tg/tg CMCs.13 The transcription of c-kit, granzyme B (Gr B), and tryptophan hydroxylase (TPH) genes is significantly reduced in mi/mi CMCs, but the reduction of transactivation of these genes is moderate in tg/tg CMCs.13 This shows that the mi-MITF possesses an inhibitory effect on the transcription of c-kit, Gr B, and TPH genes.13 The mi-MITF deletes one arginine in the basic domain, whereas MITF encoded by the miew mutant allele (ew-MITF) deletes 16 of 21 amino acids of the basic domain.10,11 In the present study, we compared the mast cell phenotypes of miew/miew mice with those of tg/tg and mi/mi mice to examine the effect of a large deletion of the basic domain.     Study design Top Abstract Introduction Study design Results and discussion References Mice and cells The C57BL/6-tg/tg and C57BL/6-mi/mi mice (hereafter tg/tg and mi/mi mice) were described previously.13 The C57BL/6-miew/+ mice16 were sent from the laboratory of M. L. Lamoreux, and C57BL/6-miew/miew mice were raised in our laboratory. C57BL/6-+/+ mice were used as a control. CMCs and P815 cells have been described.13 Northern blot analysis Total RNA prepared from CMCs was electrophoresed and blotted. The intensity of the signal for each messenger RNA (mRNA) was quantified by a densitometer (Molecular Dynamics, Sunnyvale, CA). Concentration of serotonin The concentration of serotonin was measured using high-performance liquid chromatography as previously described.17 Staining of skin mast cells Pieces of dorsal skin were removed from mice aged 20 days and embedded in paraffin. A section was stained with alcian blue and nuclear fast red, and an adjacent section was stained with berberine sulfate.18 Transient cotransfection assay The reporter plasmid that contained Gr B promoter between nucleotide 910 and +42 (pGr B-910, +1 shows a transcription initiation site) and the reporter plasmid lacking Gr B promoter (pGr B-42) have been described.13 The luciferase activity was normalized as previously described.13     Results and discussion Top Abstract Introduction Study design Results and discussion References The phenotype of miew/miew mast cells was compared with that of +/+, tg/tg, and mi/mi mast cells. First, CMCs were obtained from spleens of mice of each genotype.13 The expression of genes that were known to be affected by MITF19-23 was compared with Northern blot. The amounts of c-kit, Gr B, and TPH mRNAs in miew/miew CMCs were comparable to those of tg/tg CMCs, and the amounts were intermediate between those of +/+ and mi/mi CMCs (Figure 1A). Although the amounts of mMCP-4, mMCP-5, and mMCP-6 mRNAs were very low in all kinds of CMCs examined here, their amounts in miew/miew CMCs were comparable to those of tg/tg and mi/mi CMCs (Figure 1A). View larger version (24K): [in this window] [in a new window]   Figure 1. Comparison of abnormalities of CMCs and skin mast cells of tg/tg, mi/mi, and miew/miew mice. (A) Northern blot analysis of RNAs obtained from CMCs of +/+, tg/tg, mi/mi, or miew/miew genotype. The blot was hybridized with 32P-labeled complementary DNA probe of c-kit, Gr B, TPH, mMCP-4, mMCP-5, mMCP-6, or GAPDH as described previously.11 Twenty micrograms of total RNA was loaded in each lane, and each 32P-labeled complementary DNA was reprobed using the same membrane. A representative experiment is shown. (B) The amount of mMCP-6, mMCP-5, mMCP-4, c-kit, Gr B, and TPH mRNAs was quantified by the densitometry, and the ratio to the mRNA amount of tg/tg CMCs, which was defined as relative mRNA amount, was calculated. The bars represent the mean ± SE of 3 independent experiments. In some cases, the SE was too small to be shown by the bars. (C) The serotonin contents per 106 +/+, tg/tg, mi/mi, and miew/miew CMCs were measured. The bars represent the mean ± SE of 3 experiments. *P < .05 by t test when compared with the value of tg/tg CMCs. (D) Mast cells were counted, and the number was expressed as mast cells per centimeter of skin. Berberine sulfate-positive mast cells were also counted, and the proportion of berberine sulfate-positive cells to alcian blue-positive cells was calculated. The bars represent the mean ± SE of 5 mice. In some cases, the SE was too small to be shown by the bars. *P < .01 by t test when compared with the value of tg/tg mice. We quantified the amounts of mRNAs using densitometry and calculated the ratio of each mRNA amount of +/+, mi/mi, or miew/miew CMCs to that of tg/tg CMCs (Figure 1B). We defined the ratio as relative mRNA amount. The relative mRNA amounts of all examined genes were more than 1.0 in +/+ CMCs, indicating that the +-MITF had positive effect on transcription. In mi/mi CMCs, the relative mRNA amounts of mMCP-4, mMCP-5, and mMCP-6 genes were about 1.0, but those of c-kit, Gr B, and TPH genes were