Regulation of mechanotransduction in the intervertebral disc

研究概述：在脊椎动物中，椎间盘(IVD)对于脊柱的稳定、承载和运动是必不可少的。它由三个组织组成：中央髓核(NP)、纤维外环(AF)和软骨终板。虽然生理负荷对椎间盘的稳态是必不可少的，但过高和过低的负荷都会导致组织退化。因此，IVD是研究机械生物学的一个有趣的模型。在细胞水平上，机械负荷改变了IVD细胞的各种过程，包括能量代谢和细胞外基质合成。然而，研究表明，对机械负荷的反应是细胞类型和刺激相关的。我们以前的研究描述了新的IVD特异性遗传小鼠模型，并开发了体外模型来询问细胞机械转导。我们证明，房颤细胞对周期性拉伸应变的反应是频率依赖的，调节细胞外基质、细胞周期和促炎基因的表达。基于这些发现，这项拟议的研究将表征瞬时受体电位香草素4(TRPV4)在调节IVD机制生物学中的作用。TRPV4是一种多模式激活的钙离子通道，调节细胞对机械刺激相关肌肉骨骼组织的反应。利用新的转基因小鼠模型，我们的研究将探索TRPV4介导的钙信号介导IVD细胞对机械负荷的反应的假说。目的1：研究TRPV4在IVD中表达的时空分布特征。我们培育了一种新的报告小鼠，其内源性TRPV4基因座驱动LacZ(Trvp4LacZ/WT)的表达。使用整体和组织学分析，我们将评估与IVD发展(E8.5-17.5)和年龄(2.5-12个月龄)相关的时间点的脊柱组织。目的：探讨TRPV4在体外介导NP和AF细胞机械信号转导中的作用。我们产生了一种转基因小鼠，在整个IVD过程中从基因上敲除了TRPV4。我们将使用钙成像技术评估TRPV4在原代小鼠NP和AF细胞中的作用(在野生型和敲除细胞中)，以及它在调节IVD细胞对负荷的反应中的功能(对于AF来说是循环拉伸应变，对于NP来说是渗透应变)。目的3：在基因敲除小鼠模型中，评估TRPV4作为IVD动态平衡调节因子的作用。我们将通过检测组织特异性基因敲除小鼠在脊柱发育和衰老过程中的表型，以及体内机械负荷变化的模型来确定TRPV4在IVD中的作用。意义：建议的研究将应用独特的转基因小鼠模型来探索TRPV4作为IVD候选机械感受器的作用。了解细胞如何对复杂的IVD微环境做出反应是概括组织工程中这些组成部分的关键。

Research overview: In vertebrates, the intervertebral disc (IVD) is essential for spine stabilization, load bearing, and movement. It is composed of three tissues: the central nucleus pulposus (NP), the outer annulus fibrosus (AF), and the cartilage endplates. Although physiological loading is essential for disc homeostasis, over- and under-loading can induce tissue degradation. As such, the IVD is an intriguing model to study mechanobiology. At the cellular level, mechanical loading alters a variety of processes in IVD cells including energy metabolism and extracellular matrix synthesis. However, studies suggest that the responses to mechanical loading are cell type and stimulus dependent. Our previous studies characterized novel IVD-specific genetic mouse models and developed in vitro models to interrogate cellular mechanotransduction. We demonstrated that the response of AF cells to cyclic tensile strain is frequency-dependent, regulating extracellular matrix, cell cycle, and pro-inflammatory gene expression. Building from these findings, the proposed study will characterize the contribution of transient receptor potential vanilloid 4 (TRPV4) in regulating mechanobiology in the IVD. TRPV4 is a multi-modally activated Ca2+-permeable ion channel that regulates the cellular responses to mechanical stimuli related musculoskeletal tissues. Using novel transgenic mouse models, our study will explore the hypothesis that TRPV4-mediated Ca2+ signaling mediates the response of IVD cells to mechanical loading. Aim 1: Characterize the spatiotemporal pattern of Trpv4 expression in the IVD in situ. We generated a novel reporter mouse in which the endogenous Trpv4 locus drives expression of lacZ (Trvp4LacZ/WT). Using whole-mount and histological analyses, we will assess spinal tissues at time points associated with IVD development (E 8.5-17.5) and age (2.5-12 months of age). Aim 2: Assess the role of TRPV4 in mediating mechanotransduction in NP and AF cells in vitro. We generated a transgenic mouse strain to genetically knockout Trpv4 throughout the IVD. We will assess the role of TRPV4 in primary murine NP and AF cells using Ca2+ imaging (in wild-type and knockout cells), and its function in regulating the IVD cell response to load (cyclic tensile strain for AF; osmotic strain for NP). Aim 3: Assess the role of TRPV4 as a regulator of IVD homeostasis in knockout mouse models. We will determine the role of TRPV4 in the IVD by examining the phenotype of tissue-specific knockout mice during spine development and aging, and models of altered mechanical loading in vivo. Significance: The proposed studies will apply unique transgenic mouse models to explore the role of TRPV4 as a candidate mechanoreceptor in the IVD. Understanding how cells respond to the complex IVD microenvironment is key to recapitulating these components for tissue engineering.