Object: Increased neurotrophin activity in degenerative intervertebral discs (IVDs) is one potential cause of chronic low-back pain (LBP). The aim of the study was to assess if nerve growth factor (NGF) might alter gene expression of IVD cells and contribute to disc degeneration by enhancing expression or activity of factors that cause breakdown of IVD matrix.
Methods: Rat-tail IVD cells were stimulated by NGF and subjected to microarray analysis. Real-time polymerase chain reaction, Western blotting, and immunocytochemistry of rat and human IVD cells and tissues treated with NGF in vitro in the absence or presence of the NGF inhibitor Ro 08-2750 were used to confirm findings of the microarray studies. Phosphorylation of mitogen-activated protein kinase (MAPK) was used to identify cell signaling pathways involved in NGF stimulation in the absence or presence of Ro 08-2750.
Results: Microarray analysis demonstrated increased expression of chitinase 3-like 1 (Chi3l1), lipocalin 2 (Lcn2), and matrix metalloproteinase-3 (Mmp3) following NGF stimulation of rat IVD cells in vitro. Increased gene expression was confirmed by real-time polymerase chain reaction with a relative increase in the Mmp/Timp ratio. Increased expression of Chi3l1, Lcn2, and Mmp3 following NGF stimulation was also demonstrated in rat cells and human tissue in vitro. Effects of NGF on protein expression were blocked by an NGF inhibitor and appear to function through the extracellular-regulation kinase 1/2 (ERK1/2) MAPK pathway.
Conclusions: Nerve growth factor has potential effects on matrix turnover activity and influences the catabolic/anabolic balance of IVD cells in an adverse way that may potentiate IVD degeneration. Anti-NGF treatment might be beneficial to ameliorate progressive tissue breakdown in IVD degeneration and may lead to pain relief.
Keywords: AF = annulus fibrosus; BSA = bovine serum albumin; Chi3l1 = chitinase 3-like 1; EPK1/2 = extracellular-regulation kinase 1/2; HRP = horseradish peroxidase; IL-1β = interleukin-1β; IVD = intervertebral disc; LBP = low-back pain; Lcn2 = lipocalin 2; MAPK = mitogen-activated protein kinase; MMP3; Mmp3 = matrix metalloproteinase–3; NF-κB = neurotrophic factor κB; NGF = nerve growth factor; NGFR = nerve growth factor receptor; NP = nucleus pulposus; PBS = phosphate-buffered saline; TBST = Tris-buffered saline/Tween; VE = vertebral endplate; chitinase 3-like 1; intervertebral disc degeneration; lipocalin 2; nerve growth factor; p75NTR = p75 neurotrophin receptor.