Phosphate signaling in biomineralization

生物矿化中的磷酸盐信号传导

• 批准号: 10430021
• 负责人: Dobrawa Napierala
• 金额: $ 33.66万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10430021
• 关键词: Affect; Agonist; Binding; Blood Vessels; Calcium; Cartilage; Cell Line; Cell physiology; Cells; Chronic; Complex; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Data; Defect; Dental; Dentin; Development; Diagnosis; Disease; Extracellular Matrix; Extracellular Signal Regulated Kinases; Fluorescence Resonance Energy Transfer; Foundations; Gene Expression; Genes; Genetic; Genetic Transcription; Goals; Homeostasis; Hypophosphatasia; Hypophosphatemia; Image; Impairment; In Vitro; Isoenzymes; Knockout Mice; Knowledge; Life; MAPK3 gene; Measures; Mediating; Mediator of activation protein; Minerals; Molecular; Molecular Target; Mus; Mutate; Odontoblasts; Osteoblasts; PTH gene; Pathologic; Pathway interactions; Pharmacology; Phosphotransferases; Physiological; Physiology; Process; Protein Kinase C; Receptor Signaling; Role; Signal Pathway; Signal Transduction; Signaling Molecule; Spectroscopy, Fourier Transform Infrared; Testing; Tissues; Vesicle; base; biomineralization; bone; cell growth regulation; conditional knockout; extracellular; in vivo; inhibitor; inorganic phosphate; live cell imaging; microCT; mineralization; novel; osteogenic; parathyroid hormone-related protein; progenitor; receptor; response; skeletal; small molecule inhibitor; targeted treatment; therapeutic target

Abstract Inorganic phosphate (PO43-/Pi) is a critical regulator of the physiologic biomineralization process in skeletal and dental tissues, and is a major contributing factor to pathologic mineralization of blood vessels. The central role of Pi in biomineralization is underscored by a broad-range of mineralization defects in genetic and acquired disorders affecting systemic Pi homeostasis (hyperphosphatemia and hypophosphatemia) and local/cellular Pi availability (e.g. hypophosphatasia). Although it is well established now that Pi is a signaling molecule that can change cellular physiology, the underlying molecular mechanisms by which Pi executes this function remain largely unknown. This gap in our knowledge impedes development of targeted therapeutic approaches to diseases caused by abnormal Pi availability. Our goal is to delineate the signaling cascade that regulates biomineralization in response to extracellular Pi. We and others have shown that molecular interactions upstream of Erk1/2 kinase are central to initiating the response to extracellular Pi in the majority of analyzed cells. Our preliminary studies in cells producing mineralized extracellular matrix (osteogenic cells) identified a molecular circuit, which is required for activation of Erk1/2 and mineralization-supporting functions in response to Pi. First, our in vitro data show that osteogenic cells deficient in parathyroid hormone/parathyroid hormone related protein receptor 1 (Pth1r) do not activate Erk1/2 under high Pi conditions and have significantly impaired transcriptional response to Pi. Second, our data suggest that Pi-induced Erk1/2 activation and gene expression are dependent on the protein kinase C (PKC). Furthermore, our data suggest that the cellular response to Pi is enhanced by calcium (Ca2+). Based on these data we hypothesize that the Pi-induced signaling cascade is integrated with the Pth1r-PKC-Erk1/2 pathway in osteogenic cells. To test this hypothesis we will use in vitro and in vivo approaches focused on signaling in cells producing mineralized extracellular matrix. In the Aim 1 of this project, we will define the molecular circuit of Pth1r-dependent activation of cellular responses to Pi. In the Aim 2, we will determine the functional role of Pth1r in Pi signaling and Pi-regulated mineralization in vivo. In the Aim 3, we will determine the role of Ca2+ in mineralization-supporting functions of Pi signaling. By completing these Aims, will provide the first characterization of the Pi-induced signaling cascade and identify molecular players required for initiation of cellular responses to Pi in osteogenic cells. This will provide the foundation for identification of targets for pharmacological regulation of cellular sensitivity to available Pi.

摘要 无机磷酸盐（PO 43-/Pi）是骨骼肌生理性生物矿化过程的关键调节剂， 牙齿组织，并且是血管的病理矿化的主要促成因素。的中心作用 在生物矿化中，Pi的作用被遗传和后天的广泛矿化缺陷所强调。 影响全身Pi稳态的疾病（高磷酸盐血症和低磷酸盐血症）和局部/细胞Pi 可用性（例如低磷酸酶症）。尽管现在已经很好地确定Pi是一种信号分子， 改变细胞生理学，Pi执行这一功能的潜在分子机制仍然存在 大部分未知。我们知识上的这一差距阻碍了靶向治疗方法的发展， 由于磷供应异常而引起的疾病。我们的目标是描绘出调节 生物矿化响应于细胞外Pi。我们和其他人已经证明了分子间的相互作用 Erk 1/2激酶的上游是启动对细胞外Pi的反应的中心， 细胞我们对产生矿化细胞外基质的细胞（成骨细胞）的初步研究发现， 分子回路，这是激活Erk 1/2和矿化支持功能所必需的 到派。首先，我们的体外数据表明，甲状旁腺激素/甲状旁腺激素缺乏的成骨细胞， 相关蛋白受体1（Pth 1 r）在高Pi条件下不激活Erk 1/2， 对Pi的转录反应受损。其次，我们的数据表明，Pi诱导的Erk 1/2激活和基因表达可能与细胞凋亡有关。 表达依赖于蛋白激酶C（PKC）。此外，我们的数据表明， 对Pi的反应被钙（Ca 2+）增强。基于这些数据，我们假设Pi诱导的 信号级联与成骨细胞中的Pth 1 r-PKC-Erk 1/2通路整合。为了验证这一假设 我们将使用体外和体内的方法，集中在细胞产生矿化的细胞外信号， 矩阵在本项目的目标1中，我们将定义Pth 1 r依赖的细胞活化的分子回路， 回答Pi。在目的2中，我们将确定Pth 1 r在Pi信号传导和Pi调节中的功能作用。 体内矿化在目标3中，我们将确定Ca 2+在矿化支持功能中的作用， π信号。通过完成这些目标，将提供Pi诱导的信号传导的第一个表征 级联并鉴定成骨细胞中启动对Pi的细胞应答所需的分子参与者。 这将为确定细胞敏感性的药理学调节靶点提供基础 可用的Pi。