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.

Keywords: TRP channels; TRPA1; TRPC6; TRPV1; TRPV2; TRPV4; aggrecanases; collagen; low back pain; pro-inflammatory cytokines.

Figure 1

Gene expression of TRPA1 and TRPV1 in IVD cells treated with 5 and 10 ng/mL interleukin-1 beta (IL-1β) or tumor necrosis factor alpha (TNF-α). Gene expression of (A) TRPA1 and (B) TRPV1 in two-dimensional (2D) culture (Graph shows 2^−ddCt (mean ± SD, n = 5). Gene expression of (C) TRPA1 and (D) TRPV1 in IVD cells cultured in three-dimensional (3D) alginate beads and treated with IL-1β for 15 days. Graph shows 2^−ddCt (mean ± SD, n = 4–10). Asterisks indicate statistical significance (* p < 0.05, ** p < 0.01, Kruskal–Wallis test and Dunn’s multiple comparison test). (E) Protein expression of TRPA1 in IVD cells treated with IL-1β and untreated (DAPI = blue, TRPA1 = green). Negative control images show cells without secondary antibody. Scale bar is 50 μm.

Figure 2

Gene expression of TRP channels in IVD cells treated with 5 and 10 ng/mL IL-1β or TNF-α. Gene expression of (A) TRPC1, (B) TRPC3, (C) TRPC6, (D) TRPV2, (E) TRPV4, (F) TRPV6, and (G) TRPM7. Graphs show 2^−ddCt (mean ± SD, n = 5). Asterisks indicate statistical significance (* p < 0.05, ** p < 0.01, Kruskal–Wallis test and Dunn’s multiple comparison test).

Figure 3

Calcium flux in IVD cells untreated (control) and treated with 100 μM Allyl isothiocyanate (AITC) with and without 10 ng/mL IL-1β or 10 ng/mL TNF-α. Graph shows calcium flux as 340/380 signal ratio (mean ± SD, n = 3). Asterisks indicate statistical significance (* p < 0.05, Kruskal–Wallis test and Dunn’s multiple comparison test). The arrow indicates AITC application.

Figure 4

The effects of TRPA1 agonist allyl isothiocyanate (AITC) on the gene expression of inflammation markers and extracellular matrix (ECM) molecules in IVD cells without cytokine pre-treatment. Gene expression of (A) interleukin-6 (IL-6), (B) nerve growth factor (NGF), (C) interleukin 8 (IL-8), (D) cyclooxygenase-2 (COX-2), (E) ADAMTS4, (F) ADAMTS5, (G) tissue inhibitor of matrix metalloproteinase 1 (TIMP1), (H) matrix metalloproteinase 1 (MMP1), (I) matrix metalloproteinase 3 (MMP3), (J) tissue inhibitor of matrix metalloproteinase 2 (TIMP2), (K) COL1A1, (L) COL2A1, and (M) Aggrecan in IVD cells treated with 3 and 10 μM AITC. Graphs show gene expression and protein release calculated relative control (2^−ddCt, mean ± SD, n = 3). Asterisks indicate statistical significance (* p < 0.05, Kruskal–Wallis test and Dunn’s multiple comparison test).

Figure 5

The effects of TRPA1 agonist allyl isothiocyanate (AITC) on the expression of inflammation markers and ECM molecules in IL-1β-treated cells. Gene expression of (A) IL-6 and (D) IL-8 in IVD cells treated with 10 ng/mL IL-1β ± 3 and 10 μM AITC. Protein release of (B) IL-6 and (E) IL-8 in IVD cells that were treated with 10 ng/mL IL-1β ± 3 and 10 μM AITC. Gene expression of (C) NGF, (F) COX-2, (G) ADAMTS4, (H) ADAMTS5, (J) MMP1, (K) MMP3, (I) TIMP1 and (L) TIMP2, (M) COL1A1, (N) COL2A1, and (O) Aggrecan in IVD cells that were treated with 10 ng/mL IL-1β ± 3 or 10 μM AITC. Graphs show gene expression and protein release calculated relative to IL-1β treatment (mean ± SD, n = 3–4). Asterisks indicate statistical significance (* p < 0.05, Kruskal–Wallis test and Dunn’s multiple comparison test).

Figure 6

The effects of TRPA1 agonist allyl isothiocyanate (AITC) on gene expression of inflammation markers and ECM molecules in TNF-α-treated cells. Gene expression of (A) IL-6 and (D) IL-8 in IVD cells treated with 10 ng/mL TNF-α ± 3 or 10 μM AITC. Protein release of (B) IL-6 and (E) IL-8 in IVD cells treated with 10 ng/mL TNF-α ± 3 or 10 μM AITC. Gene expression of (C) NGF, (F) COX-2, (G) ADAMTS4, (H) ADAMTS5, (I) TIMP1, (J) MMP1, (K) MMP3, and (L) TIMP2, (M) COL1A1, (N) COL2A1, and (O) Aggrecan in IVD cells treated with 10 ng/mL TNF-α ± 3 and 10 μM AITC. Graphs show gene expression and protein release calculated relative to TNF-α treatment (mean ± SD, n = 3-4). Asterisks indicate statistical significance (* p < 0.05, Kruskal–Wallis test and Dunn’s multiple comparison test).

Figure 7

FAST staining of IVDs of TRPA1 wild-type (WT) and knock-out (KO) mice. The tail motion segments of TRPA1 young WT (A,E,I,M), TRPA1 young KO (B,F,J,N), TRPA1 old WT (C,G,K,O), and TRPA1 old KO (D,H,L,P) mice. The nucleus pulposus: NP (E–H); inner annulus fibrosus: IAF and outer annulus fibrosus: OAF (I–L); vertebral growth plate: GP (M–P) are also shown in higher magnification. Asterisks (*) indicate depletion of glycosaminoglycan deposition in IVD. Scale bars indicate 500 µm in upper panel (A–D), but 50 µm in lower panels (E–P).

Figure 8

FAST staining of IVDs of TRPA1 WT and TRPV1 KO mice. The tail motion segments of C57 BL/6 young WT (A,D,G,J), TRPV1 young KO (B,E,H,K), and TRPV1 old KO (C,F,I,L) mice. The nucleus pulposus: NP (D–F); inner annulus fibrosus: IAF and outer annulus fibrosus: OAF (G–I); and, vertebral growth plate: GP (J–L) are also shown in higher magnification. Scale bars indicate 500 µm in upper panel (A–C), but 50 µm in lower panels (D–L).