Regulation of mouse mast cell protease 6 gene expression by transcription factor encoded by the mi locus

The mi locus of mice encodes a member of the basic-helix-loop-helix-leucine zipper (bHLH-Zip) protein family of transcription factors (hereafter called MITF). Because the expression of the mouse mast cell protease 6 (MMCP-6) gene is remarkably reduced in mast cells of mi/mi mutant mice, we investigated the effect of MITF on the transcription of the MMCP-6 gene. First, we introduced the normal (+) MITF cDNA into mi/mi cultured mast cells using the retroviral vector. Overexpression of +-MITF but not mi-MITF normalized the expression of the MMCP-6 gene, indicating the involvement of +-MITF in the MMCP-6 gene transactivation. Second, we analyzed the promoter of the MMCP-6 gene by the transient cotransfection assay. The luciferase construct under the control of the MMCP-6 promoter and the cDNA encoding +-MITF or mi-MITF were cotransfected into NIH/ 3T3 fibroblasts. The coexpression of +- MITF but not mi-MITF increased the luciferase activity 10-fold. We found a CACATG and a CATCTG motif in the MMCP-6 promoter, both of which are generally recognized by bHLH-Zip-type transcription factors. We also found a GACCTG motif that was strongly bound by +-MITF. These three motifs were necessary for the 10-fold transactivation ability of the MMCP-6 promoter by +-MITF. Mutations of each motif significantly reduced the transactivation, suggesting that +-MITF directly transactivated the MMCP-6 gene through these three motifs.

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Abnormal Expression of Mouse Mast Cell Protease 5 Gene in Cultured Mast Cells Derived From Mutant mi/mi Mice

Blood ◽

10.1182/blood.v90.8.3057 ◽

1997 ◽

Vol 90 (8) ◽

pp. 3057-3066 ◽

Cited By ~ 73

Author(s):

Eiichi Morii ◽

Tomoko Jippo ◽

Tohru Tsujimura ◽

Koji Hashimoto ◽

Dae-Ki Kim ◽

...

Keyword(s):

Mast Cell ◽

Transcription Factors ◽

Mast Cells ◽

Promoter Region ◽

Leucine Zipper ◽

Consensus Sequence ◽

Mast Cell Protease ◽

Helix Loop Helix ◽

Regulation Mechanisms ◽

Mouse Mast Cell

Abstract Mast cells contain a lot of mast cell-specific proteases. We have reported that the expression of mouse mast cell protease 6 (MMCP-6) is remarkably reduced in both cultured mast cells (CMCs) and skin mast cells of mi/mi mutant mice. In the present study, we found that the expression of MMCP-5 was reduced in CMCs but not in skin mast cells of mi/mi mice, and we compared the regulation mechanisms of MMCP-5 with those of MMCP-6. The mi locus encodes a member of the basic-helix-loop-helix-leucine zipper (bHLH-Zip) protein family of transcription factors (hereafter called MITF ). The consensus sequence recognized and bound by bHLH-Zip transcription factors is CANNTG. The overexpression of the normal (+) MITF but not of mi-MITF normalized the poor expression of the MMCP-5 gene in mi/mi CMCs, indicating the involvement of +-MITF in transactivation of the MMCP-5 gene. Although +-MITF directly bound CANNTG motifs in the promoter region of the MMCP-6 gene and transactivated it, the binding of +-MITF to the CAGTTG motif in the promoter region of the MMCP-5 gene was not detectable. The +-MITF appeared to regulate the transactivation of the MMCP-5 gene indirectly. Moreover, addition of stem cell factor to the medium normalized the expression of the MMCP-5 but not of the MMCP-6 gene in mi/mi CMCs. Despite the significant reduction of both MMCP-5 and MMCP-6 expressions in mi/mi CMCs, their regulation mechanisms appeared to be different.

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Involvement of Transcription Factor Encoded by the Mouse mi Locus (MITF ) in Expression of p75 Receptor of Nerve Growth Factor in Cultured Mast Cells of Mice

Blood ◽

10.1182/blood.v90.7.2601.2601_2601_2608 ◽

1997 ◽

Vol 90 (7) ◽

pp. 2601-2608 ◽

Cited By ~ 5

Author(s):

Tomoko Jippo ◽

Eiichi Morii ◽

Kumiko Tsujino ◽

Tohru Tsujimura ◽

Young-Mi Lee ◽

...

