METALLOPROTEASES IN SKELETAL DEVELOPMENT AND GROWTH

骨骼发育和生长中的金属蛋白酶

• 批准号: 6512224
• 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/6512224
• 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，尤其是骨骼发育至关重要。 基质金属蛋白酶（MMP）在这些过程中起重要作用。 MMP-14（或MT1-MMP，I型I-MMP膜），一种整体膜MMP，在骨骼发育过程中显着表达。 在体外，MMP-14裂解ECM分子，例如胶原蛋白I和Aggrecan。 它是进场酶A和胶原酶3的细胞表面激活剂，这是最活跃的胶原蛋白蛋白酶。 这些观察结果预测了MMP-14在软骨ECM蛋白水解和骨骼发育中的重要作用。 我们已经通过胚胎干细胞中的同源重组来确认缺乏MMP-14的转基因无效小鼠。 MMP-14缺陷小鼠的异常包括软骨吸收，软骨细胞增殖降低，次级骨化中心的延迟形成和缺陷的血管生成。 这些异常的后果是严重的矮人和早亡。 导致这种表型的精确分子途径尚不清楚，并将在此提案中进行研究。我们将解决转基因对软骨细胞性能和骨骼生长参数的影响，以及与血管生成有关的阶段特异性软骨细胞标记和分子的基因表达。 描述了实验以确定MMP-14是否直接分辨软骨细胞外基质，还是通过激活其他金属蛋白酶作用。由于次级骨化中心的形成延迟，我们将在这些小鼠中使用体外和体内测定研究血管生成。 鉴于这些小鼠中干扰生长激素 - 胰岛素样生长因子（IGF）轴的初步研究，我们将研究MMP-14在通过IGF结合蛋白的蛋白水解中介导游离IGF的生物利用度中的作用。