Involvement of transcription factor encoded by the mi locus in the expression of c-kit receptor tyrosine kinase in cultured mast cells of mice.
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 of mi/mi genotype (mi/mi CMCs) did not normally respond to stem cell factor (SCF), a ligand for the c-kit receptor tyrosine kinase. The poor response of mi/mi CMCs to SCF was attributed to the deficient expression of c-kit both the mRNA and protein levels. The purpose of the present study is to investigate the effect of MITF on the transcription of the c-kit gene. First, we introduced cDNA encoding normal (+) MITF or mutant (mi) MITF into mi/mi CMCs using the retroviral vector. Overexpression of (+)-MITF but not mi-MITF normalized the expression of the c-kit and the poor response of mi/mi CMCs to SCF, indicating the involvement of (+)-MITF in the c-kit gene transactivation. Second, we analyzed the promoter of the c-kit gene. Three CANNTG motifs recognized by bHLH-Zip-type transcription factors were conserved between the mouse and human c-kit promoters. Among these three CANNTG motifs, only the CACCTG motif (nt -356 to -351) was specifically bound by (+)-MITF. When the luciferase gene under the control of the c-kit promoter was contransfected into NIH/3T3 fibroblasts with cDNA encoding (+)-MITF or mi-MITF, the luciferase activity significantly increased only when (+)-MITF cDNA was cotransfected. The deletion of the promoter region containing the CACCTG motif or the mutation of the CACCTG to CTCCAG abolished the transactivation effect of (+)-MITF, indicating that (+)-MITF transactivated the c-kit gene through the CACCTG motif. When the luciferase gene under the control of the c-kit promoter was introduced into the FMA3 mastocytoma and FEC-P1 myeloid cell lines, remarkable luciferase activity was observed only in FMA3 cells. Thus, the involvement of (+)-MITF in the c-kit transactivation appeared to be specific to the mast cell lineage.
Tsujimura
T
T
Department of Pathology, Medical School, Osaka University, Suita, Japan.
Morii
E
E
Nozaki
M
M
Hashimoto
K
K
Moriyama
Y
Y
Takebayashi
K
K
Kondo
T
T
Kanakura
Y
Y
Kitamura
Y
Y
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D017931:DNA Primers / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000502:physiology*; D005786:Gene Expression Regulation; D018257:Helix-Loop-Helix Motifs; D006801:Humans; D016350:Leucine Zippers; D008407:Mast Cells / Q000201:enzymology*; D051379:Mice; D008817:Mice, Mutant Strains; D051739:Microphthalmia-Associated Transcription Factor; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D019009:Proto-Oncogene Proteins c-kit / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D014157:Transcription Factors / Q000502:physiology*; D015533:Transcriptional Activation
D017931:DNA Primers; D004268:DNA-Binding Proteins; C497234:MITF protein, human; D051739:Microphthalmia-Associated Transcription Factor; C497235:Mitf protein, mouse; D012333:RNA, Messenger; D014157:Transcription Factors; D019009:Proto-Oncogene Proteins c-kit