Keyword(s):

Transcription Factors ◽

Nerve Growth Factor ◽

Growth Factor ◽

Mast Cells ◽

Leucine Zipper ◽

Poor Response ◽

Nerve Growth ◽

Helix Loop Helix ◽

3T3 Fibroblasts ◽

Direct Binding

The mi locus of mice encodes a member of the basic-helix-loop-helix-leucine zipper (bHLH-Zip) protein family of transcription factors (hereafter called MITF ). Cultured mast cells (CMCs) of mi/mi genotype showed a poor response to nerve growth factor (NGF ). Addition of NGF to the suboptimal dose of interleukin-3 (IL-3) increased the plating efficiency of normal (+/+) CMCs but not mi/mi CMCs. Although +/+ CMCs were berberine sulfate–negative when cultured with IL-3, +/+ CMCs became berberine sulfate–positive when cultured in the presence of both IL-3 and NGF, which suggested increased heparin content. In contrast, NGF did not influence the phenotype of mi/mi CMCs. The poor response of mi/mi CMCs to NGF was attributed to the deficient expression of p75 NGF receptor. The purpose of the present study is to examine the effect of MITF on p75 gene transcription. Overexpression of +-MITF or mi-MITF was observed in mi/mi CMCs to which cDNA encoding each type of MITF had been introduced using the retroviral vector. Overexpression of +-MITF but not of mi-MITF normalized the expression of p75 and the above-mentioned poor responses of mi/mi CMCs to NGF, indicating the involvement of +-MITF in p75 gene transactivation. Then, we analyzed the promoter of the p75 gene. Two CANNTG motifs recognized by bHLH-Zip–type transcription factors were conserved between the mouse and rat p75 promoters. One of these two CANNTG motifs was specifically bound by +-MITF. When the luciferase gene under the control of the p75 promoter was cotransfected into NIH/3T3 fibroblasts with cDNA encoding +-MITF or mi-MITF, luciferase activity increased significantly only when +-MITF cDNA was cotransfected. The mutation of this CANNTG motif abolished the transactivation effect of +-MITF, indicating that +-MITF transactivated the p75 gene, at least in part, through direct binding.

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Involvement of Transcription Factor Encoded by the Mouse mi Locus (MITF ) in Expression of p75 Receptor of Nerve Growth Factor in Cultured Mast Cells of Mice

Blood ◽

10.1182/blood.v90.7.2601 ◽

1997 ◽

Vol 90 (7) ◽

pp. 2601-2608 ◽

Cited By ~ 34

Author(s):

Tomoko Jippo ◽

Eiichi Morii ◽

Kumiko Tsujino ◽

Tohru Tsujimura ◽

Young-Mi Lee ◽

...

Keyword(s):

Transcription Factors ◽

Nerve Growth Factor ◽

Growth Factor ◽

Mast Cells ◽

Leucine Zipper ◽

Poor Response ◽

Nerve Growth ◽

Helix Loop Helix ◽

3T3 Fibroblasts ◽

Direct Binding

Abstract The mi locus of mice encodes a member of the basic-helix-loop-helix-leucine zipper (bHLH-Zip) protein family of transcription factors (hereafter called MITF ). Cultured mast cells (CMCs) of mi/mi genotype showed a poor response to nerve growth factor (NGF ). Addition of NGF to the suboptimal dose of interleukin-3 (IL-3) increased the plating efficiency of normal (+/+) CMCs but not mi/mi CMCs. Although +/+ CMCs were berberine sulfate–negative when cultured with IL-3, +/+ CMCs became berberine sulfate–positive when cultured in the presence of both IL-3 and NGF, which suggested increased heparin content. In contrast, NGF did not influence the phenotype of mi/mi CMCs. The poor response of mi/mi CMCs to NGF was attributed to the deficient expression of p75 NGF receptor. The purpose of the present study is to examine the effect of MITF on p75 gene transcription. Overexpression of +-MITF or mi-MITF was observed in mi/mi CMCs to which cDNA encoding each type of MITF had been introduced using the retroviral vector. Overexpression of +-MITF but not of mi-MITF normalized the expression of p75 and the above-mentioned poor responses of mi/mi CMCs to NGF, indicating the involvement of +-MITF in p75 gene transactivation. Then, we analyzed the promoter of the p75 gene. Two CANNTG motifs recognized by bHLH-Zip–type transcription factors were conserved between the mouse and rat p75 promoters. One of these two CANNTG motifs was specifically bound by +-MITF. When the luciferase gene under the control of the p75 promoter was cotransfected into NIH/3T3 fibroblasts with cDNA encoding +-MITF or mi-MITF, luciferase activity increased significantly only when +-MITF cDNA was cotransfected. The mutation of this CANNTG motif abolished the transactivation effect of +-MITF, indicating that +-MITF transactivated the p75 gene, at least in part, through direct binding.

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Deficient Transcription of Mouse Mast Cell Protease 4 Gene in Mutant Mice of mi/mi Genotype

Blood ◽

10.1182/blood.v93.6.1942.406k08_1942_1950 ◽

1999 ◽

Vol 93 (6) ◽

pp. 1942-1950 ◽

Cited By ~ 42

Author(s):

Tomoko Jippo ◽

Young-Mi Lee ◽

Yee Katsu ◽

Kumiko Tsujino ◽

Eiichi Morii ◽

...

