J Neurosurg Spine 21:653–661, 2014 ©AANS, 2014

Nerve promotes expression of novel genes in intervertebral disc cells that regulate tissue degradation

Laboratory investigation

Ting-Hsien Kao, M.D.,1,3,4 Yi-Jen Peng, M.D., Ph.D.,2 Hsi-Kai Tsou, M.D., Ph.D.,3,5 Donald M. Salter, M.D.,6 and Herng-Sheng Lee, M.D., Ph.D.1,2 1Graduate Institute of Medical Science, National Defense Medical Center, and 2Department of Pathology, Tri-Service General Hospital and National Defense Medical Center, Taipei; 3Department of Neurosurgery, Taichung Veterans General Hospital, Taichung; Departments of 4Acupressure Technology and 5Early Childhood Care and Education, Jen-Teh Junior College of Medicine, Nursing and Management, Miaoli County, Taiwan, Republic of China; and 6Osteoarticular Research Group, Molecular Medicine Center, Institute of Genetics and Molecular Medicine, University of Edinburgh, United Kingdom

Object. Increased 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 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 poly- merase 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 kinase (MAPK) was used to identify 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 expres- sion 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 tis- sue 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. (http://thejns.org/doi/abs/10.3171/2014.6.SPINE13756)

Key Words • intervertebral disc degeneration • nerve growth factor • chitinase 3-like 1 • lipocalin 2 • MMP3

hronic low-back pain (LBP) has become a major discs (IVDs), ligaments, muscles, sacroiliac joints, and cause of disability in industrialized societies, with lumbar facet joint degeneration.12 In patients with a dis- consequent economic and health care costs.1 LBP cogenic component to LBP, IVD degeneration is thought Ccan originate from several sources, such as intervertebral to be the initial step and plays a critical role.31 Following IVD degeneration, the biomechanical status of the ver- Abbreviations used in this paper: AF = annulus fibrosus; BSA tebral column changes, and the likelihood of facet joint = bovine serum albumin; Chi3l1 = chitinase 3-like 1; EPK1/2 = degeneration, spondylosis, spondylolisthesis, and spinal extracellular-regulation kinase 1/2; HRP = horseradish peroxidase; stenosis increases. At present, the management of LBP IL-1b = -1b; IVD = intervertebral disc; LBP = low-back consists of a variety of conservative and invasive thera- pain; Lcn2 = lipocalin 2; MAPK = mitogen-activated protein kinase; pies that are aimed at symptomatic relief. Unfortunately, Mmp3 = matrix metalloproteinase–3; NF-kB = neurotrophic fac- k many of the treatments used currently are not obviously tor B; NGF = nerve growth factor; NGFR = nerve growth factor 7 receptor; NP = nucleus pulposus; PBS = phosphate-buffered saline; effective. p75NTR = p75 neurotrophin receptor; TBST = Tris-buffered The IVD consists of a collagenous outer ring, the an- saline/Tween; VE = vertebral endplate. nulus fibrosus (AF);2 a gelatinous inner core, the nucleus

