INTRODUCTION:

The intervertebral disc is the largest avascular, low nutrient organ in the human body. Autophagy is an important cell survival mechanism by self-digestion and recycling damaged components under stress conditions, primarily nutrient deprivation. Biologically, disc cells and their extracellular matrix are stimulated by physiological range of mechanical loading, and abnormal loading can result in disc degeneration. Therefore, the mechanobiological mechanisms that govern intradiscal homeostasis need to be clarified to understand the process of disc degeneration and to develop a therapeutic strategy for disc degenerative diseases. One possible mechanosensitive regulator in disc homeostasis is Transient Receptor Potential Vanilloid 4 (TRPV4). The TRPV4 has been identified as a critical mechanosensor in cartilage, and modulating TRPV4 in cartilage has protective effects against osteoarthritis. The TRPV4 has been also reported to be activated under a physiological mechanical stimulation in disc cells in vitro. We hypothesized that TRPV4 is involved in the maintenance of intradiscal autophagy. Our objective is to elucidate the role of TRPV4 in rat intervertebral disc autophagy and extracellular matrix metabolism through loss-of-function study with the RNA interference (RNAi) technique.

METHODS:

Disc nucleus pulposus (NP) and annulus fibrosus (AF) cells harvested from 12-week-old male Sprague-Dawley rats were used. Small interfering RNA (siRNA) was applied to knockdown TRPV4 by the reverse transfection method. Three different TRPV4-siRNA sequences were used to exclude the off-target effect. Cells after transfection were cultured in DMEM with or without 10% FBS for 24 h to simulate nutrient deprivation. Expression of AMPK, mTOR, p70/S6K, LC3-II, and a substrate p62/SQSTM1 as well as TRPV4 was measured by Western blotting. Next, cells after the transfection were cultured in serum-free DMEM with 10-ng/ml interleukin-1 beta (IL-1β) for 24 h. Autophagy markers and catabolic matrix metalloproteinases (MMPs) were assessed by Western blotting. To control for protein loaded, membranes were re-probed using anti-alpha-Tubulin. The intensities of the bands were quantified using ImageJ software. Multi-way ANOVA with Tukey–Kramer post-hoc test was used to analyze the data in immunoblotting. The P-values of < 0.05 were regarded as statistically significant.

RESULTS:

In rat disc NP and AF cells, TRPV4 expression significantly decreased by TRPV4 RNAi, which proved to silence the target proteins by 70% or more (P < 0.05). The LC3-II decreased and p70/S6K and p62/SQSTM1 increased (P < 0.05), indicating autophagy suppression. In addition, AMPK decreased and mTOR increased (P < 0.05), suggesting a possible pathway between TRPV4 and autophagy. Pro-inflammatory IL-1β stimulation with TRPV4 RNAi further decreased AMPK and LC3-II and increased mTOR, p70/S6K, p62/SQSTM1 and MMPs (P < 0.05), indicating enhancement of the effect of TRPV4 knockdown.

DISCUSSION:

The TRPV4 knockdown suppressed autophagy with AMPK inhibition in rat disc NP and AF cells and developed further suppression of autophagy under pro-inflammatory IL-1β stimulation. This loss-of-function study demonstrates the involvement of TRPV4 in autophagy and extracellular matrix metabolism of rat disc NP and AF cells. The TRPV4 could be a therapeutic target for intervertebral disc diseases via modulating autophagy.