Keyword(s):

Mast Cell ◽

Leucine Zipper ◽

Granzyme B ◽

Mutant Mice ◽

Cathepsin G ◽

Mast Cell Protease ◽

Helix Loop Helix ◽

Regulation Mechanisms ◽

The Difference ◽

Mouse Mast Cell

The mi locus encodes a member of the basic-helix-loop-helix-leucine zipper (bHLH-Zip) protein family of transcription factors (hereafter called MITF). We reported that expression of the mouse mast cell protease 5 (MMCP-5) and MMCP-6 genes were deficient in cultured mast cells (CMC) derived from mutant mice ofmi/mi genotype. Despite the reduced expression of both MMCP-5 and MMCP-6, their regulation mechanisms were different. Because MMCP-5 is a chymase and MMCP-6 a tryptase, there was a possibility that the difference in regulation mechanisms was associated with their different characteristics as proteases. We compared the regulation mechanisms of another chymase, MMCP-4, with those of MMCP-5 and MMCP-6. The expression of the MMCP-4 gene was also deficient in mi/mi CMC. The overexpression of the normal (+) MITF but not of mi-MITF normalized the poor expression of the MMCP-4 gene in mi/mi CMC, indicating the involvement of +-MITF in transactivation of the MMCP-4 gene. Although MMCP-4 is chymase as MMCP-5, the regulation of MMCP-4 expression was more similar to MMCP-6 than to MMCP-5. We also showed the deficient expression of granzyme B and cathepsin G genes inmi/mi CMC. Genes encoding granzyme B, cathepsin G, MMCP-4, and MMCP-5 are located on chromosome 14. Because all these genes showed deficient expression in mi/mi CMC, there is a possibility that MITF might regulate the expression of these genes through a locus control region.

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Biochemical and immunological characterization of multiple glycoforms of mouse mast cell protease 1: comparison with an isolated murine serosal mast cell protease (MMCP-4)

Biochemical Journal ◽

10.1042/bj2940127 ◽

1993 ◽

Vol 294 (1) ◽

pp. 127-135 ◽

Cited By ~ 21

Author(s):

G F J Newlands ◽

D P Knox ◽

S R Pirie-Shepherd ◽

H R P Miller

Keyword(s):

Mast Cell ◽

Amino Acid ◽

Mast Cells ◽

Western Blotting ◽

Trichinella Spiralis ◽

Double Diffusion ◽

Terminal Amino Acid ◽

Mast Cell Protease ◽

Terminal Amino ◽

Mouse Mast Cell

Five highly soluble, chymotrypsin-like, neutral serine proteases, with molecular masses in the range 30-33 kDa, were isolated from Trichinella spiralis-infected mouse small intestine. These enzymes were closely related antigenically on Western blotting and by Ouchterlony double diffusion using a polyclonal, cross-absorbed, sheep antibody raised against mouse mast cell protease-1 (MMCP-1) and on the basis of N-terminal amino acid sequence analysis, were identified as variant forms of MMCP-1. Substrate and inhibitor analysis confirmed that the five variants (MMCP-1 A-E) had similar characteristics, although highly significant (P = 0.025 to P < 0.0001) variations in Km and kcat, were detected. Against human alpha 1-proteinase inhibitor the Ki for MMCP-1C (45 pM) was significantly (P < 0.0001) greater than those for the other proteases (0.76-2.2 pM). The differences in electrophoretic mobility are probably a result of variable glycosylation, since removal of N-linked carbohydrate produced a polypeptide of approx. 28 kDa in each case which was, like the native enzyme, immunoreactive on Western blotting. A much less soluble 28 kDa enzyme was isolated from serosal mast cells and identified as MMCP-4 by N-terminal amino acid sequencing. Like MMCP-1 it has chymotrypsin-like substrate specificities with activity at neutral pH. However, it was antigenically distinct from MMCP-1 and, using sheep anti-MMCP-1, was not detected on Western blotting or by Ouchterlony double diffusion, e.l.i.s.a. or immunohistochemistry. This last technique established that the MMCP-1 variants were uniquely present in enteric mast cells, thereby providing a highly selective means of distinguishing the mucosal and connective tissue mast cell subsets in the mouse.

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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 ◽

10.1182/blood.v80.6.1454.1454 ◽

1992 ◽

Vol 80 (6) ◽

pp. 1454-1462 ◽

Cited By ~ 6

Author(s):

Y Ebi ◽

Y Kanakura ◽

T Jippo-Kanemoto ◽

T Tsujimura ◽

T Furitsu ◽

...