less than 1.0. This suggested that the mi-MITF had negative effect on the transcription of c-kit, Gr B, and TPH genes. The relative mRNA amounts of all examined genes were about 1.0 in miew/miew CMCs, indicating that the ew-MITF had neither positive nor negative effects on transcription. Because TPH is the rate-limiting enzyme of the serotonin synthesis,24 the serotonin contents were compared among various CMCs. The serotonin content of miew/miew CMCs was comparable to that of tg/tg CMCs. Both values were significantly smaller than that of +/+ CMCs and significantly greater than that of mi/mi CMCs (Figure 1C, P < .05 by t test). Serotonin contents of +/+, tg/tg, mi/mi, and miew/miew CMCs were well correlated with the mRNA expression levels of TPH gene in CMCs of each genotype (+/+ > miew/miew ~ tg/tg > mi/mi). Second, the number of mast cells and the proportion of berberine sulfate-positive mast cells were compared among skin tissues of +/+, tg/tg, mi/mi, and miew/miew mice. The number of skin mast cells of miew/miew mice decreased to one third that of the +/+ mice and was comparable to that of tg/tg and mi/mi mice (Figure 1D). Most skin mast cells were berberine sulfate-positive in +/+ mice.25 The proportion of berberine sulfate-positive to alcian blue-positive mast cells in the skin of miew/miew mice was comparable to that of +/+ mice and to that of tg/tg mice (Figure 1D). In contrast, the proportion of berberine sulfate-positive mast cells was only 3% in the skin of mi/mi mice as reported previously.7 The abnormalities of miew/miew skin mast cells were comparable to those of tg/tg skin mast cells rather than to those of mi/mi skin mast cells. We examined the effect of ew-MITF on the transactivation of Gr B promoter using the transient cotransfection assay. The luciferase construct containing the Gr B promoter was cotransfected into P815 cells with the expression plasmid containing no insert, +-MITF, mi-MITF, or ew-MITF complementary DNA. The coexpression of +-MITF significantly increased the luciferase activity, whereas that of mi-MITF significantly reduced it as reported previously (Figure 2A).13 The expression of ew-MITF showed luciferase activity comparable to the value obtained by the expression vector alone (Figure 2A). The ew-MITF had no effect on the activity of Gr B promoter. This was consistent with the fact that the abnormalities of CMCs and skin mast cells of miew/miew mice were similar to those of tg/tg mice. The part of the basic domain that was deleted in ew-MITF (Figure 2B) appeared necessary for both positive and negative functions. View larger version (33K): [in this window] [in a new window]   Figure 2. Function and structure of +-MITF, mi-MITF, and ew-MITF. (A) The effect of coexpression of +-MITF, mi-MITF, or ew-MITF on the luciferase activity under the control of the Gr B promoter. Various reporter and effector constructs were introduced into P815 cells by electroporation. The value of the luciferase activity was divided by the value obtained with the transfection of pGr B-42 alone and was shown as the relative luciferase activity. The bars represent the mean ± SE of 3 independent experiments. In some cases, the SE was too small to be shown by the bars. *P < .01 by t test when compared with the control, in which expression vector containing no insert was cotransfected. (B) Comparison of amino acid sequence among +-MITF, mi-MITF, and ew-MITF. The amino acids are numbered from the initiation codon. Bars in mi-MITF or ew-MITF show the deleted amino acids. One of 3 arginines (amino acid 215-217) is deleted in mi-MITF. It is unclear which one has been deleted. Twenty-one amino acids that compose the basic domain are boxed.     Footnotes Submitted March 7, 2001; accepted June 22, 2001. Supported by grants from the Ministry of Education, Culture, Sports, Science and Technology and Uehara Memorial Foundation. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734. Reprints: Eiichi Morii, Dept of Pathology, Rm C2, Osaka University Medical School, Yamada-oka 2-2, Suita 565-0871, Japan; e-mail: morii{at}patho.med.osaka-u.ac.jp.     References Top Abstract Introduction Study design Results and discussion References 1. Hodgkinson CA, Moore KJ, Nakayama A, et al. Mutations at the mouse microphthalmia locus are associated with defects in a gene encoding a novel basic-helix-loop-helix-zipper protein. Cell. 1993;74:395-404[CrossRef][Medline] [Order article via Infotrieve]. 2. Kitamura Y, Morii E, Jippo T, et al. The mi transcription factor (MITF) as a regulator of mast cell differentiation. Int J Hematol. 2000;71:197-202[Medline] [Order article via Infotrieve]. 3. Kitamura Y, Morii E, Ogihara H, et al. Mutant mice: a useful tool for studying the development of mast cells. Int Arch Allergy Immunol. 