| Bone morphogenetic protein (BMP) was first discovered as a osteoinductive molecule, which proved substantial morphogenetic evidence for the theory of induction. Nearly 20 cDNAs of human BMP have been cloned since Wozney etal reported the successful cloning of BMP-1 through BMP-4 in 1988. On the basis of characteristics that include amino acid similarity, the BMPs grouped as a family within the TGF-B superfamily. BMPs not only play a key role in skeletogenesis and bone repair but also involve in dorsal/ventral pattern and extraskeletal organogenesis such as hematopoietic and nervous system during embryonic development. Natural BMPs can be extracted from animal or human bones, but the extraction is labor- and time-consuming process and the amounts of BMPs obtained from bones are often less productive. Fortunately, different recombinant human BMPs (rhBMP) have been expressed in mammalian cells, insects cells or E.coli and all of them can induce cartilage and bone formation in in vivo assay, so the recombinant DNA technology will allow a supply of almost unlimited amounts of rhBMP for furtherDepartment of Biochemistry and Molecular Biology. FMMU- 7 -study and clinical applications. Especially the use of bacterial expression system for the production of rhBMPs might facilitate the introduction of them for clinical applications, as this system offers an extremely high protein output at relatively low cost. In the near future, rhBMPs can potentially replace conventional autogenous bone grafts in the repair of nonunions, large bone fractures, and bone defects.But there is not an ideal quantitative bioassay method for BMP so far, for BMPs are cell differentiation factors. Their activities are always judged in vivo in mouse or other animals by a standard if they can induce ectopic bone formation. Although this in vivo rodent assay system is reliable and classical, it needs a long time and is affected by many factors. So it is necessary to develop a new in vitro assay method for BMP. A lot of efforts have been applied, including detection of ALP, osteocalcin (OC) and glycogan etc, but the results are not satisfied, for they are not specific or sensitive for BMP's effects. We also analyzed the changes of ALP, OC and synthesis of protein in bone morrow stromal cells (MSCs), NIH3T3 fibroblast and C2C12 myoblast cells stimulated by rhBMP-2. The results showed that concentrations of OC and proteins were gently added in all 3 cells, while the level of ALP was only gone up in the MSCs in dose-dependant effect. Therefore, the direct detection of ALP or OC is not suitable for the quantitative assay of rhBMPs' biological abilities.Meanwhile the mechanism of osteoblast differentiation is well progressed and the Cbfal (Core binding factor al) is cloned as an essential transcription factor required for osteoblast differentiation. The gene knockout or mutation of Cbfal will lead to a total lack of bone or abnormal skeleton. Cbfal stim Dlates osteoblast-specific gene expression, such as ALP, osteocalcin (OC), type I collagen, osteopontin, and bone sialoprotein. The consensus DNA binding sequence for Cbfal is found in the promoters of these genes as PuACCPuCA, which is regarded as osteoblast-specific element (OSE). More importantly, BMPs' up-regulation of the expression of ALP and OC is associated with the expression of Cbfal, which is prior to the expression of osteoblastic phenotype. It is suggested that Cbfal is one of BMPs targeting key transcriptors which then activate the expression of osteoblast-Department of Biochemistry and Molecular Biology, FMMU- 8 -specific genes. So we can take advantage of the findings that effects of BMPs on osteoblast differentiation are partly mediated by Cbfal, and augment the effects of BMPs by increasing numbers of OSE (Cbfal activating osteoblast-specific element) in order to quantitatively assay the biological abilities of rhBMP. Then we focus on the OC promoter and Luciferase reporter gene as OC is one of osteoblastic phenotype and Luciferase reporter gen...
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