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Unauthenticated | Downloaded 10/07/21 04:34 PM UTC T. H. Kao et al. pulposus (NP); and the adjacent vertebral endplate (VE).5 sultant cell suspension was centrifuged at 1,000 rpm for The healthy NP is avascular, and its nutritional supply de- 10 minutes; the supernatant was discarded, and the pel- pends on diffusion via the AF and VE. In a normal disc let was resuspended in PBS. After further centrifugation the NP is also devoid of nerve fibers, whereas the outer for 10 minutes, cells were resuspended and seeded in 10 AF and VE contain nerve fibers derived from branches ml DMEM/Nutrient Mixture F-12 Ham medium (Gibco) of sympathetic trunk and sinuvertebral nerves.22 During containing 10% fetal bovine serum (Gibco), 100 IU/ml IVD degeneration, increased nerve ingrowth is found in penicillin (Gibco), and 100 mg/ml streptomycin (Gibco). the NP and has been suggested as a potential contribu- The cells were then cultured in a humidified 5% CO2 in- tor to LBP.9,13,14 Nociceptive such as cal- cubator at 37°C until confluent. Cells between passages 3 citonin gene–related peptide and substance P, which are and 5 were used. present within the nerve fibers of the outer AF and , have been associated with discogenic pain Experimental Protocol 2,6,18 transmission. Therapies targeting neuronal transmis- Cells were seeded at a density of 2 × 105 cells/dish in sion pathways can reasonably be expected to be use- 4,5,11,42 60-mm dishes, and the culture medium was changed to ful and relieve pain. such as nerve 3% fetal bovine serum. The control group was incubated growth factor (NGF), brain-derived neurotrophic fac- with PBS, and the experimental group was incubated with tor, and neuropeptides (substance P) have a wide range NGF at a concentration of 100 ng/ml/day for a total of 5 of activities, including roles in tissue development37 and 26,45 3 days. For assay, cells were stimulated repair, modulation of inflammatory responses, and for 0, 6, 12, and 24 hours. bone and cartilage metabolism.20–24 We hypothesized that NGF, whose local production Tetrazolium Methylthiotetrazole Assay is increased in degenerated IVD,13 could modify gene expression of IVD cells and thereby influence tissue re- Cytotoxicity of NGF was assessed by the tetrazolium modeling and contribute to enhanced matrix breakdown. methylthiotetrazole assay. A total of 1 × 104 cells were Increased knowledge of the basic mechanisms involved seeded onto 96-well microtiter plates in 0.2 ml of growth in IVD degeneration and chronic LBP will allow devel- medium. In triplicate experiments, NGF (100 ng/ml) was opment of therapies that may delay or obviate surgical added to cell-seeded wells and incubated for 0, 1, 3, and 5 intervention and may improve postsurgical outcomes. days. Tetrazolium (100 ml; Sigma) was added to each well and incubated for 3 hours at 37°C, followed by the addi- tion of 150 ml dimethyl sulfoxide. The plate was shaken Methods for 10 minutes, and then optical density at 570 nm was Experimental Animals and Ethics Statement determined using a microplate reader (Dynatech Labo- ratories Inc.). Two-month-old skeletally mature male Sprague- Dawley rats (380–420 g) were obtained from the National Microarray Assays Applied Research Laboratories and National Laboratory Gene expression of rat-tail disc cells with or without Animal Center. All experimental procedures were ap- NGF stimulation for 5 days was analyzed. Briefly, fluo- proved by the Institutional Animal Care and Use Com- rescent-amplified RNA targets were hybridized to the Rat mittee of Taichung Veterans General Hospital. Human Whole Genome OneArray (Phalanx Biotech Group), and IVD tissue was obtained with full informed written con- the signals were scanned by an 4000 scanner (Mo- sent under approval from the ethics committee of Tai­ lecular Devices, LLC). The fluorescent intensity of each chung Veterans General Hospital from patients undergo- spot was analyzed by GenePix 4.1 software (Molecular ing discectomy. Devices). The signal intensity of each spot was corrected Reagents by subtracting background signal. Spots with a signal-to- noise ratio < 1 or from control probes were filtered out. Human and rat beta-nerve growth factor (NGF) were Spots that passed these criteria were normalized by the R purchased from R&D System Inc. Stock solution of NGF program (TIBOC Software Inc.). The fold change of gene (100 mg/ml) was prepared in phosphate-buffered saline expression was calculated by dividing the normalized sig- (PBS) containing 0.1% bovine serum albumin (BSA). nal intensities of genes in NGF-treated cells by those in The NGF inhibitor Ro 08-2750 was purchased from untreated cells. Genes with more than 2-fold or less than Tocris Bioscience; a 10-mM stock solution was prepared 2-fold changes were analyzed by Kyoto Encyclopedia of in dimethyl sulfoxide. Gene and Genomes pathways on the Tree Machine website, a respected Web-based and tree-based Isolation and Expansion of Rat Disc Cells data mining environment for gene sets. The gene Set Test Annulus fibrosus tissue of rat-tail IVDs was dissect- function implemented in the limma R package was used to ed carefully with minimal NP or other tissue contami- test significant Kyoto Encyclopedia of Gene and Genomes nation by an experienced spinal surgeon. The dissected pathways. The Web Gestalt tool was used to test significant tissue was then cut into small pieces, incubated with an- Gene Ontology terms. The microarray data comply with timicrobial solution for 4 hours, and then washed with MIAME (Minimum Information About a Microarray Ex- sterile PBS before with 3 mg/ml collagenase periment) guidelines, and the raw data have been deposited type H (Sigma-Aldrich Inc.) at 37°C for 6 hours. The re- in a MIAME-compliant database.

