Reduced tissue osmolarity increases TRPV4 expression and pro-inflammatory cytokines in intervertebral disc cells

Abstract

The mechanical behaviour and cellular metabolism of intervertebral discs (IVDs) and articular cartilage are strongly influenced by their proteoglycan content and associated osmotic properties. This osmotic environment is a biophysical signal that changes with disease and may contribute to the elevated matrix breakdown and altered biologic response to loading observed in IVD degeneration and osteoarthritis. This study tested the hypothesis that changes in osmo-sensation by the transient receptor potential vallinoid-4 (TRPV4) ion channel occur with disease and contribute to the inflammatory environment found during degeneration. Immunohistochemistry on bovine IVDs from an inflammatory organ culture model were used to investigate if TRPV4 is expressed in the IVD and how expression changes with degeneration. Western blot, live-cell calcium imaging, and qRT-PCR were used to investigate whether osmolarity changes or tumour necrosis factor α (TNFα) regulate TRPV4 expression, and how altered TRPV4 expression influences calcium signalling and pro-inflammatory cytokine expression. TRPV4 expression correlated with TNFα expression, and was increased when cultured in reduced medium osmolarity and unaltered with TNFα-stimulation. Increased TRPV4 expression increased the calcium flux following TRPV4 activation and increased interleukin-1β (IL-1β) and IL-6 gene expression in IVD cells. TRPV4 expression was qualitatively elevated in regions of aggrecan depletion in degenerated human IVDs. Collectively, results suggest that reduced tissue osmolarity, likely following proteoglycan degradation, can increase TRPV4 signalling and enhance pro-inflammatory cytokine production, suggesting changes in TRPV4 mediated osmo-sensation may contribute to the progressive matrix breakdown in disease.

Fig. 1

TRPV4 is expressed and functional in the IVD. (a) Immunohistochemistry for TRPV4 in the nucleus pulposus (NP) and annulus fibrosus (AF) regions of bovine caudal IVDs. Positive immunostaining appears brown. (b) Calcium traces of bovine NP and AF cells in response to the TRPV4 specific agonist GSK101 confirming the presence and functionality of the TRPV4 ion channels. Scale bar = 100 μm.

Fig. 2

TRPV4 is correlated with TNFα expression in an IVD organ culture model of inflammatory degeneration.( a) Schematic of the experimental set-up of the bovine whole IVD organ culture model of degeneration involving TNFα stimulation. (b) Schematic of the transverse cross-section of the IVD demonstrating the location of tissue used for immunohistochemistry and immunofluorescence. c) Immunohistochemical and immunofluorescence staining for TNFα and TRPV4, respectively, in the nucleus pulposus region of the control and TNFα groups. The images in each row are from the same tissue but a different area within each region; scale bar = 50 μm. Staining showed greater TNFα and TRPV4 expression in the TNFα-treated group.( correlation between d)There was a significant corrleation between TNFα and TRPV4 positive cells demonstrating a relationship between TNFα and TRPV4 expression in the IVD. Rabbit control IgG antibody used as a negative control.

Fig. 3

Hypo-osmotic environment but not TNFα increased TRPV4 expression in NP cells. (a) Western blot for TRPV4 in bovine NP cell culture experiments demonstrated no change in TRPV4 expression after 24 h of exposure to varying doses of TNFα. TRPV4 protein expression was increased when cultured for (b) 24 h and (c) 72 h in a reduced media osmolarity compared to the physiologic osmotic environment within the IVD (~434 mOsm/kgH₂O). Together, results suggest that increased TRPV4 expression observed in the organ culture model was due to reduced tissue osmolarity associated with TNFα-induced aggrecan degradation and not a direct result of TNFα stimulation. Bar = p < 0.05.

Fig. 4

Elevated TRPV4 expression increased TRPV4 signalling in NP cells. Representative traces and quantification of the calcium response of bovine NP cells treated with or without the TRPV4 agonist (GSK101) after 24 h of pre-culture in (a) hypo-osmolar (334 mOsm/kgH₂O) culture medium or (b) physiologic (434 mOsm/kgH₂O) culture medium. (c) Comparison of the calcium responses under different osmolarities for both GSK treated groups. Hypo-osmolarity conditions increased the magnitude of calcium flux in cells cultured in a lower osmolarity medium (334 mOsm/kgH₂O). (d) Conceptual model depicting a mechanism for greater TRPV4-mediated calcium flux in hypo-osmotic conditions. Bar = p < 0.05.

Fig. 5

TRPV4 signalling mediates IVD cell expression of pro-inflammatory cytokines. Gene expression for the pro-inflammatory cytokines ( a) interleukin-1β and (b) interleukin-6 were increased after 24 h under hypo-osmotic conditions (334 mOsm/kgH₂O). TRPV4 inhibition using the TRPV4 antagonist (GSK205) decreased the pro- inflammatory gene expression indicating that TRPV4 signalling was an important regulator of pro-inflammatory gene expression in the IVD. Bar = p < 0.05 and ǂ = p < 0.1.

Fig. 6

TRPV4 expression in human IVD tissue. Immunohistochemistry for TRPV4 in nucleus pulposus and annulus fibrosus regions of (a) a healthy IVD from a 44 year old male and (b) a degenerated IVD from a 93 year old male; scale bars = 50μm . The images in the top row were taken at low magnification and the bottom row was taken at high magnification under polarised light. Picrosirius red and alcian blue staining of the same (c) healthy 44 year old male IVD and (d) degenerated 93 year old male demonstrating reduced alcian blue staining intensity, indicative of proteoglycan loss, in the degenerated IVD; scale bars = 5 mm. Whole IVD images are modified from (Walter et al., 2014). Results suggest that the changes in TRPV4 expression seen in organ culture and osmolarity experiments are likely to be relevant to the changes that occur in human IVD degeneration.

Fig. 7

Proposed hypothetical mechanism for inflammation-induced aggrecan degradation leading to TRPV4-mediated pro-inflammatory cytokine production. Schematic demonstrating the potential feedback loop where (a) a healthy nucleus pulposus tissue is exposed to pro-inflammatory cytokines that induce ( b) glycosaminoglycan (GAG) degradation and loss which reduces the tissue osmolarity within the NP. (c) Reduced osmolarity stimulates increased TRPV4 expression that (d) mediated expression of additional pro-inflammatory cytokines thereby leading to a potential feedback loop.