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.

成骨细胞形成骨，它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 在一个孤立的隔间里。磷酸盐生产过程中的矿物沉积会产生酸。因此，成骨细胞必须 去除矿物沉积产生的酸。我们的初步数据包括直接证明矩阵 成骨细胞上皮内的pH与骨中的细胞外pH无关。我们的工作支持 强烈的前提是成骨细胞使骨基质碱化，尽管理解上的差距仍然存在。许多 胶原蛋白的分泌、磷酸盐的产生和钙的运输等方面都得到了很好的研究，但质子 与我们的工作不同，我们对成骨细胞上皮细胞的转运进行了最低限度的研究。 我们将使用创新的方法，膜转运，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值和矿化情况。我们希望能够描述组件的特征 关于成骨细胞质子运输的详细信息，并清楚地定义矿物质沉积在骨骼上的性质 胶原蛋白基质。当我们开发这些运输途径的分子基础时，我们预计分子 治疗干预的靶点将变得可用于在体外和体内操纵骨矿化。 目的1.调节活性和非活性成骨细胞中的酸运输将直接解决这一假说 酸运输是维持骨矿物质所必需的，而更高的运输活性被调节为 使骨骼矿化发生。我们将通过从兔体内分离活性和非活性成骨细胞来研究这一点 脊椎按大小分为活动和非活动。我们将测量酸运输的数量和活性 膜蛋白，以及酸运输过程的调节蛋白。此外，我们还将生产 在体外，成骨细胞在骨形成后，根据活性从细胞中分离运输蛋白。 这将在正常细胞和破骨细胞中进行，NHE1、ClC-3或两者都不表达或过度表达。它是 预计这些转运蛋白的过度表达将刺激骨的形成和活性。 目的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 质子在成骨细胞上的矢量传输建立了与细胞外pH的pH梯度 碱化是由于基侧膜上的氯/氢交换分子ClC-3的活性所致。成骨细胞分泌 基质钙和pH传感器，可实现矿物和质子通量的时空检测。 目的3.影响I型胶原上矿物质沉积的参数将使用表面来确定 等离子激元共振。我们将使用生物矿化的胶原蛋白(第一类)或不正常矿化的胶原蛋白(第二类) 探讨对胶原结构的影响及骨桥蛋白、骨钙素等的作用。 监管影响。时间、H+、Ca~(2+)和P~(2+)将成为主要的自变量。 我们专注于支持矿化骨基质形成的新机制，特别是酸运输。 这些都是重要的、研究甚少的骨形成元素，也是潜在的新治疗靶点。