Regulation of mechanotransduction in the intervertebral disc

研究概述：在脊椎动物中，椎间盘 (IVD) 对于脊柱稳定、承重和运动至关重要。它由三个组织组成：中央髓核（NP）、外纤维环（AF）和软骨终板。尽管生理负荷对于椎间盘稳态至关重要，但负荷过度和负荷不足会导致组织退化。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 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取决于细胞类型和刺激。我们之前的研究描述了新型 IVD 特异性遗传小鼠模型，并开发了体外模型来研究细胞力转导。我们证明 AF 细胞对循环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。基于这些发现，拟议的研究将描述瞬时受体电位香草酸 4 (TRPV4) 在调节 IVD 机械生物学方面的贡献。 TRPV4 是一种多模式激活的 Ca2 渗透性离子通道，可调节细胞对机械刺激相关肌肉骨骼组织的反应。 使用新型转基因小鼠模型，我们的研究将探索 TRPV4 介导的 Ca2 信号传导介导 IVD 细胞对机械负荷的反应的假设。 目标 1：原位表征 IVD 中 Trpv4 表达的时空模式。 我们生成了一种新型报告小鼠，其中内源 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 发育 (E 8.5-17.5) 和年龄 (2.5-12 月龄) 相关的时间点的脊柱组织。目标 2：评估 TRPV4 在体外介导 NP 和 AF 细胞机械转导中的作用。 我们培育了一种转基因小鼠品系，在整个 IVD 过程中基因敲除 Trpv4。我们将使用 Ca2 成像（在野生型和敲除细胞中）评估 TRPV4 在原代小鼠 NP 和 AF 细胞中的作用，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。目标 3：评估 TRPV4 在敲除小鼠模型中作为 IVD 稳态调节剂的作用。我们将通过检查组织特异性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改变的模型来确定 TRPV4 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 TRPV4 作为 IVD 中候选机械感受器的作用。了解细胞如何响应复杂的 IVD 微环境是概括这些组织工程成分的关键。

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.