| Co-Investigator(Kenkyū-buntansha) |
INAGI Reiko Tokai University , Medical Research Institute, Assistant professor, 総合医学研究所, 講師 (50232509)
MIYATA Toshio Tokai University , Medical Research Institute, Associate professor, 総合医学研究所, 助教授 (10222332)
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| Research Abstract |
Elucidation of the transcriptional regulation of megsin should help us to understand its tissue specificity and provide important insights into the mechanisms of cell-type dependent gene expression. We determined the transcriptional start site of megsin by primer extension analysis, and showed that the site lied 391 bp upstream from the start codon. The sequence and reporter analyses on 4021 bp-length 5'-flanking region of megsin gene demonstrated a consensus promoter segment within this region and a relatively strong promoter activity in human mesangial cells.
Furthermore, site-directed and deletion mutagenesis analyses in combination with electrophoretic mobility shift assay identified one positive regulatory motif, an incomplete activator protein-1 (AP-1) binding motif (CTGATTCAC) within -120 to -112 region. PDTC, a dominant activator of AP-1, activated the megsin promoter. In contrast, our studies with deletion mutants of megsin promoter vectors showed that the YB-1 binding site, which was previously shown to be a major, cell type-specific transactivator of matrix metalloprotease-2 (MMP-2) gene expression in glomerular mesangial cells, is not important for megsin gene transcription. These results suggest that this cis-acting element in the 5'-flanking region of megsin is involved in activation of megsin, and that AP- 1 is a good candidate as a transcriptional factor of megsin.
However, the reporter construct with the 5'-flanking region also induced a mild expression of a reporter gene in non-mesangial cells, suggesting that the cell type specific transcriptional regulation in not located solely in this promoter region. We are currently performing extensive investigation of more upstream of megsin promoter region as well as introns to find out the mechanism of mesangium-predominant expression of megsin.
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