Regulation of mechanotransduction in the intervertebral disc

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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.