Abstract
Nerve growth factor (NGF) contributes to the development of chronic pain associated with degenerative connective tissue pathologies, such as intervertebral disc degeneration and osteoarthritis. However, surprisingly little is known about the regulation of NGF in these conditions. Toll-like receptors (TLR) are pattern recognition receptors classically associated with innate immunity but more recently were found to be activated by endogenous alarmins such as fragmented extracellular matrix proteins found in degenerating discs or cartilage. In this study we investigated if TLR activation regulates NGF and which signaling mechanisms control this response in intervertebral discs. TLR2 agonists, TLR4 agonists, or IL-1β (control) treatment increased NGF, brain-derived neurotrophic factor (BDNF), and IL-1β gene expression in human disc cells isolated from healthy, pain-free organ donors. However, only TLR2 activation or IL-1β treatment increased NGF protein secretion. TLR2 activation increased p38, ERK1/2, and p65 activity and increased p65 translocation to the cell nucleus. JNK activity was not affected by TLR2 activation. Inhibition of NF-κB, and to a lesser extent p38, but not ERK1/2 activity, blocked TLR2-driven NGF up-regulation at both the transcript and protein levels. These results provide a novel mechanism of NGF regulation in the intervertebral disc and potentially other pathogenic connective tissues. TLR2 and NF-κB signaling are known to increase cytokines and proteases, which accelerate matrix degradation. Therefore, TLR2 or NF-κB inhibition may both attenuate chronic pain and slow the degenerative progress in vivo.
Highlights
— — NP only — — — AF only ever, little is known about their regulation in pathologies such as painful disc degeneration
To determine if TLR activation induces neurotrophin expression, NP and AF cells from healthy human discs were treated with the TLR agonists PGN (TLR2 agonist) and LPS (TLR4 agonist) for 6, 12, 24, and 48 h
In some experiments baseline IL-1 expression was undetectable in untreated cells, and IL-1 expression could not be normalized to baseline levels, rendering 2Ϫ⌬⌬Ct values impossible to calculate for those experiments
Summary
— — NP only — — — AF only ever, little is known about their regulation in pathologies such as painful disc degeneration. A possible mechanism that has not been explored is that TLR signaling directly increases neurotrophin expression. In vivo injection of fibronectin fragments into rabbit discs induces degenerative changes [37], and exposure of NP cells to fibronectin fragments decreases proteoglycan synthesis and increases proteoglycan degradation [38] These studies did not investigate the mechanism responsible for these changes. The potential role of TLR signaling in the early stages of disc degeneration led us to hypothesize that TLR activation induces neurotrophin expression either directly or as a secondary effect via cytokines. In this study we investigated TLR agonist- and cytokine-induced neurotrophin induction in human disc cells and determined that TLRs directly regulate neurotrophin expression. This study has identified novel mechanisms contributing to NGF regulation, offering alternative strategies to target NGF in disc degeneration to treat chronic pain
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