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初始响应是细胞内Ca2+的迅速上升，这取决于细胞外Ca2+进入和细胞内Ca2+释放。我们已经证明，通过L型电压敏感的Ca2+通道（L-VSCC）进入Ca2+对于成骨细胞增殖，体外和机械诱导的骨形成很重要。我们还发现，通过激活磷脂酶C/IP3的细胞内Ca2+释放负责剪切诱导的NFKB易位和随后的基因表达变化。最后，我们的数据表明，剪​​切激活磷脂酶C是通过机械敏感通道的激活和诱导Ca2+依赖性核苷酸释放的L-VSCC介导的。反过来，这种释放导致嘌呤能受体（P2）激活细胞内Ca2+释放。这些数据以及我们实验室的其他数据，使我们假设Ca2+在单元内离散的亚细胞域中升高。在这种情况下，细胞外Ca2+进入将刺激因信号扩增重要的因素（例如ATP和/或UTP）的释放，而细胞内Ca2+通过P2受体的ATP Activaton释放会导致基因表达的变化。我们提供的数据表明，在成骨细胞对机械刺激的反应中，L-VSCC A1同工型CAV 1.2很重要。为了研究L-VSCC与ATP释放的相互作用以及P2受体的激活，我们将同时使用细胞生物学和体内遗传基因敲除研究来检查CAVL.2 A1 L-VSCC以及P2受体在机械转导中的作用。我们的具体目的是：1）定义L-VSCC CAVL.2 A1亚基在骨骼和成骨细胞对机械刺激的响应中的作用； 2）检查P2受体激活在[Ca2+] I，Prostaglandin分泌因子，转录因子激活和基因表达中的骨细胞中的变化的响应[Ca2+] I的响应中的影响。这些目标的完成将为最初的感知和对成骨细胞的机械刺激的快速反应提供更多的了解，并可能为改变骨形成的药理目标提供。