Function and Regulation of Osteonectin in Bone

描述（由申请人提供）：骨连接素是骨中最丰富的非胶原基质成分之一，骨连接素缺失的小鼠出现进行性、低周转率的骨质减少。骨连接素缺失小鼠成骨细胞数量和骨形成率下降，因此，这种糖蛋白对骨重塑和骨量的维持至关重要。体外对骨连接素缺失小鼠细胞的分析表明，骨连接素支持成骨细胞的形成、成熟和存活。此外，骨连接素缺失的成骨细胞更容易转分化为脂肪细胞。体外研究表明，Notch信号通路在骨连接素缺失的成骨细胞中发生了改变。Notch-1转录物在骨连接蛋白缺失的细胞中增加，构成型Notch信号抑制成骨细胞分化。此外，骨连接素支持细胞在应激条件下存活。促凋亡和抗凋亡信号之间的比例在决定细胞是否会发生凋亡或能够抵抗这种命运方面起着重要作用，初步研究表明，对照和骨连接素缺失的成骨细胞具有不同比例的促凋亡和抗凋亡蛋白。本建议的第一个具体目的是确定骨连接素支持细胞存活的机制。这将通过定义骨连接素支持细胞存活的条件，以及通过使用微阵列方法确定骨连接素缺失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接素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素缺失突变对成骨细胞分化和成熟的影响。这一假设将通过确定在骨连接素缺失的细胞中下调Notch-1是否可以修复成骨细胞分化的缺陷来验证。此外，将使用微阵列方法研究影响控制和骨连接素缺失成骨细胞细胞命运的信号通路，并且由于骨连接素调节成骨细胞中Notch-1 mRNA的表达，因此将确定其发生的机制。这些研究将为骨连接素影响细胞存活和细胞命运的机制提供数据，也可能为细胞如何检测骨连接素提供线索。

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.