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Extraction of RNA and Real-Time Polymerase Chain with ice-cold PBS containing 100 mM Na3VO4 (Sigma) Reaction and lysed in situ with ice-cold lysis buffer at 4°C for 15 m Total RNA from cell cultures was extracted us- min. Lysis buffer contained 1% Igepal (Sigma), 100 M ing TRIzol RNA isolation reagents (Invitrogen). For Na3VO4, and protease inhibitor cocktail tablet (Roche Di- first-strand cDNA synthesis, 2 mg total RNA was used agnostics Corporation). Whole-cell lysates were collected in a single-round reverse-transcription reaction with the after centrifugation at 13,000 rpm for 15 min. Protein con- High-Capacity cDNA Reverse Transcription Kit (Applied centrations were determined by the Lowry method. Equal Biosystems). Complementary DNAs were amplified using amounts of protein were loaded onto 10% sodium dodecyl the Power SYBR Green PCR Master Mix (Applied Bio- sulfate–polyacrylamide gels and subsequently transferred to polyvinylidene fluoride membranes (EMD Millipore). systems) and a StepOne Real-Time PCR System (Applied ° Biosystems) under the following conditions: 95°C for 10 Membranes were blocked overnight at 4 C with 2% BSA minutes, 40 cycles of 95°C for 15 minutes, and 60°C for in TBST. After washing 3 times with TBST, blots were 60 seconds. Amplification of glyceraldehyde-3-phosphate incubated overnight with primary antibody (anti-Chi3l1, dehydrogenase (Gapdh) simultaneously served as an in- 1/1,000; anti-Lcn2, 1/1,000; anti-MMP3, 1/1,000) diluted ternal control and allowed normalization of the various in 2% BSA. After washing 6 times with TBST, blots were mRNA levels against the total mRNA content in the sam- then incubated with the HRP-labeled secondary antibody ples, which was used to calculate changes in gene expres- for 1 hour at room temperature. Membranes were rewashed sion by 2-DDCt. Table 1 lists the specific primers used. extensively, and antibody binding was detected using the Amersham Biosciences Enhanced Chemiluminescence Immunocytochemistry plus Western Blotting Detection System according to the manufacturer’s instructions. The membranes were scanned After incubation with NGF for 5 days, cells were and analyzed by densitometry (model LAS 3000; Fujifilm washed twice with ice-cold PBS and then fixed using 2 ml ° Corporation). expression assessed with the mouse of a 1:1 methanol/acetone mixture for 5 minutes at –20 C. monoclonal antibody tubulin Ab-4 (1/5,000; secondary an- For immunocytochemistry, cells were washed twice with tibody, 1/20,000) served as internal control. PBS before incubation with 1% BSA for 30 minutes at room temperature. The solution was removed, and cells Immunohistochemistry were incubated sequentially