| Co-Investigator(Kenkyū-buntansha) |
TAKAOKA Kunio OSAKA MUNCIPAL UNIVERSITY, SCHOOL OF MEDICINE, DEPARTMENT OF ORTHOPAEDIC SUEGERY, PROFESSOR & CHAIRMAN, 医学部, 教授 (30112048)
NAKAYAMA Kohzo SHINSHU UNIVERSITY, SCHOOL OF MEDICINE, DEPARTMENT OF THE 1ST ANATOMY, ASSOCIATE PROFESSOR, 医学部, 講師 (70192680)
NIKAIDO Toshio SHINSHU UNIVERSITY, GRADUATE SCHOOL OF MEDICINE, DEPARTMENT OF ORGAN REGENERATION, INSTITUTE OF ORGAN TRANSPLANTS, ASSOCIATE PROFESSOR, 大学院・医学研究科, 助教授 (50180568)
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| Budget Amount *help |
¥5,400,000 (Direct Cost: ¥5,400,000)
Fiscal Year 2002: ¥2,600,000 (Direct Cost: ¥2,600,000)
Fiscal Year 2001: ¥2,800,000 (Direct Cost: ¥2,800,000)
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| Research Abstract |
We analyzed the promoter activity of murine bone morpbogenetic protein-4 (mBMP-4) and determined that the 5'-flanking region of exon I is involved mainly in transcriptioiial regulation and position -265 to -246 of 5 '-flanking region of mBMP-4 gene acts as a cis-element important for the regulation of BMP4 transcription in MC3T3E1 cells. By use of site-directed mutagenesis, we have established that the E-box, CACGTG, located within this short region of promoter, is essential for transcriptional activation of the mBMP-4 gene. Upstream stimulatory factor (USF), a member of helix-loop-helix (HLH) group of regulatory proteins, was found to bind to this E-box using supershift gel mobility analysis. We also analyzed the promoter activity of mouse BMP-4 gene in three different types of cell lines (MC3T3E1, ST2 and C6 glioma cells) and demonstrated that position -786 to -691 of the 5'-flanking region of exon I plays a critical role for regulation of tissue specific expression of mouse BMP-4 ge
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ne in cells of an osteogenic lineage. By use of site-directed mutagenesis, we have established that the E-box, CATCTG, located within this 5'-flanking region, is essential for tissue specific transcriptional activation of mouse BMP-4 gene and demonstrated that an osteogenic lineage-specific novel transcriptional factor(s) recognizes this E-box. It is proposed that the HLH transcription regulatory proteins play an important role in mBMP-4 gene transcription in this osteoblastic cell line, like that of Myo-D in myogenic differentiation. Thorough these transcriptional study, we seek and assess the effect of some chemical substrates in BMP expression and in also osteoblastic differentiation in osteoblast-like and other mesenchymal cells. Retinoic acid also enhanced the mRNA level of BMP-4 gene (under submission). FK506, immnunosuppressant often used clinically, induced a dose- and time-dependent increase in alkaline phosphatase (ALP) activity, one of the markers of osteoblast differentiation, in cells derived from mesenchymal. In the presence of BMP-4, ALP activity, mRNA levels of ALP and osteocalcin increased. FK506 was found to not only stimulate osteoblastic differentiation, but also to enhance BMP-4 induced osteoblastic differentiation. Thsese results suggest that FK506 promotes differentiation of osteoblastic cells. Less
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