Analysis of osteoblast proliferation, differentiation and apoptosis in Col3.6-p20C/EBP beta transgenic mice

C/EBP transcription factors have been recognized as key regulators of cellular differentiation in a variety of cells, including adipocytes, hepatocytes, granulocytes and macrophages. A growing literature also suggests an important role for C/EBP family members in the growth and differentiation of osteoblasts. To better comprehend the role of C/EBP in osteogenesis, a transgenic mouse model has been generated in which the function of C/EBP in cells of the stromal and osteoblast lineage has been blocked by overexpression of a dominant negative isoform of C/EBP/3, known as p20C/EBP/3. Preliminary studies of these p20C/EBP/3 transgenic mice demonstrated varying degrees of osteopenia. Although only three out of the four lines of transgenic mouse models created exhibited an osteopenic bone phenotype, all four possessed alterations in osteoblastic marker gene expression. The analysis of such markers in femurs and calvaria of transgenic mice indicated two possible sites of action at which C/EBP/3 affected the osteoblast lineage. The first site of action occurred late in osteoblast differentiation, manifested by a decrease in terminal markers of osteoblastic differentiation such as osteocalcin and pOBCol 2.3 GFP. The latter is a transgenic reporter of osteoblast differentiation in which the visible GFP protein is expressed under control of a 2.3 kb Colalpromoter construct. The second site appears to be in early osteoblast differentiation characterized an increase in early differentiation markers such as bone sialoprotein (BSP) and alkaline phosphatase. These preliminary studies have also shown an increase in cell number in transgenic mouse stromal cells cultures. The same phenomenon was observed in primary cell cultures, with an increase in cell number in the TG cultures after day 7, reaching a plateau at day 15. It was unknown if the increase in cell number in TG cultures was due to an increase in cell proliferation or a decrease in cell apoptosis. Therefore, in the present study, primary osteoblast and BMSC cultures from TG mice were be used to measure proliferation and apoptosis in the early stages of culture. Studies were performed on a pOBCol3.6-GFP genetic background to provide a real-time marker of the stromal-osteoprogenitor pool and to allow changes in this cell population to be specifically tracked within a heterogeneous population of cells. Proliferation and apoptosis were assessed by flow cytometry analysis. In order to measure cell proliferation, cells were pulsed with BrdU, because it can be incorporated into DNA in place of thymidine. Cells were pulsed in a range from 2 to 12 hours, and detected using a monoclonal antibody against BrdU. Apoptosis was assessed using annexin V, a marker of early apoptosis, and 7AAD, a marker of late apoptosis, as measured by flow cytometry analysis. By analyzing the results of these experiments we will be determine if the role of eC/EBP transcription factors in early osteoblast differentiation and whether they act to increase proliferation or as a protective factor decreasing the number of cells undergoing programmed cell death.