Related Experiment Videos
Abnormal expression of mouse mast cell protease 5 gene in cultured mast cells derived from mutant mi/mi mice
E Morii1, T Jippo, T Tsujimura
1Department of Pathology, Osaka University Medical School, Suita, Japan.
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.
Insights
The study reveals distinct regulatory mechanisms for mouse mast cell proteases 5 and 6 (MMCP-5 and MMCP-6) in mi/mi mice. While MITF directly regulates MMCP-6, its control over MMCP-5 is indirect, highlighting differential gene expression pathways.
Area of Science:
- Immunology
- Molecular Biology
- Genetics
Background:
- Mast cells synthesize specific proteases, including mouse mast cell protease 6 (MMCP-6), whose expression is reduced in mi/mi mutant mice.
- The mi locus encodes the microphthalmia-associated transcription factor (MITF), a basic-helix-loop-helix-leucine zipper (bHLH-Zip) protein crucial for regulating gene expression.
Purpose of the Study:
- To investigate and compare the distinct regulatory mechanisms of MMCP-5 and MMCP-6 expression in cultured mast cells (CMCs) from mi/mi mice.
- To elucidate the role of MITF in the differential expression of MMCP-5 and MMCP-6.
Main Methods:
- Comparative analysis of MMCP-5 and MMCP-6 expression in mi/mi CMCs and skin mast cells.
- Overexpression of normal (+) MITF and mutant (mi-MITF) to assess transactivation of MMCP-5 and MMCP-6 genes.
- Analysis of MITF binding to promoter regions of MMCP-5 and MMCP-6 genes.
- Evaluation of stem cell factor's effect on MMCP-5 and MMCP-6 expression in mi/mi CMCs.
Main Results:
- MMCP-5 expression was reduced in mi/mi CMCs but not skin mast cells, unlike MMCP-6 which was reduced in both.
- +-MITF normalized MMCP-5 gene expression in mi/mi CMCs, indicating its involvement in MMCP-5 transactivation.
- +-MITF directly bound and transactivated the MMCP-6 gene via CANNTG motifs but did not bind to the MMCP-5 promoter's CAGTTG motif, suggesting indirect regulation of MMCP-5.
- Stem cell factor normalized MMCP-5 expression but not MMCP-6 in mi/mi CMCs.
Conclusions:
- The regulation of MMCP-5 and MMCP-6 expression in mast cells involves distinct mechanisms, despite both being downregulated in mi/mi mice.
- +-MITF directly regulates MMCP-6, while its regulation of MMCP-5 appears to be indirect.
- Stem cell factor plays a role in MMCP-5 regulation, further differentiating its control from MMCP-6.