Regulation of mechanotransduction in the intervertebral disc

研究概述：在脊椎动物中，椎间盘（IVD）对脊柱稳定、承重和运动至关重要。它由三种组织组成：中央髓核（NP），外部纤维环（AF）和软骨终板。虽然生理负荷对于椎间盘内环境稳定是必不可少的，但过度负荷和负荷不足可诱导组织降解。因此，IVD是研究机械生物学的有趣模型。在细胞水平上，机械负荷改变了IVD细胞中的多种过程，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依赖性的。我们以前的研究特征在于新的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。我们证明了AF细胞对周期性拉伸应变的反应是频率依赖性的，调节细胞外基质、细胞周期和促炎基因表达。根据这些发现，拟议的研究将表征瞬时受体电位香草酸4（TRPV 4）在IVD中调节机械生物学的贡献。TRPV 4是一种多模式激活的钙离子通道，调节肌肉骨骼组织对机械刺激的细胞反应。 我们的研究将使用新型转基因小鼠模型，探讨TRPV 4介导的Ca 2+信号转导介导IVD细胞对机械负荷的反应的假设。 目的1：表征原位IVD中Trpv 4表达的时空模式。 我们产生了一种新的报告小鼠，其中内源性Trpv 4基因座驱动lacZ的表达（Trvp 4LacZ/WT）。使用整体包埋和组织学分析，我们将在与IVD发展（E 8.5-17.5）和年龄（2.5-12月龄）相关的时间点评估脊柱组织。目的2：研究TRPV 4在体外介导NP和AF细胞机械力信号转导中的作用。 我们产生了转基因小鼠品系以在整个IVD中遗传敲除Trpv 4。我们将使用Ca 2+成像（在野生型和敲除细胞中）评估TRPV 4在原代鼠NP和AF细胞中的作用，以及其在调节IVD细胞对负荷反应（AF的循环拉伸应变; NP的渗透应变）中的功能。目的3：在基因敲除小鼠模型中评估TRPV 4作为IVD稳态调节剂的作用。我们将通过研究脊柱发育和衰老过程中组织特异性基因敲除小鼠的表型以及体内机械负荷改变的模型来确定TRPV 4在IVD中的作用。意义：拟议的研究将应用独特的转基因小鼠模型来探索TRPV 4作为IVD中的候选机械受体的作用。了解细胞如何对复杂的IVD微环境做出反应是重现组织工程这些组件的关键。

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.