with primary antibody anti- Chi3l1 (1:100; AVIVA Systems Biology Corporation), anti- Annulus fibrosus tissue from 3 human IVDs was in- MMP3 (1:100; Epitomics), or anti-Lcn2 (1:100; Abcam plc) cubated in DMEM/F12 serum-free medium with either for 60 minutes, biotinylated secondary antibody (1:200) for 100 ng/ml NGF or 10 nM Ro 08-2750 for 12 hours. The 45 minutes, and horseradish peroxidase (HRP)–conjugat- tissues were embedded in OCT, and frozen sections cut ed streptavidin for 20 minutes. Following each incubation, and fixed. Sections were rinsed in PBS and incubated in cells were washed 3 times with Tris-buffered saline/Tween 0.3% H2O2 at room temperature for 10 min before incu- (TBST; 2.5 mM Tris/HCl, pH 7.6; 137 mM NaCl; 0.1% bation with 1% BSA for 30 minutes at room temperature. Tween 20). Diaminobenzidine (Invitrogen) was then added Sections were then incubated sequentially with anti- for 15 minutes before counterstaining with Mayer’s hema- Chi3l1 (1:100), anti-MMP3 (1:100), or anti-Lcn2 (1:100) toxylin and visualizing under light microscopy. for 60 minutes, biotinylated secondary antibody (1:200) for 45 minutes, and HRP-conjugated streptavidin for 20 Protein Extraction and Western Blotting minutes. Between each incubation, slides were washed 3 Following stimulation, cells were immediately washed times with TBST. Diaminobenzidine was then added for 15 minutes, and sections were then counterstained with TABLE 1: Oligonucleotide primers for rat real-time polymerase Mayer’s hematoxylin. chain reaction analysis Statistical Analysis Gene Accession No. Sequence All values were expressed as mean ± standard de- viation. Statistical evaluation of the quantification data of Gapdh NM_017008.4 5′-CCCATCACCATCTTCCAGGAG-3′ mRNA and protein expression levels was performed by 5′-GTTGTCATGGATGACCTTGGC-3′ Student t-tests. For the ratios of Timp1 to Mmp3, Lcn2 Chi3l1 NM_053560.1 5′-TGGGTGCACAGGGAATGGTCG-3′ to Mmp9, and Chi3l1 to Col1a1, Wilcoxon matched pairs 5′-CTTCAGCTGCCAGGCTTTGCG-3′ test was used, allowing for the nonsymmetrical distribu- Lcn2 NM_130741.1 5′-TCCATCCTCGTCAGGGGCCA-3′ tion data. The results were considered significant at a p 5′-AGTGTCGGCCACTTGCACATCG-3′ value < 0.05. Mmp3 NM_133523.2 5′-ACCCGCTGAGAGCAGTGCAGA-3′ ′ ′ 5 -ACAGCCACAGCAGGACTGGGA-3 Results Mmp9 NM_031055.1 5′-TGGCCTTTAGTGTCTCGCTGT-3′ 5′-CACACAGCTGGCAGAGGATT-3′ Effect of NGF on AF Cell Gene Expression Col1a1 NM_053304.1 5′-GACTGTCCCAACCCCCAAAA-3′ 5′-CTTGGGTCCCTCGACTCCTA-3′ Nerve growth factor had no effect on cell viability over the time course of the experiment at the concentra- Timp1 NM_053819.1 5′-TGCCCCAACCCACCCACAGA-3′ tion used, as assessed by a tetrazolium methylthiotetra- 5′-GCTGGGGTGTAGGCGAACCG-3′ zole assay (data not shown).

