Abstract
Introduction
Intervertebral disc degeneration is a leading cause of chronic low back pain (LBP), but how degeneration contributes to LBP is poorly understood. Nerve growth factor (NGF) levels increase during disc degeneration and evidence suggests NGF promotes disc innervation, neuronal sensitization and low back pain, making NGF a possible therapeutic target. However, current anti-NGF therapeutics have limited efficacy and safety concerns. Mechanisms leading to increased NGF in the disc are poorly understood and the molecular signaling mechanisms regulating NGF in inflammatory mediator rich environments, such as a degenerating disc, are unknown. During degeneration, proteoglycans and extra-cellular matrix (ECM) proteins are degraded and fragmented. ECM fragments can act as endogenous danger signal ligands for toll-like receptors (TLR). TLR activation induces cytokine and chemokine expression, and could thus regulate expression of inflammatory mediators, such as IL-1β or TNFα, during disc degeneration. NGF is often increased in environments with elevated levels of inflammatory mediators. Therefore, we hypothesized that TLR activation in the disc induces NGF expression.
Materials and Methods
Non-degenerate human intervertebral discs from organ donors without a history of low back pain were collected for cell isolation. NP and AF tissues were separated and enzymatically digested. Cells were treated with IL-1β (control), peptidoglycan (PGN, TLR2 agonist) and lipopolysaccharide (LPS, TLR4 agonist). Neutralizing antibodies against TLR2 were used to prevent TLR2 activation. Activated cell-signaling pathways following IL-1β and PGN treatment were evaluated by western blot and immunofluorescence. Small molecule inhibitors blocking p38 MAPK (SB203580) and NF-κB (BMS-345541) activity were added to cell cultures in combination with IL-1β and PGN. NGF gene expression was evaluated by qRT-PCR after 6, 12 and 24 hours, and NGF protein levels were examined by western blot and ELISA after 48 hours.
Results
TLR 2 activation significantly increased NGF gene expression in NP and AF cells following 6, 12 and 24 hours of treatment, while TLR 4 activation had little effect on NGF expression. TLR 2 activation significantly increased NGF protein secretion after 48 hours in NP and AF cells while TLR 4 activation did not increase NGF protein levels. TLR 2 neutralization with antibodies showed that TLR 2 is required for PGN induced NGF expression. TLR 2 activation of the p38 MAPK, ERK1/2, JNK and NF-κB signaling pathways was analyzed using phosphorylation specific antibodies. TLR activation increased p38 MAPK, ERK1/2 and p65 (NF- κB pathway) phosphorylation in disc cells compared with untreated cells. Furthermore, immunofluorescence found that TLR activation induces p65 translation to the nucleus, indicative of NF-κB signaling. Inhibiting NF-κB signaling decreased TLR 2 induced NGF expression.
Conclusion
This study found that TLR 2 activation directly regulates NGF expression via NF-κB signaling in human intervertebral disc cells. These findings represent a novel regulatory mechanism of NGF in the IVD. In the central nervous system NF-κB distinct signaling pathways regulate NGF. Therefore, these finding may provide therapeutic strategies to target NGF and low back pain without affecting the central nervous system.