title: 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.
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.
authors.affiliation: Second Department of Internal Medicine, Osaka University Medical School, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Ebi
authors.forename: Y
authors.initials: Y
authors.lastname: Kanakura
authors.forename: T
authors.initials: T
authors.lastname: Jippo-Kanemoto
authors.forename: T
authors.initials: T
authors.lastname: Tsujimura
authors.forename: T
authors.initials: T
authors.lastname: Furitsu
authors.forename: H
authors.initials: H
authors.lastname: Ikeda
authors.forename: S
authors.initials: S
authors.lastname: Adachi
authors.forename: T
authors.initials: T
authors.lastname: Kasugai
authors.forename: S
authors.initials: S
authors.lastname: Nomura
authors.forename: Y
authors.initials: Y
authors.lastname: Kanayama
mesh_terms: D016475:3T3 Cells; D000818:Animals; D002454:Cell Differentiation; D002455:Cell Division; D002478:Cells, Cultured; D053818:Chymases; D005260:Female; D005347:Fibroblasts / Q000166:cytology; D005838:Genotype; D008297:Male; D008407:Mast Cells / Q000166:cytology*; D008415:Mastocytosis / Q000235:genetics; D051379:Mice; D008817:Mice, Mutant Strains / Q000235:genetics*; D010022:Osteopetrosis / Q000235:genetics*; D010641:Phenotype; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D019009:Proto-Oncogene Proteins c-kit; D012333:RNA, Messenger / Q000032:analysis; D011956:Receptors, Cell Surface / Q000235:genetics; D012697:Serine Endopeptidases / Q000235:genetics; D014158:Transcription, Genetic
chemical_list: D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D019009:Proto-Oncogene Proteins c-kit; D012697:Serine Endopeptidases; D053818:Chymases