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: 10335222
• 负责人: Harry C. Blair
• 金额: $ 60.43万
• 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/10335222
• 关键词: Acids; Address; Affect; Aging; Air; Alkaline Phosphatase; Alkalinization; Apatites; 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; 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.

成骨细胞产生骨骼，这是一种致密的细胞外基质，主要由 I 型胶原蛋白和羟基磷灰石矿物质组成 在一个隔离的隔间里。磷酸盐生产过程中的矿物质沉积产生酸。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 值的变化与骨细胞外 pH 值无关。我们的工作支持 强烈的前提是成骨细胞碱化骨基质，尽管理解上仍然存在差距。许多 胶原蛋白分泌、磷酸盐产生和钙运输等方面已得到充分研究，但质子 除了我们的工作之外，对成骨细胞上皮转运的研究很少。 我们将使用创新方法、膜运输、器官外植体的活细胞成像以及表面 等离子共振，研究骨基质的 pH 值和矿化。我们期望表征组件 详细研究成骨细胞质子传输，并明确定义骨骼上矿物质沉积的性质 胶原蛋白基质。当我们开发这些转运途径的分子基础时，我们期望分子 治疗干预的目标将可用于在体外和体内操纵骨矿化。 目标 1. 活性和非活性成骨细胞中酸转运的调节将直接解决这一假设 维持骨矿物质需要酸运输，并且调节更高的运输活性 允许骨矿化发生。我们将通过从兔子身上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非活动。我们将测量酸转运的量和活性 膜蛋白，以及酸转运过程的调节蛋白。此外，我们还将生产 体外成骨细胞在骨形成后，根据活性从细胞中分离转运蛋白。 这将在不过度表达 NHE1、ClC-3 或两者的正常细胞和破骨细胞中进行。这是 预计这些转运蛋白的过度表达将刺激骨形成和活动。 目标 2. 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与细胞外 pH 值建立 pH 梯度 由于基底外侧膜上 Cl/H 交换剂 ClC-3 的活性而碱化。成骨细胞分泌 基质钙和 pH 传感器能够时空检测矿物质和质子通量。 目标 3. 将使用表面确定影响 I 型胶原上矿物质沉积的参数 等离子共振。我们将使用通常会进行生物矿化（I 型）或不会进行生物矿化（II 型）的胶原蛋白 探讨胶原蛋白结构的影响以及骨桥蛋白、骨钙素等的作用介绍 监管影响。时间、H+、Ca2+ 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，特别是酸运输。 这些是骨形成的重要的、研究不足的元素，也是潜在的新治疗靶点。