TRPV4 Inhibition and CRISPR-Cas9 Knockout Reduce Inflammation Induced by Hyperphysiological Stretching in Human Annulus Fibrosus Cells

Abstract

Mechanical loading and inflammation interact to cause degenerative disc disease and low back pain (LBP). However, the underlying mechanosensing and mechanotransductive pathways are poorly understood. This results in untargeted pharmacological treatments that do not take the mechanical aspect of LBP into account. We investigated the role of the mechanosensitive ion channel TRPV4 in stretch-induced inflammation in human annulus fibrosus (AF) cells. The cells were cyclically stretched to 20% hyperphysiological strain. TRPV4 was either inhibited with the selective TRPV4 antagonist GSK2193874 or knocked out (KO) via CRISPR-Cas9 gene editing. The gene expression, inflammatory mediator release and MAPK pathway activation were analyzed. Hyperphysiological cyclic stretching significantly increased the IL6, IL8, and COX2 mRNA, PGE2 release, and activated p38 MAPK. The TRPV4 pharmacological inhibition significantly attenuated these effects. TRPV4 KO further prevented the stretch-induced upregulation of IL8 mRNA and reduced IL6 and IL8 release, thus supporting the inhibition data. We provide novel evidence that TRPV4 transduces hyperphysiological mechanical signals into inflammatory responses in human AF cells, possibly via p38. Additionally, we show for the first time the successful gene editing of human AF cells via CRISPR-Cas9. The pharmacological inhibition or CRISPR-based targeting of TRPV4 may constitute a potential therapeutic strategy to tackle discogenic LBP in patients with AF injury.

Multiscale Regulation of the Intervertebral Disc: Achievements in Experimental, In Silico, and Regenerative Research

“…They respond to these loads via a number of mechanotransduction mechanisms, which have been reviewed previously [ 130 , 131 , 132 ]. For example, TRP channels, whereby TRPV4 as well as TRPC6, TRPM2, and TRPML1 stand out due to fundamental roles in osmo- and mechano-sensing [ 118 , 133 , 134 ]. NP cells are more responsive to hydrostatic pressure, while AF cells respond better to cyclic strain [ 135 ].…”

“…The p38 signaling pathway is generally activated by stressors and is known to regulate inflammation, autophagy, apoptosis, and differentiation [ 241 ]. Numerous studies have investigated p38 in the IVD, thereby identifying hypoxia [ 245 ], hyperosmolarity [ 120 ], hyperphysiological mechanical loads [ 133 ], ER stress [ 258 ], acidity [ 257 ], high glucose levels [ 256 ], and IL-1 [ 253 ] as potent activators. Interestingly, p38 is connected to TPRV4 [ 133 ], which has previously been described to transduce mechanical, inflammatory, and pain signals in cartilage [ 259 ].…”

“…Numerous studies have investigated p38 in the IVD, thereby identifying hypoxia [ 245 ], hyperosmolarity [ 120 ], hyperphysiological mechanical loads [ 133 ], ER stress [ 258 ], acidity [ 257 ], high glucose levels [ 256 ], and IL-1 [ 253 ] as potent activators. Interestingly, p38 is connected to TPRV4 [ 133 ], which has previously been described to transduce mechanical, inflammatory, and pain signals in cartilage [ 259 ]. Different research fields have shown extensive cross talk between p38 and other signaling pathways, e.g., ERK [ 258 ], TGF-β/Smad, [ 260 ] or Akt [ 261 ], which should be investigated in IVD cells.…”

Genetic Therapy for Intervertebral Disc Degeneration

“…Similarly, Cambria et al [ 79 ] reported that the transient receptor potential vanilloid type 4 (TRPV4) gene was successfully knocked out in vitro by the CRISPR/Cas9 gene-editing system in AF-injured patients with chronic LBP. By using CRISPR, they investigated the role of the TNFR4 gene in the regulation of IL6 and IL8 using CRISPR and the reduction in inflammation induced by hyper physiological stretching of AF cells.…”

TRPV4 mediates cell damage induced by hyperphysiological compression and regulates COX2/PGE2 in intervertebral discs

“…The TRP super‐family is composed of six sub‐families in mammals: TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPP (polycystin), TRPML (mucolipin), and TRPA (ankyrin) 19 . Several studies have recently investigated the expression and function of TRP channels in the IVD 20‐24 . The mechanosensitive member 4 of the vanilloid subfamily (TRPV4) is especially intriguing for the study of load‐mediated IVD degeneration and LBP.…”