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Using purified RNA samples from the AF cells, we ent in human tissue, we collected AF tissue from 3 pa- found that 97 of the 24,358 transcripts (< 0.4%) covered tients undergoing discectomy (Pfirrmann Grade 2 and 3), by the Rat OneArray showed > 2-fold change after NGF stimulated these ex vivo with human NGF, and undertook stimulation. After the initial screen, 3 genes were chosen immunohistochemical analysis for CHI3L1, LCN2, and for further study because of their involvement in matrix MMP3. NGF-treated tissue showed increased expression turnover regulation: Chi3l1 (chitinase 3-like 1), Lcn2 (lipo- of CHI3L1, LCN2, and MMP3, which was inhibited by calin 2), and Mmp3 (matrix metalloproteinase 3) (Table 2). the presence of Ro 08-2750 (Fig. 4). Real-time polymerase chain reaction was performed and protein expression was analyzed in rat and human IVD cells focusing on these genes and related pathways Discussion (Fig. 1). Real-time polymerase chain reaction showed in- Discogenic back pain has been associated with IVD creased expression of Chi3l1, Lcn2, and Mmp3 follow- degenerative changes, including desiccation, space nar- ing NGF stimulation (Fig. 1A). Similarly, Western blot- rowing, and annular tears.25 Advanced changes such as ting showed increased expression of CHI3L1, LCN2, and Modic lesions have been associated with an increased MMP3 in NGF-stimulated cells (Fig. 1B), which was also incidence of LBP.25 The pathophysiology of disc degen- shown by immunocytochemistry (Fig. 1C). eration has been linked to an imbalance of catabolic and anabolic that leads to matrix degradation and Nerve Growth Factor Influences the Balance of Catabolic/ subsequent height loss. Many recent investigations have Anabolic Activity of AF Cells postulated that altering the biochemical factors within Because NGF appears to influence expression of disc tissue can slow the progression of IVD degeneration. genes thought to have an influence on matrix integrity, In this study we tested the hypothesis that NGF, a neu- we next assessed effects on the catabolic/anabolic activity rotrophin that stimulates outgrowth from neuronal of AF cells. Because Chi3l1 modulates the rate of type cells,38 influences expression of genes involved in degrada- I collagen fibril formation and Lcn2 has been shown to tion of the IVD matrix and thereby promotes IVD degener- protect Mmp9 from degradation by forming an Lcn2/ ation and chronic LBP. The results demonstrate previously Mmp9 complex, we focused on expression of these genes/ unreported effects of NGF on AF cells that would lead to molecules in addition to Mmp3/Timp1. The results are enhanced tissue breakdown and progressive IVD degen- shown in Fig. 2. There was no significant change in the eration. If these observations are replicated in vivo, NGF ratio of Chi3l1/Col1a1 (Fig. 2A) or Lcn2/Mmp9 (Fig. 2B) or downstream mediators of its potential adverse effects gene expression following NGF stimulation. In contrast, on IVD structure would be candidate molecules that could the ratio of Mmp3/Timp1 expression was increased (Fig. be targeted to slow down IVD degeneration and delay the 2C), suggesting a trend to a more catabolic phenotype. need for surgical intervention. In addition, targeting NGF may have benefit in the postoperative period by decreasing Nerve Growth Factor Signals Through ERK1/2 in AF Cells pain and enhancing tissue regeneration. Treatment of AF cells with NGF led to altered ex- To determine the signal pathway by which NGF may pression of less than 0.4% of genes, indicating likely be influencing gene expression, we stimulated rat AF specific effects. Importantly, among these significantly cells with NGF for 0, 6, 12, or 24 hours and examined k k upregulated genes, Lcn2 (lipocalin 2), Mmp3 (matrix activation of neurotrophic factor B (NF- B), p38, and metalloproteinase–3), and Chi3l1 (chitinase 3-like 1) are extracellular-regulation kinase (ERK1/2) mitogen-acti- of particular interest because of their novelty and known vated protein kinase (MAPK) by Western immunoblot- activity in regulating matrix turnover. We are the first to ting with 24-hour incubation. The results showed that report that Lcn2 is upregulated by NGF in AF cells. The NGF significantly increased ERK1/2 phosphorylation at Chi3l1 protein, also known as YKL-40, has previously 12 hours after stimulation (Fig. 3A), while there was no k been shown to be released following in vitro culture of significant activation of either NF- B or p38 (protein lev- degenerate IVD tissue35 but has never been associated el of the molecular signal examined showed no change). with dependence on NGF for expression. Activation of ERK1/2 by NGF was inhibited by Ro 08- The LCN2 protein is multifunctional, with roles 2750 (Fig. 3B), a nonpeptide inhibitor of NGF that binds in innate immunity and tumorigenesis. Also known the NGF dimer. as neutrophil gelatinase-associated lipocalin, LCN2 is now recognized as an because it is produced Effects of NGF on Human AF Cells predominantly by white .40 It has recently To assess whether similar activity of NGF was pres- been identified in growth plate chondrocytes,34 where ex-

