Relationship Of Growth Factor Expression Of The Osteoblast-specific Transcription Factor Cbfal Gene And Cbfal Gene Polymorphism And Osteoporosis

ObjectiveOsteoblasts originate from common progenitors, which are capable of differentiating into other mesenchymal cell lineages such as chondrocytes, myoblasts, and bone marrow stromal cells including adipocytes. During the differentiation process from mesenchymal progenitors, various hormones and cytokines regulate osteoblast differentiation. Recently, an osteoblast specific transcription factor Cbfal was coloned. It is essential but not sufficient for osteoblast differentiation and bone formation. Cbfal- deficient mice completely lacked bone formation due to maturational arrest of osteoblasts. The phenotype of the heterozygous Cbfal mutation is similar to that of CCD. Patients suffering from this disease exhibit Cbfal mutations. Cleidocranial dysplasia (CCD) is an autosomal-dominant disease.Cbfal is an essential transcription factor for osteoblast differentiation and bone formation, however, the sigaling pathways regulating Cbfal has not been clarified. Xiao et al showed that Cbfal can be phosphorylated and activaled by the mitogen-activated protein kinase (MAPK) pathway. This pathway can be stimulated by a variety of signals including those initiated by extracellular matrix (ECM), mechanical loading and osteogenic growth factors. So, the present study was undertaken to further explore the involvement of the MAPK pathway in Cbfal gene expression affected by growth factors IGF-I, GM-CSF, EGF, VEGF and FGF-9. Methods1. Construction of reported Cbfal promoter (p696-Luc): To generate a targeting vector, genomic fragment containing Cbfal promoter (-641bp—+55bp) region were isolated from the genome of C57BJ mouse white blood cells by PCR. The A-tailing PCR fragment was ligated into pGEM-T Easy Vector, named p696-Teasy. Then we got the Cbfal promoter (-641bp— +55bp) region from the p696-Teasy using a 5'primer built in the MluI site and a 3'primer with a XhoI site and digested the product with MluI and XhoI. The gel-purified fragment was ligated into MluI/XhoI sites of pGL3-Basic vector. The resulting product contains nucleotides -641— +55 (696bp) of the reported Cbfal promoter, named p696-Luc.2. Determination of luciferase activity of p696-Luc: MC3T3-E1 and C2C12 cells were...