2001;124:16-19[CrossRef][Medline] [Order article via Infotrieve]. 4. Silvers WK. The Coat Colors of Mice: A Model for Mammalian Gene Action and Interaction. New York, NY: Springer-Verlag; 1979. 5. Ebi Y, Kasugai T, Seino Y, et al. Mechanism of mast cell deficiency in mutant mice of mi/mi genotype: an analysis by co-culture of mast cells and fibroblasts. Blood. 1990;75:1247-1251[Abstract/Free Full Text]. 6. Ebi Y, Kanakura Y, Jippo-Kanemoto T, et al. Low c-kit expression of cultured mast cells of mi/mi genotype may be involved in their defective responses to fibroblasts that express the ligand for c-kit. Blood. 1992;80:1454-1462[Abstract/Free Full Text]. 7. Kasugai T, Oguri K, Jippo-Kanemoto T, et al. Deficient differentiation of mast cells in the skin of mi/mi mice: usefulness of in situ hybridization for evaluation of mast cell phenotype. Am J Pathol. 1993;143:1337-1347[Abstract]. 8. Isozaki K, Tsujimura T, Nomura S, et al. Cell type-specific deficiency of c-kit gene expression in mutant mice of mi/mi genotype. Am J Pathol. 1994;145:827-836[Abstract]. 9. Jippo T, Ushio H, Hirota S, et al. Poor response of cultured mast cells derived from mi/mi mutant mice to nerve growth factor. Blood. 1994;84:2977-2983[Abstract/Free Full Text]. 10. Moore KJ. Insight into the microphthalmia gene. Trends Genet. 1995;11:442-448[CrossRef][Medline] [Order article via Infotrieve]. 11. Steingrimsson E, Moore KJ, Lamoreux ML, et al. Molecular basis of mouse microphthalmia (mi) mutations helps explain their developmental and phenotypic consequences. Nat Genet. 1994;8:256-263[CrossRef][Medline] [Order article via Infotrieve]. 12. Hemesath TJ, Streingrimsson E, McGill G, et al. Microphthalmia, a critical factor in melanocyte development, defines a discrete transcription factor family. Gene Dev. 1994;8:2770-2780[Abstract/Free Full Text]. 13. Ito A, Morii E, Kim DK, et al. Inhibitory effect of the transcription factor encoded by the mi mutant allele in cultured mast cells of mice. Blood. 1999;93:1189-1196[Abstract/Free Full Text]. 14. Ogihara H, Morii E, Kim DK, et al. Inhibitory effect of the transcription factor encoded by the mutant mi microphthalmia allele on transactivation of mouse mast cell protease 7 gene. Blood. 2001;97:645-651[Abstract/Free Full Text]. 15. Morii E, Ogihara H, Kim DK, et al. Importance of leucine zipper domain of mi transcription factor (MITF) for differentiation of mast cells demonstrated using mice/mice mice of which MITF lacks the zipper domain. Blood. 2001;97:2038-2044[Abstract/Free Full Text]. 16. Wood BC, Miner G. Mouse News Lett. 1969;40:32. 17. Kim DK, Morii E, Ogihara H, et al. Different effect of various mutant MITF encoded by mi, Mior, or Miwh allele on phenotype of murine mast cells. Blood. 1999;93:4179-4186[Abstract/Free Full Text]. 18. Nakano T, Sonoda T, Hayashi C, et al. Fate of bone marrow-derived cultured mast cells after intracutaneous, intraperitoneal, and intravenous transfer into genetically mast cell deficient W/Wv mice: evidence that cultured mast cells can give rise to both "connective tissue-type" and "mucosal" mast cells. J Exp Med. 1985;162:1025-1043[Abstract/Free Full Text]. 19. Jippo T, Lee YM, Katsu Y, et al. Deficient transcription of mouse mast cell protease 4 gene in mutant mice of mi/mi genotype. Blood. 1999;93:1942-1950[Abstract/Free Full Text]. 20. Morii E, Jippo T, Tsujimura T, et al. Abnormal expression of mouse mast cell protease 5 gene in cultured mast cells derived from mutant mi/mi mice. Blood. 1997;90:3057-3066[Abstract/Free Full Text]. 21. Morii E, Tsujimura T, Jippo T, et al. Regulation of mouse mast cell protease 6 gene expression by transcription factor encoded by the mi locus. Blood. 1996;88:2488-2494[Abstract/Free Full Text]. 22. Tsujimura T, Morii E, Nozaki M, et al. Involvement of transcription factor encoded by the mi locus in the expression of c-kit receptor tyrosine kinase in cultured mast cells of mice. Blood. 1996;88:1225-1233[Abstract/Free Full Text]. 23. Ito A, Morii E, Maeyama K, et al. Systematic method to obtain novel genes that are regulated by mi transcription factor (MITF): impaired expression of granzyme B and tryptophan hydroxylase in mi/mi cultured mast cells. Blood. 1998;91:3210-3221[Abstract/Free Full Text]. 24. Jequier E, Lovenberg W, Sjoerdsma A. Tryptophan hydroxylase inhibition: the mechanism by which p-chlorophenylalanine depletes rat brain serotonin. Mol Pharmacol. 1967;3:274-278[Abstract/Free Full Text]. 25. Kitamura Y. Heterogeneity of mast cells and phenotypic change between subpopulations. Annu Rev Immunol. 1989;7:59-76[CrossRef][Medline] [Order article via Infotrieve]. © 2001 by The American Society of Hematology.   CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? 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