| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2002: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2001: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Research Abstract |
MITF is a basic-helix-loop-helix-leucine zipper-type transcription factor. The mutant mi and Mi^<wh> alleles encode MITFs with deletion and alteration of a single amino acid, respectively, while the tg is a null mutation. When cultured mast cells (CMCs) are established from spleens of C57BL/6 (B6) wild-type (+/+) mice and cocultured on a monolayer ofNIH/3T3 fibroblasts, a considerable number of CMCs adhered to NIH/3T3 fibroblasts. In contrast, the number of CMCs derived from C57BL/6 (B6)-mi/mi, B6-Mi^<wh>/Mi^<wh> and B6-tg/tg mice that adhered to NIH/3T3 fibroblasts was one third that ofB6-+/+ CMCs. We subtracted a cDNA library ofB6-+/+ CMCs with messenger RNAs expressed by B6-mi/mi CMCs and found a clone encoding SgIGSF, a recently identified member of the immunoglobulin superfamily. Northern and Western blot analyses revealed that SgIGSF was expressed in B6-+/+ CMCs but not in CMCs derived from MITF mutants. Immunocytochemical analysis showed that SgIGSF localized to the cell-cell contact areas between B6-+/+ CMCs and NIH/3T3 fibroblasts. Transfection of B6-mi/mi and B6-tg/tg CMCs with SgIGSF cDNA normalized their adhesion to NIH/3T3 fibroblasts. NIH/3T3 fibroblasts did not express SgIGSF, indicating that SgIGSF acts as a heterophilic adhesion molecule. Transfection of B6-tg/tg CMCs with normal MITF cDNA elevated their SgIGSF expression to normal levels. Wild-type MITF, but not mi-type MITF, could bind to a motif in the SgIGSF gene promoter and this association resulted in a significant transactivation of the promoter. These results indicated that SgIGSF mediated the adhesion of CMCs to fibroblasts and that the transcription of SgIGSF was critically regulated by MITF.
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