Keyword(s):

Mast Cell ◽

Mast Cells ◽

Tyrosine Phosphorylation ◽

Messenger Rna ◽

Tissue Type ◽

Phenotypic Change ◽

3T3 Fibroblasts ◽

Mouse Mast Cell ◽

And Function

Abstract Mutant mice of mi/mi genotype are osteopetrotic and deficient in tissue mast cells due to a defect in osteoclasts and mast cells. In an effort to further understand the mechanisms behind why mi/mi mouse-derived cultured mast cells (mi/mi-CMC) responded to interleukin-3 (IL-3), but not to the proliferative stimuli presented by fibroblasts, mi/mi-CMC and congenic normal (+/+) mouse-derived CMC (+/+-CMC), both of which expressed the phenotypic characteristics of immature mast cells, were cocultured with Swiss albino/3T3 fibroblasts in a medium containing IL- 3. In the in vitro CMC/fibroblast coculture, mi/mi-CMC did not acquire the phenotypes of connective tissue-type mast cells (CTMC), while +/+- CMC did. In addition, attachment of mi/mi-CMC to the fibroblasts was found to be significantly lower than that of +/+-CMC. Because the interaction of c-kit product with its ligand (stem cell factor [SCF]) is known to play an important role not only in proliferation and differentiation of mast cells but also in attachment of CMC to fibroblasts, the expression and function of c-kit were investigated in mi/mi-CMC and +/+-CMC. Recombinant rat SCF (rrSCF164) induced a dose- dependent proliferation of +/+-CMC. Also, rrSCF164 induced +/+-CMC to acquire the phenotypes of CTMC in the medium containing IL-3. By contrast, rrSCF164 did not stimulate the proliferation of mi/mi-CMC nor induce a phenotypic change of the cells from immature mast cells to mature, CTMC-like mast cells. Immunoblotting with antiphosphotyrosine antibody showed that rrSCF164 induced considerable tyrosine phosphorylation of 145- to 165-Kd protein, the product of c-kit, in +/+- CMC, whereas tyrosine phosphorylation of the protein was barely detectable in mi/mi-CMC. Northern blot and flow cytometry analyses showed that mi/mi-CMC expressed much less c-kit at both protein and message levels than +/+-CMC. Further, mi/mi-CMC were found to differ from +/+-CMC in the expression of mouse mast cell protease-6 (MMCP-6) and MMCP-2 messenger RNA transcripts. These results suggest that the gene product of the mi locus may be important in regulating the expression of gene products such as c-kit, and that mast cell deficiency of mi/mi mice appears to be due, at least in part, to impaired signaling through the c-kit receptor because of the low c-kit expression.

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Inhibitory Effect of the Transcription Factor Encoded by themi Mutant Allele in Cultured Mast Cells of Mice

Blood ◽

10.1182/blood.v93.4.1189.404k32_1189_1196 ◽

1999 ◽

Vol 93 (4) ◽

pp. 1189-1196 ◽

Cited By ~ 2

Author(s):

Akihiko Ito ◽

Eiichi Morii ◽

Dae-Ki Kim ◽

Tatsuki R. Kataoka ◽

Tomoko Jippo ◽

...

Keyword(s):

Transcription Factor ◽

Mast Cells ◽

Leucine Zipper ◽

Tryptophan Hydroxylase ◽

Cdna Probe ◽

Inhibitory Effect ◽

Northern Analysis ◽

Helix Loop Helix ◽

Genes Encoding ◽

Mouse Mast Cell

The mi locus of mice encodes a transcription factor of the basic-helix-loop-helix-leucine zipper protein family (MITF). The MITF encoded by the mutant mi allele (mi-MITF) deletes 1 of 4 consecutive arginines in the basic domain. The mice of mi/migenotype express mi-MITF, whereas the mice of tg/tggenotype have a transgene at the 5′ flanking region of themi gene and do not express any MITF. To investigate the function of mi-MITF in cultured mast cells (CMCs), we took two approaches. First, mRNA obtained from mi/mi CMCs ortg/tg CMCs was subtracted from complementary (c) DNA library of normal (+/+) CMCs, and the (+/+-mi/mi) and (+/+-tg/tg) subtraction libraries were obtained. When the number of clones that hybridized more efficiently with +/+ CMC cDNA probe than with mi/mi or tg/tg CMC cDNA probe was compared using Southern analysis, the number was larger in the (+/+-mi/mi) library than in the (+/+-tg/tg) library. Second, we compared mRNA expression of six genes betweenmi/mi and tg/tg CMCs by Northern analysis. The transcription of three genes encoding mouse mast cell proteases was impaired in both mi/mi and tg/tg CMCs. On the other hand, the transcription of three genes encoding c-kit receptor, tryptophan hydroxylase, and granzyme B was markedly reduced inmi/mi CMCs, but the reduction was significantly smaller intg/tg CMCs. These results indicated the inhibitory effect ofmi-MITF on the transactivation of particular genes in CMCs.

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