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（由申请人提供）：前蛋白转化酶位点-1蛋白酶（S1 P）已成为哺乳动物骨骼发育的关键调节因子。它是调节脂质代谢和内质网应激反应的膜内蛋白水解系统的不可或缺的组成部分。我们的研究表明S1 P是定义所有结缔组织的蛋白质分泌装置的必要组成部分。我们使用Col 2-Cre、Col 2-CreER（T）和Osx-Cre小鼠研究了S1 P在一组进行性S1 P敲除小鼠模型中的功能。除了脂质表型，对软骨祖细胞中S1 P消融的研究（S1 Pcko模型）已经证明了软骨发育不良，其特征在于IIB型前胶原的特异性内质网（ER）截留、软骨基质发育不良和小鼠软骨内骨形成丧失。我们最近的研究表明，沿着IIB型前胶原，软骨细胞也不能分泌Adamts 3，即II型前胶原N-蛋白酶。我们在Adamts 3的C-末端发现了一个高度保守的S1 P共有序列，这表明S1 P对Adamts 3的C-末端进行了强制性加工。软骨细胞中的出生后S1 P消融（使用Col 2-CreER（T））证明了肥大软骨细胞分化的完全破坏。当我们通过使用Osx-Cre小鼠消融前肥大软骨细胞和成骨细胞中的S1 P来进行观察时，青春期小鼠表现出脊柱后凸和脊柱侧凸，并伴有软骨发育不良和脆弱的长骨。这些观察结果证实了S1 P对整体骨骼发育的需求，并表明S1 P具有潜在的细胞特异性功能。我们对S1 Pcko模型的机制表征表明，S1 Pcko软骨细胞中的脂质代谢被下调。脂质组成的变化引起的ER膜功能的破坏可能会将IIB型前胶原困在ER中。鉴于这些发现，在特定目标1中，我们将通过质谱法研究S1 Pcko ER膜脂质组成的偏差，并通过钙成像分析破坏的ER膜功能。在具体目标2中，我们将研究Adamts 3作为一种新的S1 P底物，并分析IIB型前胶原的运输是否受与Adamts 3的物理关联的调节。我们还将研究小鼠中Adamts 3的软骨特异性消融，以分析其在软骨发育和维持中的作用。在特定目标3中，从我们以前的敲除模型中吸取教训，我们将描述由于S1 P消融导致的软骨细胞和成骨细胞功能的破坏如何导致脊柱异常，以确定所需的生物学触发因素 脊柱后凸和脊柱侧凸的发展。这种方法将允许研究S1 P的多种新功能;此外，它还将为蛋白质分泌途径的调节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提供新的见解。