Mechanotransduction: Puringergic Signaling in Bone

机械转导：骨中的普林能信号传导

• 批准号: 7119541
• 负责人: RANDALL L DUNCAN
• 金额: $ 32.49万
• 依托单位: UNIVERSITY OF DELAWARE
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-05 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7119541
• 关键词: adenosine triphosphate; biological signal transduction; biomechanics; bone; calcium channel; computed axial tomography; densitometry; gene expression; gene targeting; laboratory mouse; mechanical stress; osteoblasts; phenotype; prostaglandins; protein isoforms; purinergic receptor; receptor expression; shear stress; transcription factor

DESCRIPTION (provided by applicant): The initial response of osteoblasts to mechanical stimulation is a rapid rise in intracellular Ca2+ that is dependent on both extracellular Ca2+ entry and intracellular Ca2+ release. We have demonstrated that Ca2+ entry via L-type voltage-sensitive Ca2+ channels (L-VSCC) is important to osteoblast proliferation, in vitro, and mechanically-induced bone formation, in vivo. We have also found that intracellular Ca2+ release through activation of phospholipase C/IP3 is responsible for shear-induced NFkB translocation and subsequent changes in gene expression. Finally, our data suggest that activation of phospholipase C by shear is mediated by activation of mechanically-sensitive channels and the L-VSCC that induces Ca2+ dependent nucleotide release. This release, in turn, results in activation of purinergic receptors (P2) to alter intracellular Ca2+ release. These data, coupled with other data from our lab, have led us to hypothesize that Ca2+ is elevated in discrete subcellular domains within the cell. In this scenario, extracellular Ca2+ entry would stimulate release of factors, such as ATP and/or UTP, that are important in signal amplification, while intracellular Ca2+ release via ATP activaton of P2 receptors would result in changes in gene expression. We present data suggesting the L-VSCC a1 isoform, Cav 1.2, is important in this response of osteoblasts to mechanical stimulation. To study the interaction of the L-VSCC with ATP release and the resulting activation of P2 receptors, we will use both cell biologic and in vivo genetic knockout studies to examine the role of the Cavl.2 a1 L-VSCC as well as P2 receptors in mechanotransduction. Our specific aims are to: 1) define the role of the L- VSCC Cavl.2 a1 subunit in the response of bone and osteoblasts to mechanical stimulation and 2) examine the effects of P2 receptor activation in the response of [Ca2+]i, prostaglandin secretion, transcription factor activation and changes in gene expression in osteoblastic cells subjected to fluid shear. Completion of these aims will provide added insight into the initial perception and rapid responses to mechanical stimulation of osteoblasts and may provide pharmacologic targets for alteration of bone formation.

描述（由申请人提供）：成骨细胞对机械刺激的最初反应是细胞内Ca2+的快速升高，这取决于细胞外Ca2+进入和细胞内Ca2+释放。我们已经证明，Ca2+ 通过 L 型电压敏感 Ca2+ 通道 (L-VSCC) 进入对于体外成骨细胞增殖和体内机械诱导的骨形成非常重要。我们还发现，通过激活磷脂酶 C/IP3 释放细胞内 Ca2+，是剪切诱导的 NFkB 易位和随后的基因表达变化的原因。最后，我们的数据表明剪切对磷脂酶 C 的激活是通过机械敏感通道和诱导 Ca2+ 依赖性核苷酸释放的 L-VSCC 的激活介导的。这种释放反过来会导致嘌呤能受体 (P2) 的激活，从而改变细胞内 Ca2+ 的释放。这些数据与我们实验室的其他数据相结合，使我们推测细胞内离散的亚细胞域中的 Ca2+ 升高。在这种情况下，细胞外 Ca2+ 进入将刺激信号放大中重要的因子（例如 ATP 和/或 UTP）释放，而细胞内 Ca2+ 通过 ATP 激活 P2 受体释放将导致基因表达的变化。我们提供的数据表明 L-VSCC a1 亚型 Cav 1.2 在成骨细胞对机械刺激的这种反应中很重要。为了研究 L-VSCC 与 ATP 释放的相互作用以及由此产生的 P2 受体激活，我们将使用细胞生物学和体内基因敲除研究来检查 Cavl.2 a1 L-VSCC 以及 P2 受体的作用在力传导中。我们的具体目标是：1) 定义 L-VSCC Cavl.2 a1 亚基在骨和成骨细胞对机械刺激的反应中的作用，2) 检查 P2 受体激活在 [Ca2+]i 反应中的影响，流体剪切作用下成骨细胞中前列腺素的分泌、转录因子的激活和基因表达的变化。这些目标的完成将为成骨细胞机械刺激的初始感知和快速反应提供更多见解，并可能为改变骨形成提供药理学靶点。