METALLOPROTEASES IN SKELETAL DEVELOPMENT AND GROWTH

骨骼发育和生长中的金属蛋白酶

• 批准号: 6167519
• 负责人: SUNEEL S APTE
• 金额: $ 32.3万
• 依托单位: CLEVELAND CLINIC FOUNDATION
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-07-15 至 2004-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6167519
• 关键词: SDS polyacrylamide gel electrophoresis; angiogenesis; binding proteins; bone development; cell proliferation; chondrocytes; densitometry; extracellular matrix; gene expression; genetically modified animals; high performance liquid chromatography; immunocytochemistry; in situ hybridization; insulinlike growth factor; laboratory mouse; metalloendopeptidases; normal ossification; phenotype; protein deficiency; protein structure function; proteolysis; statistics /biometry

Cartilage extracellular matrix (ECM) degradation and angiogenesis are critical for skeletal development in general and for endochondral ossification in particular. Matrix metalloproteinases (MMPs) play an important role in these processes. MMP-14 (or MT1-MMP, membrane type I-MMP), an integral membrane MMP, is prominently expressed during skeletal development. In vitro, MMP-14 cleaves ECM molecules such as collagen I and aggrecan. It is the cell surface activator of progelatinase A and collagenase-3, the most active collagen II protease. These observations predicted a significant role for MMP-14 in cartilage ECM proteolysis and skeletal development. We have confirmed this by generation of transgenic null mice lacking MMP-14, via homologous recombination in embryonic stem cells. The abnormalities in MMP-14 deficient mice include defective cartilage resorption, decreased chondrocyte proliferation, delayed formation of secondary ossification centers, and defective angiogenesis. The consequences of these abnormalities are severe dwarfism and early death. The precise molecular pathways leading up to this phenotype are unknown and will be investigated in this proposal. We will address the effects of the transgene on parameters of chondrocyte performance and bone growth, and on expression of genes for stage specific chondrocyte markers and molecules related to angiogenesis. Experiments are described to determine whether MMP-14 directly resorbs cartilage extracellular matrix or whether it acts through activation of other metalloproteases. Since secondary ossification center formation is delayed, we will study angiogenesis using in vitro and in vivo assays in these mice. In view of preliminary studies which suggest interference with the growth hormone-insulin-like growth factor (IGF) axis in these mice, we will investigate a role for MMP-14 in mediating the bioavailability of free IGF via proteolysis of IGF binding proteins.

软骨细胞外基质 (ECM) 降解和血管生成对于一般骨骼发育，尤其是软骨内骨化至关重要。 基质金属蛋白酶（MMP）在这些过程中发挥着重要作用。 MMP-14（或 MT1-MMP，膜型 I-MMP）是一种完整的膜 MMP，在骨骼发育过程中显着表达。 在体外，MMP-14 裂解 ECM 分子，例如 I 型胶原蛋白和聚集蛋白聚糖。 它是原明胶酶 A 和胶原酶 3（最活跃的 II 型胶原蛋白酶）的细胞表面活化剂。 这些观察结果预测 MMP-14 在软骨 ECM 蛋白水解和骨骼发育中发挥重要作用。 我们通过胚胎干细胞中的同源重组产生缺乏 MMP-14 的转基因无效小鼠，证实了这一点。 MMP-14缺陷小鼠的异常包括软骨吸收缺陷、软骨细胞增殖减少、次级骨化中心形成延迟和血管生成缺陷。 这些异常的后果是严重的侏儒症和早逝。 导致这种表型的精确分子途径尚不清楚，将在本提案中进行研究。我们将探讨转基因对软骨细胞性能和骨生长参数的影响，以及对特定阶段软骨细胞标记物和与血管生成相关分子的基因表达的影响。 实验旨在确定 MMP-14 是否直接吸收软骨细胞外基质，或者是否通过激活其他金属蛋白酶发挥作用。由于次级骨化中心形成被延迟，我们将使用这些小鼠的体外和体内测定来研究血管生成。 鉴于初步研究表明这些小鼠的生长激素-胰岛素样生长因子 (IGF) 轴受到干扰，我们将研究 MMP-14 在通过 IGF 结合蛋白的蛋白水解介导游离 IGF 的生物利用度中的作用。