Development of high-throughput screening system for osteogenic drugs using a cell-based sensor.
To effectively treat osteoporosis and other bone-loss disorders, small compounds that potently induce bone formation are needed. The present study initially attempted to establish a monitoring system that could detect osteogenic differentiation easily, precisely, and noninvasively. For this purpose, we established pre-osteoblastic MC3T3E1 cells stably transfected with the GFP reporter gene driven by a 2.3 kb fragment of rat type I collagen promoter (Col1a1GFP-MC3T3E1). Among these cells, we selected a clone that fluoresced upon osteogenic stimulation by BMP2. The GFP fluorescence intensity corresponded well to the intensity of alkaline phosphatase (ALP) staining and to the level of osteocalcin (Oc) mRNA. Using this system, we screened natural and synthetic compound libraries and thus identified an isoflavone derivative, glabrisoflavone (GI). GI induced ALP staining and Oc mRNA in a dose-dependent manner. The Col1a1GFP-MC3T3E1 system may be useful for identifying novel osteogenic drugs.
Hojo
Hironori
H
Japan Society for the Promotion of Science, 8 Ichibancho, Chiyoda-ku, Tokyo 102-8472, Japan.
Igawa
Kazuyo
K
Ohba
Shinsuke
S
Yano
Fumiko
F
Nakajima
Keiji
K
Komiyama
Yuske
Y
Ikeda
Toshiyuki
T
Lichtler
Alexander C
AC
Woo
Je-Tae
JT
Yonezawa
Takayuki
T
Takato
Tsuyoshi
T
Chung
Ung-il
UI
D000469:Alkaline Phosphatase / Q000032:analysis / Q000096:biosynthesis; D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000494:pharmacology; D002460:Cell Line; D024042:Collagen Type I / Q000235:genetics; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005453:Fluorescence; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D007529:Isoflavones / Q000494:pharmacology; D051379:Mice; D010006:Osteoblasts / Q000187:drug effects*; D015675:Osteocalcin / Q000032:analysis / Q000096:biosynthesis; D010012:Osteogenesis / Q000187:drug effects*; D010024:Osteoporosis / Q000188:drug therapy*; D051381:Rats; D016212:Transforming Growth Factor beta / Q000494:pharmacology
C441109:5,7,4'-trihydroxy-6-(3-hydroxymethyl-2-butenyl)isoflavone; C525719:Bmp2 protein, mouse; C525720:Bmp2 protein, rat; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D024042:Collagen Type I; D007529:Isoflavones; D016212:Transforming Growth Factor beta; D015675:Osteocalcin; D049452:Green Fluorescent Proteins; D000469:Alkaline Phosphatase