Function and Regulation of Osteonectin in Bone

描述(申请人提供)：骨连蛋白是骨骼中含量最丰富的非胶原基质成分之一，骨连蛋白缺失的小鼠会出现进行性、低周转性骨量减少。Osteonectin缺失的小鼠的成骨细胞数量和骨形成率降低，因此，这种糖蛋白对骨重建和骨量的维持至关重要。对骨连蛋白缺失小鼠细胞的体外分析表明，骨连蛋白支持成骨细胞的形成、成熟和存活。此外，骨连蛋白缺失的成骨细胞更有可能转分化为脂肪细胞。体外研究表明，在骨连蛋白缺失的成骨细胞中，Notch信号通路(已知的直接细胞命运)发生了变化。Notch-1转录本在骨连接蛋白缺失的细胞中增加，结构性Notch信号抑制成骨细胞的分化。此外，骨连蛋白还支持细胞在应激条件下存活。促凋亡信号和抗凋亡信号之间的比例在决定细胞是否会发生凋亡或是否能够抵抗这一命运方面发挥了作用，初步研究表明，对照和骨连接蛋白缺失的成骨细胞具有不同的促凋亡和抗凋亡蛋白比例。这项建议的第一个具体目标是确定骨连蛋白支持细胞存活的机制。这将通过定义骨连蛋白支持细胞存活的条件，并通过使用微阵列方法确定在骨连蛋白缺失的细胞中哪些细胞死亡途径被修改来实现。第二个具体目标是确定骨连蛋白支持成骨细胞形成和成熟的机制。我的假设是，Notch信号通路的失调至少部分地介导了骨连接蛋白零突变对成骨细胞分化和成熟的影响。这一假说将通过确定在骨连蛋白缺失细胞中下调Notch-1是否可以挽救成骨细胞分化中的缺陷来检验。此外，在对照和骨连接蛋白缺失的成骨细胞中，影响细胞命运的信号通路将用微阵列方法进行研究，由于骨连接蛋白调节成骨细胞中Notch-1mRNA的表达，因此将确定发生这种情况的机制。这些研究将提供有关骨连蛋白影响细胞存活和细胞命运的机制的数据，也可能提供细胞如何检测骨连蛋白的线索。

DESCRIPTION (provided by applicant): 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.