Expression and Activity of TRPA1 and TRPV1 in the Intervertebral Disc: Association with Inflammation and Matrix Remodeling

Abstract

Transient receptor potential (TRP) channels have emerged as potential sensors and transducers of inflammatory pain. The aims of this study were to investigate (1) the expression of TRP channels in intervertebral disc (IVD) cells in normal and inflammatory conditions and (2) the function of Transient receptor potential ankyrin 1 (TRPA1) and Transient receptor potential vanilloid 1 (TRPV1) in IVD inflammation and matrix homeostasis. RT-qPCR was used to analyze human fetal, healthy, and degenerated IVD tissues for the gene expression of TRPA1 and TRPV1. The primary IVD cell cultures were stimulated with either interleukin-1 beta (IL-1β) or tumor necrosis factor alpha (TNF-α) alone or in combination with TRPA1/V1 agonist allyl isothiocyanate (AITC, 3 and 10 µM), followed by analysis of calcium flux and the expression of inflammation mediators (RT-qPCR/ELISA) and matrix constituents (RT-qPCR). The matrix structure and composition in caudal motion segments from TRPA1 and TRPV1 wild-type (WT) and knock-out (KO) mice was visualized by FAST staining. Gene expression of other TRP channels (A1, C1, C3, C6, V1, V2, V4, V6, M2, M7, M8) was also tested in cytokine-treated cells. TRPA1 was expressed in fetal IVD cells, 20% of degenerated IVDs, but not in healthy mature IVDs. TRPA1 expression was not detectable in untreated cells and it increased upon cytokine treatment, while TRPV1 was expressed and concomitantly reduced. In inflamed IVD cells, 10 µM AITC activated calcium flux, induced gene expression of IL-8, and reduced disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) and collagen 1A1, possibly via upregulated TRPA1. TRPA1 KO in mice was associated with signs of degeneration in the nucleus pulposus and the vertebral growth plate, whereas TRPV1 KO did not show profound changes. Cytokine treatment also affected the gene expression of TRPV2 (increase), TRPV4 (increase), and TRPC6 (decrease). TRPA1 might be expressed in developing IVD, downregulated during its maturation, and upregulated again in degenerative disc disease, participating in matrix homeostasis. However, follow-up studies with larger sample sizes are needed to fully elucidate the role of TRPA1 and other TRP channels in degenerative disc disease.

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

“…Moreover, cartilage-specific TRPV4 knockout in mice reduced age-related osteoarthritis [22]. The exploration of TRP channels in the IVD is at its infancy, with only few published studies [23][24][25][26][27]. Most of the known TRP channel genes are expressed in the IVD [26].…”

“…Interestingly, TRPV4 has been associated with reduced osmolarity and pro-inflammatory cytokines in bovine nucleus pulposus cells [23]. Additionally, treatment with IL1β increases TRPV4 gene expression in human IVD cells, thus revealing a potential inflammatory role of the channel in IVDs [27]. Nevertheless, the roles of TRPV4 in mechanosensing and mechanotransduction in the IVD remain to be established.…”

Advanced Strategies for the Regeneration of Lumbar Disc Annulus Fibrosus

“…A decrease in the AF cell density, as frequently observed in degenerated IVDs, alters the balance of anabolism:catabolism ratio in the extracellular matrix (ECM), changing the AF mechanical, biological, and structural properties [ 69 , 70 , 71 ]. Therefore, regenerative strategies aim to repopulate a degenerated IVD with healthy cells (cell therapy) or stimulating the existing cells to regulate the ECM reaching an enhanced composition, quantity, and structure (biomolecule and gene therapy) [ 81 , 82 , 83 , 84 , 85 ]. ECM remodeling is crucial to control the action of biochemical mediators such as cytokines (interleukins), growth factors, and enzymes (metalloproteinases and heparanase) to ultimately stop or reverse the IVD degeneration process [ 86 , 87 , 88 , 89 ].…”

TRPV4 and TRPM8 as putative targets for chronic low back pain alleviation

“…To understand the players involved in peripheral pain hypersensitivity, the altered expression of selected membrane receptors was investigated in specimens from patients of different ages and genders, with confirmed diagnosis of CLBP. TRPV1-4, TRPA1, and TRPM8 were selected because of their expression in peripheral sensory neurons as molecular nociceptors, actively transducing thermal, chemical, and mechanical stimuli [13,24], thus being involved in CLBP. For example, TRPV4 acts as a sensor of mechanical or osmotic signals and it is present not only in the nervous system but also in several musculoskeletal tissues, including cartilage, bone, and synovium.…”

“…Also, TRPV1 is known to be stimulated by several inflammatory neuropeptides and signaling molecules [14] and overexpressed in osteoarthritis [38,39]; thus, it is reasonable to expect a similar pattern of expression in specimens retrieved from patients affected by CLBP. Moreover, in inflammatory tissue, it is often co-expressed with TRPA1 [5] that can be equally stimulated by an array of molecules present in inflammation and in acute mechanical hypersensitivity [2,13,41]. Finally, many papers reported that also TRPM8 is expressed on both Aδ and C fiber and overexpressed in pain conditions, where it plays a role in amplifying pain sensation after injury, particularly in models of neuropathic pain.…”