Abstract
Nerve growth factor (NGF) expression and activity is important in chronic lower back pain but may also act as a pro-catabolic factor in the pathogenesis of intervertebral disc (IVD) degeneration. Lipocalin 2 (Lcn2) expression in IVD was upregulated by NGF stimulation in our previous study. The current study was undertaken to identify potential mechanisms of the latter effect including potential interactions between Lcn2 and matrix metalloproteinase 9 (MMP9). Rat annulus fibrosus (AF) cells were stimulated by NGF and subjected to microarray analysis, subsequent real-time PCR, western immunoblotting, and immunofluorescence. Cells were treated with NGF in the absence or presence of the NGF inhibitor Ro 08-2750. Zymography and functional MMP9 assays were used to determine MMP9 activity, whilst the dimethyl-methylene blue assay was used to quantify the release of glycosaminoglycans (GAGs) reflecting catabolic effects following NGF treatment. Immunoprecipitation with immunoblotting was used to identify interactions between MMP9 and Lcn2. Increased expression of Lcn2 gene and protein following NGF stimulation was confirmed by microarray analysis, real-time PCR, western blot and immunofluorescence. Zymography showed that NGF enhanced 125-kDa gelatinase activity, identified as a Lcn2/MMP9 complex by immunoprecipitation and immunoblotting. Functional assays showed increased MMP9 activity and GAG release in the presence of NGF. The effects of NGF were neutralized by the presence of Ro 08-2750. NGF upregulates Lcn2 expression and increases MMP9 activity in AF cells; processes which are likely to potentiate degeneration of AF tissue in vivo. Anti-NGF treatment may have benefit for management of pain relief and slowing down progression of AF tissue degeneration.
Highlights
Chronic low back pain (LBP) is a relatively common condition with a number of possible etiologies including intervertebral disc (IVD) degeneration, spondylolisthesis, diskitis or spondylitis [1, 2]
After washing six times with Tris-buffered saline/Tween (TBST), blots were incubated with the HRP-labeled secondary antibody for 1 h at room temperature
Equal amounts of 500 lg protein were incubated with anti-matrix metalloproteinase 9 (MMP9) antibody (1 mg/mL, 1 lL) overnight and immunoprecipitated with a Protein G Immunoprecipitation Kit (Sigma)
Summary
Chronic low back pain (LBP) is a relatively common condition with a number of possible etiologies including intervertebral disc (IVD) degeneration, spondylolisthesis, diskitis or spondylitis [1, 2]. During IVD degeneration, nerve fiber ingrowth into the nucleus pulposus (NP) is seen and is believed to possibly contribute to chronic LBP [5]. Consequent therapies targeting neuronal transmission pathways are increasingly being thought to be useful for alleviating pain [6, 7]. It is becoming clear, that neurotrophins such as NGF and brain-derived neurotrophic factor (BDNF) have a wide range of activities other than neurotransmission that include roles in tissue development, repair, modulation of inflammatory responses and bone and cartilage metabolism [8,9,10,11]
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