The Dynamic Range of Site-1 Protease Functions in Skeletal Development

骨骼发育中 Site-1 蛋白酶功能的动态范围

• 批准号: 9032449
• 负责人: Debabrata Patra
• 金额: $ 33.55万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-01 至 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9032449
• 关键词: Ablation; Adolescent; Antibodies; Binding Proteins; Biological; Biological Process; Bone Density; Bone Development; Bone Growth; C-terminal; Calcium; Cartilage; Cartilage Matrix; Cells; Chondrocytes; Collagen Type II; Complex; Connective Tissue; Consensus Sequence; Coupled; Defect; Deformity; Degenerative polyarthritis; Development; Down-Regulation; Dwarfism; Embryo; Endoplasmic Reticulum; Enzymes; Exhibits; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Foundations; Fura-2; Future; Genes; Growth and Development function; Health; Homeostasis; Image; Intervertebral disc structure; Investigation; Knock-out; Knockout Mice; Knowledge; Kyphosis deformity of spine; Laboratories; Lead; Light; Lipids; Maintenance; Mass Spectrum Analysis; Membrane; Membrane Lipids; Microarray Analysis; Modeling; Molecular; Mus; Osteoblasts; Osteocalcin; Osteogenesis; Pathology; Pathway interactions; Peptide Hydrolases; Phenotype; Phosphorylation; Play; Process; Procollagen; Property; Proprotein Convertases; Proteins; Proteolysis; Regulation; Regulatory Element; Role; Shapes; Signal Pathway; Skeletal Development; Skeleton; Spectrometry, Mass, Electrospray Ionization; Staging; Sterols; System; Transforming Growth Factor beta; Type II Procollagen; Vertebral column; Vertebrates; biological adaptation to stress; bone; cartilage development; chondrodysplasia; clinical phenotype; design; endoplasmic reticulum stress; falls; gel electrophoresis; insight; lipid metabolism; long bone; mouse model; novel; novel strategies; novel therapeutics; parathyroid hormone (1-34); postnatal; programs; response; scoliosis; secretion process; secretory protein; site-1 protease; skeletal; skeletal abnormality; spine bone structure; trafficking; transcription factor; two-dimensional

 DESCRIPTION (provided by applicant): The proprotein convertase Site-1 protease (S1P) has emerged as a critical regulator of mammalian skeletal development. It is an indispensable component of the regulated intramembrane proteolysis systems that govern lipid metabolism and endoplasmic reticulum stress response. Our studies implicate S1P as a mandatory component of the protein secretory apparatus that defines all connective tissues. We have studied the functions of S1P in a progressive set of S1P knock-out mouse models using Col2-Cre, Col2-CreER(T) and Osx-Cre mice. In addition to a lipid phenotype, studies on S1P ablation in chondroprogenitor cells (S1Pcko model) have demonstrated chondrodysplasia characterized by the specific endoplasmic reticulum (ER) entrapment of type IIB procollagen, poor cartilage matrix development, and loss of endochondral bone formation in mice. Our recent studies demonstrated that along with type IIB procollagen, chondrocytes are also unable to secrete Adamts3, the type II procollagen N-proteinase. We have discovered a well-conserved S1P consensus sequence in the C-terminus of Adamts3, which suggests mandatory C-terminal processing of Adamts3 by S1P. Post-natal S1P ablation in chondrocytes (using Col2-CreER(T)) demonstrated complete disruption of hypertrophic chondrocyte differentiation. When we followed this observation by ablating S1P in prehypertrophic chondrocytes and osteoblasts using Osx-Cre mice, adolescent mice exhibited kyphosis and scoliosis coupled to chondrodysplasia and fragile long bones. These observations confirm the requirement of S1P to overall skeletal development and suggest potential cell-specific functions for S1P. Our mechanistic characterization of the S1Pcko model has demonstrated that lipid metabolism is down-regulated in S1Pcko chondrocytes. Disrupted ER membrane functions induced by changes in lipid composition may trap the type IIB procollagen in the ER. In light of these findings, in Specific Aim 1 we will investigate deviations in S1Pcko ER membrane lipid composition by mass spectrometry and analyze disrupted ER membrane functions by calcium imaging. In Specific Aim 2 we will investigate Adamts3 as a novel S1P substrate and analyze whether type IIB procollagen trafficking is regulated by a physical association with Adamts3. We will also study cartilage-specific ablation of Adamts3 in mice to analyze its role in cartilage development and maintenance. In Specific Aim 3, taking the lead from our previous knock-out models, we will characterize how disruptions in chondrocyte and osteoblast functions due to S1P ablation could lead to spine abnormalities in order to identify the biological trigger required for kyphosis and scoliosis development. This approach will allow investigation of multiple novel functions of S1P; additionally it will also provide novel insights into the regulation of protein secretory pathways and how their disruptions lead to the clinical phenotypes of chondrodysplasia and spine deformities.

 描述（由应用程序提供）：促蛋白转化酶Site-1蛋白酶（S1P）已成为哺乳动物骨骼发育的关键调节剂。它是控制脂质代谢和内质网应激反应的受调节膜内蛋白水解系统的必不可少的组成部分。我们的研究暗示S1P是定义所有连接组织的蛋白质秘密设备的强制性组成部分。我们使用COL2-CRE，COL2-CREER（T）和OSX-CRE小鼠研究了S1P的功能。除了脂质表型外，对软骨元素细胞中S1P消融的研究（S1PCKO模型）表明，软骨增生到具有特定内质网（ER）特异性的软骨膜膜片（ER）的特征。我们最近的研究表明，与IIB型Procollagen一起，软骨细胞也无法秘密秘密ADAMTS3，即II型胶原蛋白酶N-蛋白酶。我们已经在ADAMTS3的C端发现了保存良好的S1P共识序列，这表明S1P对ADAMTS3进行了强制性的C末端处理。在软骨细胞中（使用COL2-CREER（T））中产后S1P消融表现出肥大软骨细胞分化的完全破坏。当我们通过使用OSX-CRE小鼠的植物性软骨细胞和成骨细胞进行消融S1P进行观察时，青少年小鼠暴露于脊柱侧弯和脊柱侧弯与软骨浮肿和脆弱的长骨相连。这些观察结果证实了S1P对总体骨骼发育的要求，并提出了S1P的潜在细胞特异性功能。我们对S1PCKO模型的机械表征表明，脂质代谢在S1PCKO软骨细胞中被下调。脂质组合物变化引起的破坏ER膜功能可能会捕获ER中的IIB型Procollagen。鉴于这些发现，在特定的目标1中，我们将通过质谱法研究S1PCKO ER膜脂质组成中的出发，并通过钙成像分析了破坏的ER膜功能。在特定的目标2中，我们将研究ADAMTS3作为新型S1P底物，并分析IIB型Procollagen贩运是否受到与ADAMTS3的物理关联调节。我们还将研究小鼠ADAMTS3的软骨特异性消融，以分析其在软骨发育和维持中的作用。在特定的目标3中，从我们以前的淘汰模型中脱颖而出，我们将表征由于S1P消融而引起的软骨细胞和成骨细胞功能的破坏可能导致脊柱异常，以识别所需的生物学触发因素 用于脑脊柱和脊柱侧弯的发展。这种方法将允许研究S1P的多个新功能。此外，它还将提供有关蛋白质分泌途径调节的新见解，以及它们的破坏如何导致软骨发育不良和脊柱畸形的临床表型。