Transient Receptor Potential Vanilloid 4 Knockdown Decreases Extracellular Matrix Synthesis via Autophagy Suppression in the Rat Intervertebral Disc

Abstract

Background

Transient receptor potential vanilloid 4 (TRPV4) has been identified as a Ca²⁺-permeable channel and is activated under physiological mechanical stimulation in disc nucleus pulposus (NP) cells. Meanwhile, the Ca²⁺-dependent AMP-activated protein kinase (AMPK)/mTOR pathway activates autophagy in notochordal cells. We hypothesized that TRPV4 is involved in the maintenance of intradiscal homeostasis via autophagy. Our objective was to elucidate the role of TRPV4 in extracellular matrix (ECM) metabolism and autophagy in the rat intervertebral disc through a loss-of-function study with the RNA interference (RNAi) technique.

Methods

In vitro study: Small interfering RNA (siRNA) was applied to knockdown TRPV4 by the reverse transfection method in rat disc NP cells. Expression of TRPV4, AMPK/mTOR pathway-related markers, and autophagy markers were measured by Western blotting (WB). Next, ECM metabolism was assessed under serum starvation and/or proinflammatory interleukin-1 beta (IL-1β) stimulation. In vivo study: TRPV4 and control siRNAs were injected into rat discs. To confirm in vivo transfection, WB for TRPV4 was conducted in rat disc NP-tissue protein extracts 2, 28, and 56 days after injection. Furthermore, 24-h temporary static compression-induced disruption of TRPV4 siRNA-injected discs was observed by radiography, histomorphology, and immunofluorescence.

Results

In vitro study: In disc cells, three different TRPV4 siRNAs consistently suppressed autophagy with TRPV4 protein knockdown (mean 33.2% [95% CI: −50.8, −15.5], 44.1% [−61.7, −26.4], 58.3% [−76.0, −40.7]). ECM metabolism was significantly suppressed by TRPV4 RNAi under proinflammatory IL-1β stimulation. In vivo study: The WB displayed sustained decreases in TRPV4 protein expression 2, 28, and 56 days after injection. Under the loaded condition, TRPV4 siRNA-injected discs presented radiographic height loss ([−31.7, −7.75]), histomorphological damage ([0.300, 4.70]), and immunofluorescent suppression of autophagy ([1.61, 20.5]) and ECM metabolism ([−25.2, −6.41]) compared to control siRNA-injected discs at 56 days.

Conclusions

The TRPV4 could be a therapeutic target for intervertebral disc diseases via modulating autophagy.