Induction of nerve growth factor expression and release by mechanical and inflammatory stimuli in chondrocytes: possible involvement in osteoarthritis pain

Abstract

IntroductionNerve growth factor (NGF) level is increased in osteoarthritis (OA) joints and is involved in pain associated with OA. Stimuli responsible for NGF stimulation in chondrocytes are unknown. We investigated whether mechanical stress and proinflammatory cytokines may influence NGF synthesis by chondrocytes.MethodsPrimary cultures of human OA chondrocytes, newborn mouse articular chondrocytes or cartilage explants were stimulated by increasing amounts of IL-1β, prostaglandin E2 (PGE2), visfatin/nicotinamide phosphoribosyltransferase (NAMPT) or by cyclic mechanical compression (0.5 Hz, 1 MPa). Before stimulation, chondrocytes were pretreated with indomethacin, Apo866, a specific inhibitor of NAMPT enzymatic activity, or transfected by siRNA targeting visfatin/NAMPT. mRNA NGF levels were assessed by real-time quantitative PCR and NGF released into media was determined by ELISA.ResultsUnstimulated human and mouse articular chondrocytes expressed low levels of NGF (19.2 ± 8.7 pg/mL, 13.5 ± 1.0 pg/mL and 4.4 ± 0.8 pg/mL/mg tissue for human and mouse articular chondrocytes and costal explants, respectively). Mechanical stress induced NGF release in conditioned media. When stimulated by IL-1β or visfatin/NAMPT, a proinflammatory adipokine produced by chondocytes in response to IL-1β, a dose-dependent increase in NGF mRNA expression and NGF release in both human and mouse chondrocyte conditioned media was observed. Visfatin/NAMPT is also an intracellular enzyme acting as the rate-limiting enzyme of the generation of NAD. The expression of NGF induced by visfatin/NAMPT was inhibited by Apo866, whereas IL-1β-mediated NGF expression was not modified by siRNA targeting visfatin/NAMPT. Interestingly, PGE2, which is produced by chondrocytes in response to IL-1β and visfatin/NAMPT, did not stimulate NGF production. Consistently, indomethacin, a cyclooxygenase inhibitor, did not counteract IL-1β-induced NGF production.ConclusionsThese results show that mechanical stress, IL-1β and extracellular visfatin/NAMPT, all stimulated the expression and release of NGF by chondrocytes and thus suggest that the overexpression of visfatin/NAMPT and IL-1β in the OA joint and the increased mechanical loading of cartilage may mediate OA pain via the stimulation of NGF expression and release by chondrocytes.

The role of transient receptor potential channels in joint diseases

“…The same cells treated with IL-1β show an impaired regulatory volume decrease in hypotonic conditions, being unable to reduce their size to counteract hypotonic swelling. Interestingly, this IL-1β-induced effect is eliminated upon TRPV4 activation, indicating that TRPV4 may be involved in modulating inflammation in response to osmotic stress (Phan et al, 2009). In NP cells, reduced osmolarity activates the expression of TRPV4 and at the same time induces TRPV4-mediated Ca 2+ signalling and gene expression of pro-inflammatory cytokines.…”

“…In porcine articular chondrocytes, TRPV4-mediated Ca 2+ signalling regulates osmolarity-induced volume change and subsequent release of prostaglandin E2 (PGE2), a mediator of hyperalgesia (Phan et al, 2009). The same cells treated with IL-1β show an impaired regulatory volume decrease in hypotonic conditions, being unable to reduce their size to counteract hypotonic swelling.…”

“…TRPV4 could also be involved in mediating thermal hyperalgesia and inflammatory swelling in inflamed joints (Keeble et al, 2005). Apart from hyperalgesia, TRPV4 regulates transduction of mechanical and osmotic signals activated by load and swelling in chondrocytes and IVD cells, as outlined in detail below (Guler et al, 2002;Kohler and Hoyer, 2007;Phan et al, 2009;Strotmann et al, 2000b;Todaka et al, 2004).…”

“…Interleukin-1 beta (IL-1β) and tumour necrosis factor alpha (TNF-α) contribute to disease progression and pain, by acting directly not only as nociceptive triggers, but also by inducing generation of other potentially nociceptive molecules, including nitric oxide and prostaglandin E (Liu et al, 2016;. IL-1β and TNF-α are secreted by cells of the immune system, such as macrophages, and by a variety of other cells types, including chondrocytes, annulus fibrosus (AF) and nucleus pulposus (NP) cells, regulating host responses to stress, inflammation, infection or trauma (Berenbaum, 2013;Johnson et al, 2015;Oda et al, 2004;Vo et al, 2016). IL-1β and TNF-α are known to activate nuclear factor κB (NF-κB), c-Jun N-terminal protein kinase (JNK) and/ or p38 mitogen-activated protein kinase (MAPK) (Hoyland et al, 2008;McNulty et al, 2009;Vo et al, 2013;Wang et al, 2015;Wilusz et al, 2008).…”

“…The presence and severity of pain in OA and DDD is associated with cellular decline, ECM degradation and activity of inflammatory cytokines (Aoki et al, 2014;Grassel, 2014;Halliday et al, 1998;Pecchi et al, 2014). It has www.ecmjournal.org · impaired responses to noxious cold and oxidative stress · reduced nociception hypersensitivity in arthritic joints · suppressed inflammatory and fibrogenic reaction in cornea after chemical injury · decreased basal levels of pro-inflammatory mediators Bessac et al, 2008;Horvath et al, 2016;Karashima et al, 2009;Kun et al, 2014;Kwan et al, 2006;Okada et al, 2014 …”