Epithelial Osteoblast Function: The Role of Acid Transport

上皮成骨细胞功能：酸转运的作用

• 批准号: 10155434
• 负责人: Harry C. Blair
• 金额: $ 58.51万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10155434
• 关键词: Acids; Address; Affect; Aging; Air; Alkaline Phosphatase; Alkalinization; Apatites; Biological; Bone Matrix; Breathing; Calcium; Carbonates; Carrier Proteins; Cell membrane; Cells; Chlorides; Collagen; Collagen Type I; Collagen Type II; Complex; Data; Defect; Deposition; Detection; Dyes; Elements; Epithelial; Extracellular Fluid; Extracellular Matrix; Glycocalyx; Hydrogen; Hydroxyapatites; In Vitro; Ions; Levamisole; Measures; Membrane Proteins; Membrane Transport Proteins; Methods; Minerals; Molecular; NHE1; Nature; Nodule; Normal Cell; Organ; Oryctolagus cuniculus; Osteoblasts; Osteocalcin; Osteoclasts; Osteogenesis; Osteon; Osteoporosis; Pathway interactions; Physiologic calcification; Production; Protein Overexpression; Proteins; Protons; Regulation; Role; Sodium; Structure; Surface Plasmon Resonance; Testing; Therapeutic Intervention; Time; Transmembrane Transport; Transport Process; Vertebral column; Vertebrates; Visualization; Work; alkalinity; antiporter; basolateral membrane; bone; cariporide; cortical bone; extracellular; fluorexon; genetic regulatory protein; in vivo; inhibitor/antagonist; innovation; inorganic phosphate; knock-down; live cell imaging; mineralization; molecular targeted therapies; new therapeutic target; novel; osteopontin; overexpression; pH gradient; phosphatase inhibitor; sensor; sodium-hydrogen exchanger regulatory factor; spatiotemporal; therapeutic target; uptake; vector

Osteoblasts make bone, a dense extracellular matrix of mainly type I collagen and hydroxyapatite mineral in an isolated compartment. Mineral deposition by phosphate production yields acid. Thus, osteoblasts must remove the acid created by mineral deposition. Our preliminary data include direct demonstration that matrix pH inside the osteoblast epithelium varies independently of extracellular pH in bone. Our work supports strongly the premise that osteoblasts alkalinize the bone matrix, although gaps in understanding persist. Many aspects of collagen secretion, phosphate production, and calcium transport are well studied, but proton transport across osteoblast epithelium is studied minimally other than in our work. We will use innovative methods, membrane transport, live cell imaging of organ explants, and surface plasmon resonance, to study the pH and mineralization of bone matrix. We expect to characterize components of the osteoblast proton transport in detail, and to define clearly the nature of mineral deposition on the bone collagen matrix. As we develop the molecular basis for these transport pathways we expect that molecular targets for therapeutic intervention will become available to manipulate bone mineralization in vitro and in vivo. Aim 1. Regulation of acid transport in active and inactive osteoblasts will directly address the hypothesis that acid transport is required to maintain bone mineral, and that much higher transport activity is regulated to allow bone mineralization to occur. We will study this by isolating active and inactive osteoblasts from rabbit spine separating activie and inactive by size. We will measure the amount and activity of acid transporting membrane proteins, as well as regulatory proteins for the acid transport process. In addition, we will produce osteoblasts in vitro, following bone formation, isolating transport proteins from cells as a function of activity. This will be done in normal cells and osteoclasts without and with over-expression of NHE1, ClC-3 or both. It is expected that bone formation and activity will be stimulated by over-expression of these transport proteins. Aim 2. Fluorescent visualization of live cell osteoblast proton fluxes will directly test the hypothesis that vectorial transport of protons across the osteoblast epithelium establishes a pH gradient with extracellular pH alkalinization due to the activity of the Cl/H exchanger ClC-3 at the basolateral membrane. Osteoblast secreted matrix calcium and pH sensors with enable spatiotemporal detection of mineral and proton fluxes. Aim 3. Parameters that affect mineral deposition on type I collagen will be determined using surface plasmon resonance. We will use collagens that do (Type I) or do not normally mineralize biologically (Type II) to explore the influence of collagen structure and the effects of osteopontin, osteocalcin and others to introduce regulatory influences. Time, H+, Ca2+ and phosphate will be primary independent variables. We focus on novel mechanisms supporting formation of mineralized bone matrix, specifically acid transport. These are important poorly studied elements of bone formation, and also potential novel therapeutic targets.

成骨细胞制造骨，骨是一种致密的细胞外基质，主要由I型胶原和羟基磷灰石矿物组成 在一个隔离的隔间里磷酸盐生产过程中的矿物沉积产生酸。因此，成骨细胞必须 去除矿物质沉积产生的酸。我们的初步数据包括直接证明矩阵 成骨细胞上皮内的pH独立于骨中的细胞外pH而变化。我们的工作支持 强烈的前提是成骨细胞碱化骨基质，虽然在理解的差距持续存在。许多 胶原蛋白分泌、磷酸盐产生和钙转运方面的研究很好，但质子 除了我们的工作外，很少研究穿过成骨细胞上皮的转运。 我们将使用创新的方法，膜运输，器官外植体的活细胞成像，以及表面 等离子体共振，以研究骨基质的pH和矿化。我们希望能够描述 的成骨细胞质子运输的详细情况，并明确定义骨上的矿物质沉积的性质 胶原基质当我们发展这些转运途径的分子基础时，我们预计分子 治疗干预的靶点将可用于在体外和体内操纵骨矿化。 目标1.调节活跃和不活跃的成骨细胞中的酸转运将直接解决这一假设 酸转运是维持骨矿物质所必需的，并且更高的转运活性受到调节， 允许骨矿化发生。我们将通过从家兔中分离活性和非活性成骨细胞来研究这一点 脊柱按大小区分活动和不活动。我们将测量酸运输的量和活性 膜蛋白以及酸转运过程的调节蛋白。此外，我们还将生产 体外成骨细胞，骨形成后，从细胞中分离转运蛋白作为活性的函数。 这将在没有和有NHE 1、ClC-3或两者过表达的正常细胞和破骨细胞中完成。是 预期这些转运蛋白的过度表达将刺激骨形成和活性。 目标二。活细胞成骨细胞质子通量的荧光可视化将直接检验以下假设： 质子穿过成骨细胞上皮的矢量运输建立了pH梯度， 由于基底外侧膜上Cl/H交换器ClC-3的活性而导致的碱化。成骨细胞分泌 矩阵钙和pH传感器，使矿物质和质子通量的时空检测。 目标3。影响I型胶原蛋白上矿物质沉积的参数将使用表面活性剂测定。 等离子共振我们将使用具有（I型）或通常不具有生物矿化作用（II型）的胶原蛋白 探讨骨桥蛋白、骨钙素等对胶原结构的影响， 监管影响。时间、H+、Ca 2+和磷酸盐将是主要自变量。 我们专注于支持矿化骨基质形成的新机制，特别是酸转运。 这些是重要的骨形成的研究不足的元素，也是潜在的新的治疗靶点。