Phosphate signaling in biomineralization

摘要 无机磷(PO43-/PI)是骨骼和骨骼中生理生物矿化过程的关键调节因子。 牙科组织，是导致血管病理性矿化的主要因素。核心作用 在生物矿化中的PI的作用被广泛的遗传和获得性矿化缺陷所强调 影响全身PI动态平衡(高磷血症和低磷血症)和局部/细胞PI的疾病 可获得性(如低磷血症)。尽管现在已经确定PI是一种信号分子，可以 改变细胞生理，PI执行这一功能的潜在分子机制仍然存在 很大程度上是未知的。我们知识上的这种差距阻碍了靶向治疗方法的发展 磷供应异常引起的疾病。我们的目标是描绘出调节 响应胞外PI的生物矿化。我们和其他人已经证明了分子相互作用 在大多数被分析的患者中，ERK1/2的上游是启动对胞外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 激活ERK1/2和支持矿化功能所需的分子回路 敬圆周率。首先，我们的体外数据显示，成骨细胞缺乏甲状旁腺激素/甲状旁腺激素 相关蛋白受体1(Pth1r)在高等电点条件下不激活ERK1/2，并显著 对PI的转录反应受损。第二，我们的数据表明，PI诱导的ERK1/2激活和基因 表达依赖于蛋白激酶C(PKC)。此外，我们的数据表明，细胞 钙离子(Ca~(2+))可增强对PI的反应。基于这些数据，我们假设PI诱导 在成骨细胞中，信号级联与Pth1r-PKC-ERK1/2通路整合。为了检验这一假说 我们将使用体外和体内方法，着重于细胞内产生矿化细胞外信号的方法。 矩阵。在本项目的目标1中，我们将定义Pth1r依赖的细胞激活的分子回路 对PI的反应。在目标2中，我们将确定Pth1r在PI信号和PI调节中的功能作用 体内矿化作用。在目标3中，我们将确定钙离子在矿化支持功能中的作用。 PI信令。通过完成这些目标，将提供PI诱导信号的第一个特征 级联并鉴定成骨细胞中启动细胞对PI反应所需的分子成员。 这将为确定药物调节细胞敏感性的靶点奠定基础。 到可用的PI。

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.