KITANO Yoshikazu Kitasato University, School of Medicine, Assistant, 医学部, 助手 (90265603)
IZUMI Tohru Kitasato University, School of Medicine, Professor, 医学部, 教授 (80143775)
|Budget Amount *help
¥6,400,000 (Direct Cost: ¥6,400,000)
Fiscal Year 2004: ¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 2003: ¥3,300,000 (Direct Cost: ¥3,300,000)
We established the in vitro differentiation system of vascular smooth muscle cells. Using this system, we cloned a novel transcription factor, Mrf2 (Modulator Recognition Factor 2) as an important key molecule for the differentiation of the smooth muscle cells. Our research has focused on the role of Mrf2.
1, The expression of Mrf2
We made a rabbit polyclonal antibody using the half portion of the Mrf2 protein, which was prepared using the E.Coli. With this antibody, we could observe the nuclear localization of Mrf2 protein, which is transfected into 293T cells by the expression vector, but it did not seem to work for the immunohistochemistry. Next, we made additional three antibodies using the specific oligopeptides designed from the Mrf2 amino acid sequence. The one would detect only beta-isoform of Mrf2, and the others should detect both alpha and beta isoforms. Moreover, we prepared the constructs for in situ hybridization. We are now analysing the expression pattern of Mrf2 from the both results.
2, The regulation of the smooth muscle cell specific promoters by Mrf2
To test if Mrf2 can regulate smooth muscle cell specific promoters, we prepared luciferase constructs with the CArG box, or SM22 α promoter et al. in their promoter regions, and performed the promoter assays. But, the activities of these promoters were not affected by Mrf2 expression. The co-expression of Mrf2 and SRF could not regulate the promoter activity. We made several deletion mutants of Mrf2 and are now testing if they could change the promoter activities, as dominant negative or positive mutants.
3, The construction of the virus vectors of Mrf2
We have constructed the retro-virus and adeno-virus constructs of Mrf2.
Recently, the role of Mrf2 in the differentiation of adepocytes was reported. We are very interested in the role of Mrf2 during the differentiation of both smooth muscle cells and adepocytes.