GROWTH FACTOR SIGNAL TRANSDUCTION IN OSTEOBLASTS

骨质流失是一种口腔健康问题，发生在两个牙周 疾病和无牙颌患者的牙槽嵴。因此我们 感兴趣的因素已被证明增加骨吸收。 表皮生长因子（EGF）是一种生长速度较快的因子。 新生小鼠的牙齿萌出。EGF的作用也越来越大。 长骨和颅骨培养系统中的吸收和EGF受体 表皮生长因子受体（EGFR）位于牙周膜周围的前成骨细胞上， 对大鼠EGF可能在牙齿萌出中起作用， 控制牙槽骨沉积和流失。EGFR，以及 其它骨吸收剂的受体，如甲状旁腺激素， 位于成骨细胞上。为了了解成骨细胞如何响应 针对这些因素，我们计划研究骨的信号转导机制， 成骨细胞中的吸收剂。 在已经研究的不同细胞中，EGF引起细胞增殖。 磷脂酰肌醇水解和随后的细胞内 游离钙已报道EGFR激活磷脂酶C（PLC） 在不同的细胞类型中，同工酶通过多种机制而变化。EGFR具有 通过百日咳毒素敏感的G 它可以直接磷酸化PLC γ同工酶。虽然 已知对毒素不敏感的G蛋白激活PLC γ， EGF对肝癌的作用还有待进一步研究。我们发现 原代培养新生大鼠颅骨成骨细胞， 克隆性成骨细胞骨肉瘤细胞系G292和SaOS-2具有 对EGF的反应。所有这些细胞都有 这种生长因子的受体，因此我们假设， 它们的信号转导通路的差异。这是他们的目的。 EGF信号级联反应的早期事件 培养成骨细胞。第一步：确定EGF 激活百日咳毒素敏感型、霍乱毒素敏感型或 使用放射性标记的GTP γ-S结合的毒素不敏感的G蛋白 比色法在具体目标二中，EGF通过毒素敏感性和 将通过免疫共沉淀法评估毒素不敏感的G蛋白 实验这些研究将着眼于EGFR-G蛋白的变化， 复合物和G-蛋白-PLC复合物作为EGF刺激的功能。在 具体目标三，将确定是否EGFR直接 磷酸化PLC γ。这些实验将寻找EGF刺激 EGFR PLC γ复合物以及PLC γ酪氨酸增加 磷酸化这些研究将有助于了解吸收性骨水泥 药剂通过成骨细胞作用以引起骨损失。

Bone loss is an in oral health problem which occurs in both periodontal disease and the alveolar ridges of edentulous patients. We are therefore interested in factors which have been shown to increase bone resorption. Originally, epidermal growth factor (EGF) was found to cause accelerated tooth eruption in neonatal mice. In addition, EGF causes increased resorption in long bone and calvarial culture systems and the EGF receptor (EGFR) is located on preosteoblasts surrounding the periodontal ligament in rats. It is possible that EGF plays a role in tooth eruption and control of alveolar bone deposition and loss. The EGFR, as well as receptors for other bone resorptive agents, such as parathyroid hormone, are located on osteoblasts. In order to understand how osteoblasts respond to these factors we plan to study signal transduction mechanisms of bone resorptive agents in osteoblastic cells. In the different cells which have been studied, EGF causes an increase in phosphatidylinositol hydrolysis and a subsequent increase in intracellular free calcium. The EGFR has been reported to activate phospholipase C (PLC) isozymes by a number of mechanisms in different cell types. The EGFR has been associated with PLC activation via pertussis toxin-sensitive G proteins and it can directly phosphorylate the PLCgamma isozyme. Although the toxin-insensitive G proteins are known to activate PLCgamma, their role in EGF activation of PLC remains to be examined. We have found that primary culture osteoblasts isolated from neonatal rat calvaria, and the clonal osteoblastic osteosarcoma cell lines G292 and SaOS-2 have differences in their mitogenic response to EGF. All of these cells have receptors for this growth factor, therefore we assume that there must be differences in their signal transduction pathways. It is the aim of these studies to examine early events in the EGF signal cascade in primary culture osteoblastic cells. In specific aim one, it will determined if EGF activates either pertussis toxin-sensitive, cholera toxin-sensitive or toxin-insensitive G proteins using a radiolabeled GTPgamma-S binding assay. In specific aim two, EGF activation of PLC by toxin-sensitive and toxin-insensitive G proteins will be assessed by coimmunoprecipitation experiments. These studies will look at changes in EGFR-G protein complexes and G-protein-PLC complexes as a function of EGF stimulation. In specific aim three, it will be determined if the EGFR directly phosphorylates PLCgamma. These experiments will look for EGF stimulated EGFR PLCgamma complexes as well as an increase in PLCgamma tyrosine phosphorylation. These studies will help to understand how resorptive agents act via osteoblasts to bring about bone loss.