Involvement of Ets transcription factors and targets in osteoblast differentiation and matrix mineralization.

The osteoblast-like MC3T3-E1 cell line provides an excellent in vitro model of bone development. This system undergoes three orderly time-dependent phases characterized by proliferating preosteoblasts, matrix accumulation by postmitotic differentiating osteoblasts, and mineralization of the matrix, which results in the formation of multilayered bone nodules. The Ets family transcription factors regulate genetic programs that affect the proliferation and differentiation of osteoblasts. Of the eight Ets family transcription factors examined by our laboratory, only Etsl and Ets2 were found to be expressed at significant levels in this osteogenic system. Etsl is expressed in proliferating preosteoblastic cells, whereas Ets2, silent during this phase, is expressed by differentiating and mature osteoblasts. In addition, the expression of Etsl can be induced in MC3T3-E1 and fetal rat calvaria cells by retinoic acid (RA) which is known to exert profound effects on skeletal growth and development and bone turnover and induce specific cellular responses in bone cells. Thus, the multiple functions of RA in bone cells are likely to be mediated in part by Etsl. We show that the expression of Ets2 precedes and then parallels osteopontin expression and that the OPN promoter contains Ets binding sites and is a transcriptional target of Ets2. In order to identify other potential Ets target genes, we analyzed promoter regions of genes revealed by serial analysis of gene expression as present in the differentiation stage. The functional analysis of these genes has the potential to provide much needed information as to their function in osteogenesis and mineralization of the extracellular matrix and in bone-related diseases.