Independent influence of strain difference and mi transcription factor on the expression of mouse mast cell chymases

Abstract

Expression of mouse mast cell protease (mMCP) genes was examined with particular attention to the transactivation effect of mi transcription factor (MITF) and the expression differences between C57BL/6 (B6) and WB strains. We had reported the enhancing effect of MITF on the expression of mMCP-4, -5, and -6 genes in cultured mast cells (CMCs) of B6 strain, and in the present study we demonstrated the enhancing effect on the expression of mMCP-2 and -9 genes as well. The enhancing effect of MITF on the expression of mMCP-2, -4, -5, -6, and -9 genes was also detected in CMCs of the WB strain. The regulation of mMCP-2, -4, and -9 genes was localized to a specific promoter element (CANNTG) which was recognized and bound by MITF and which was conserved between the B6 and WB strains. On the other hand, the expression of mMCP-2, -4, and -9 genes was smaller in CMCs of the B6 strain when compared to their expression in CMCs of the WB strain. Although mMCP-5 is a chymase as mMCP-2, -4, and -9, and genes encoding all of the chymases are located on chromosome 14, the mMCP-5 gene was regulated in a manner distinct from mMCP-2, -4, and -9 genes.

Figure 1.

Expression of mMCP-2, -4, -5, -6, and -9 mRNA transcripts in CMCs of B6-+/+ and B6-mi/mi mice. Total RNAs were extracted from CMCs of B6-+/+ and B6-mi/mi mice, and 20 μg of total RNA was electrophoresed and hybridized with each probe. Three similar experiments were done, and a representative result is shown. Comparable results were obtained in two other experiments.

Figure 2.

Reduced expression of mMCP-2 and -9 mRNAs in B6-mi/mi CMCs and normalization of the mMCP-2 and -9 expression in B6-mi/mi CMCs by the introduction of the +-MITF cDNA but not of mi-MITF cDNA. The blot was hybridized with ³²P-labeled cDNA probe of MITF, mMCP-2, -9, or of β-actin. Three similar experiments were done, and a representative result is shown. Comparable results were obtained in two other experiments.

Figure 3.

Luciferase reporter gene promoter assay in the IC-2 cell line which expressed +-MITF. The luciferase gene under control of the normal, deleted, or mutated mMCP-2 promoter was introduced into the IC-2 cells with electroporation. Bars indicate the SE of three assays.

Figure 4.

EGMSA using GST-+-MITF fusion protein. The labeled 5′-GCTCTCCCCCACATGCTCATA-3′ oligonucleotide (oligo 1, nucleotide −386 to −366) in the mMCP-2 promoter was used as a probe (CANNTG motif is boxed). The sequences of the mutated oligonucleotide is shown as oligo 2. The mutated nucleotides are underlined. The excess amount (200×) of unlabeled oligo 1 or oligo 2 was added as a competitor. The DNA-protein complexes are indicated by an arrow. Three similar experiments were done, and a representative result is shown. Comparable results were obtained in two other experiments.

Figure 5.

Luciferase reporter gene promoter assay in the IC-2 cell line that expressed +-MITF. The luciferase gene under control of the normal, deleted, or mutated mMCP-9 promoter was introduced into the IC-2 cells with electroporation. Bars indicate the SE of three assays.

Figure 6.

EGMSA using GST + MITF fusion protein. The labeled 5′-CCTCTCACCCATATGCTCATA-3′ oligonucleotide (oligo 1, nucleotide −390 to −369) in the mMCP-9 promoter was used as a probe (CANNTG motif is boxed). The sequences of the mutated oligonucleotide is shown as oligo 2. The mutated nucleotides are underlined. The excess amount (200×) of unlabeled oligo 1 or oligo 2 was added as a competitor. The DNA-protein complexes are indicated by an arrow. Three similar experiments were done, and a representative result is shown. Comparable results were obtained in two other experiments.

Figure 7.

Expression of mMCP-2, -4, -5, -6, and -9 mRNA transcripts in CMCs of WB-+/+ and B6-+/+ mouse origin. Total RNA was extracted from CMCs of WB-+/+ and B6-+/+ mice, and 1, 10, and 50 μg of total RNA was electrophoresed and hybridized with each probe. Three similar experiments were done, and a representative result is shown. Comparable results were obtained in two other experiments.

Figure 8.

Comparison of the nucleotide sequence of mMCP-2 cDNA among BALB/c, B6, and WB strains. Numbering of the nucleotides begins at the transcription initiation site of the mMCP-2 cDNA of BALB/c strain. Each sequence was confirmed three times. Dashes indicate identical nucleotides. *, Translation-initiation site. **, Translation-termination site. The repetitive UGXCCCC sequence is underlined.

Figure 9.

Comparison of the nucleotide sequences of mMCP-4 cDNA obtained from the KiSV-MC1 mastocytoma cell line of DBA/2 strain origin, CMCs of B6 strain origin, and CMCs of WB strain origin. Numbering of the nucleotides begins at the transcription initiation site of the mMCP-4 cDNA of DBA/2 strain. Each sequence was confirmed three times. Dashes indicate identical nucleotides. Blank indicates the deletion in the sequence. *, Translation-initiation site. **, Translation-termination site. The repetitive UGXCCCC sequence is underlined.

Figure 10.

Expression of mMCP-2, -4, -5, -6, and -9 mRNA transcripts in CMCs of a B6-(P2^B6/P2^B6, P4^B6/P4^B6, P9^B6/P9^B6, +/+) mouse, a N2 × N2-(P2^B6/P2^B6, P4^B6/P4^B6, P9^B6/P9^B6, +/+) mouse, a B6- (P2^B6/P2^B6, P4^B6/P4^B6, P9^B6/P9^B6, mi/mi) mouse, a N2 × N2-(P2^B6/P2^B6, P4^B6/P4^B6, P9^B6/P9^B6, mi/mi) mouse, a WB-(P2^WB/P2^WB, P4^WB/P4^WB, P9^WB/P9^WB, +/+) mouse, two N2 × N2-(P2^WB/P2^WB, P4^WB/P4^WB, P9^WB/P9^WB, +/+) mice, and two N2 × N2-(P2^WB/P2^WB, P4^WB/P4^WB, P9^WB/P9^WB, mi/mi) mice. Total RNA was extracted from CMCs of each genotype, and 20 μg of total RNA was electrophoresed and hybridized with mMCP-2, -4, -5, -6, and -9 probes. Three similar experiments were done, and a representative result is shown. Comparable results were obtained in two other experiments.

Figure 11.

Semiquantitative RT-PCR analysis of expression of MITF mRNA in CMCs of WB-+/+ and B6-+/+ mouse origin. PCR products from RNAs of WB-+/+ CMCs (lanes 1 to 3) and from B6-+/+ CMCs (lanes 4 to 6) were electrophoresed in 1.0% agarose gel containing ethidium bromide. The amounts of RNA used for reverse transcription were 5 μg (lanes 1 and 4), 0.5 μg (lanes 2 and 5), and 0.05 μg (lanes 3 and 6), respectively. Three similar experiments were done, and a representative result is shown. Comparable results were obtained in two other experiments.