C/EBP transcription factors are known to stimulate adipocyte differentiation, and mounting evidence suggests that osteoblasts and adipocytes share a common pluripotent progenitor in the bone marrow. We prepared transgenic mice with Col1a1 promoter-targeted expression of FLAG-tagged p20C/EBPbeta in an effort to enhance osteoblast differentiation at the expense of adipogenesis. Surprisingly, however, we found that all four lines of transgenic pOBCol3.6-FLp20C/EBPbeta mice exhibit osteopenia. Preliminary histological evidence suggests that low bone mass may be due to impaired osteoblast differentiation or function. We hypothesize that mice with targeted expression of p20C/EBPbeta have osteopenia resulting from decreased bone formation, and that p20C/EBPbeta inhibits osteoblast differentiation in vivo.
In Specific Aim 1, we will characterize the bone phenotype of transgenic mice using microcomputed tomography, static and dynamic histomorphometry, and measurement of transgene expression by Northern blotting and immunohistochemistry. C/EBP gene knock-out mice will be screened for the presence of a similar phenotype.
In Specific Aim 2, we will determine the effects of p20C/EBPbeta expression on osteoblast differentiation and function using ex vivo bone marrow stromal and primary calvarial cell culture models. Cell proliferation will be determined in primary calvarial cultures by cell number and by (3H)thymidine incorporation. Osteoblast differentiation will be assessed in both models by the formation of mineralized bone nodules and the expression of osteoblast markers such as Col1a1, bone sialoprotein and osteocalcin. As a first step toward understanding the mechanism of p20C/EBPbeta action, its dimerization partners in primary osteoblastic cells will be identified by immunoprecipitation and electrophoretic mobility shift analysis. The proposed studies should help to elucidate role of transcription factors, specifically members of the C/EBP family, in the control of osteoblast differentiation and bone mass.
