Osteonectin is one of the most abundant non-collagen matrix components in bone, and osteonectin-null mice develop progressive, low turnover osteopenia. Osteonectin-null mice have decreased osteoblast numbers and bone formation rate, hence, this glycoprotein is critical for bone remodeling and the maintenance of bone mass. In vitro analysis of cells from osteonectin-null mice shows that osteonectin supports osteoblast formation, maturation, and survival. In addition, osteonectin-null osteoblastic cells are more likely to transdifferentiate into adipocytes. In vitro studies indicate that the Notch signaling pathway, known to direct cell fate, is altered in osteonectin-null osteoblastic cells. Notch-1 transcripts are increased in osteonectin-null cells, and constitutive Notch signaling inhibits osteoblastic differentiation. In addition, osteonectin supports cell survival under conditions of stress. The ratio between pro- and anti-apoptotic signals plays a role in determining whether a cell will undergo apoptosis or be able to resist this fate, and preliminary studies suggest that control and osteonectin-null osteoblasts have different ratios of pro- and anti-apoptotic proteins. The first Specific Aim of this proposal is to determine the mechanisms by which osteonectin supports cell survival. This will be accomplished by defining the conditions under which osteonectin can support cell survival, and by determining which cell death pathways are modified in osteonectin-null cells, using a microarray approach. The second Specific Aim is to determine the mechanisms by which osteonectin supports osteoblast formation and maturation. It is my hypothesis that dysregulation of the Notch signaling pathway mediates, at least in part, the effect of the osteonectin-null mutation on osteoblast differentiation and maturation. This hypothesis will be tested by determining whether down-regulation of Notch-1 in osteonectin-null cells can rescue the defect in osteoblastic differentiation. In addition, the signaling pathways impacting cell fate in control and osteonectin-null osteoblasts will be investigated using a microarray approach, and because osteonectin regulates Notch-1 mRNA expression in osteoblasts, the mechanisms by which this occurs will be determined. These studies will provide data on the mechanisms by which osteonectin affects cell survival and cell fate, and may also provide clues as to how cells detect osteonectin. ? ?
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