TABLE 2: Microarray analysis

Gene Symbol Log Ratio (2) Gene Name Gene Function Chi3l1 6.643856 chitinase 3-like 1 remodeling or degradation of the extracellular matrix Lcn2 6.643856 lipocalin 2 iron trafficking Mmp3 5.930552 matrix metalloproteinase–3 can degrade fibronectin, laminin, gelatins of types I, III, IV, & V; collagens III, IV, X, & IX; & cartilage proteoglycans; activates procollagenase

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Fig. 1. Confirmation of microarray findings. A: Real-time polymerase chain reaction showed that selected expression of Chi3l1 (31.82 ± 7.57-fold, p = 0.0001), Lcn2 (29.86 ± 9.06-fold, p = 0.0001), and Mmp3 (24.67 ± 3.63-fold, p = 0.0001) significantly increased expression after NGF stimulation for 5 days (n = 8). B: Western blot verification of microarray data. The genes Chi3l1 (5.46 ± 0.87-fold, p = 0.0001), Lcn2 (5.1216 ± 1.09-fold, p = 0.0001), and Mmp3 (4.66 ± 1.23-fold, p = 0.0001) were significantly upregulated by NGF stimulation (n = 5). ***Significant at p < 0.001. C: Immunocytochemical detection of Chi3l1, Lcn2, and Mmp3 showing increased expression in cells following NGF stimulation (n = 3). No stimulation (i) and NGF treatment (ii) (100 ng/ ml for 5 days), original magnification ×200. pression is modulated by interleukin-1b (IL-1b), , similar to that in cartilage. The CHI3L1 protein is abun- , lipopolysaccharide, and . dantly produced by cultured chondrocytes and is gener- The function of LCN2 in cartilage and IVD tissue is not ated in cultures of degenerate IVD explants.16,35 It is over- known. Dimerization of LCN2 with pro-MMP9 appears expressed in osteoarthritic cartilage,43 but expression in to enhance activation of the enzyme with plasma kal- noncultured IVD tissues has not been previously report- likrein and protects the enzyme from degradation.41 In ed. The CHI3L1 protein is a recognized biomarker in os- patients with osteoarthritis, LCN2 levels are increased in teoarthritis, with expression correlating with disease pro- the synovial fluid, where it also acts to protect MMP9 gression,19 but the role of CHI3L1 in cartilage and IVD from degradation and enhance MMP9-induced cartilage tissues has not yet been clarified. Both tumor necrosis breakdown.15 Although the function of LCN2 in IVD tis- factor-a and IL-1b stimulate the expression of CHI3L1 in sue is unknown, similar activity, if present, would be ex- articular chondrocytes.36 Interestingly, CHI3L1 inhibits pected to enhance tissue degeneration. cellular responses induced by IL-1b and tumor necrosis The function of CHI3L1 in the IVD is likely to be factor-a,30 raising the possibilities that the induction of

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Fig. 2. NGF influences balance of catabolic/anabolic activity of AF cells. Time-course analysis of expression ratios of Chi3l1/ Col1a1, Lcn2/Mmp9, and Mmp3/Timp1 from Day 1 to Day 5. The cells were incubated in the absence or presence of NGF for 1, 3, or 5 days (n = 8). A: Ratio of Chi3l1 to Col1a1 (p = 0.81). B: Ratio of Lcn2 to Mmp9 (p = 0.074). C: Ratio of Mmp3 to Timp1 revealed balance significantly shifted toward catabolic potential on Day 5 (3.79 ± 0.53-fold, p = 0.022). *Significant at p < 0.05.

Fig. 3. Nerve growth factor induced ERK1/2 phosphorylation in rat-tail disc cells. A: Increased phosphorylation of ERK1/2 is seen at 12 hours (1.5 ± 0.095-fold, p = 0.027) after NGF treatment (n = 5). *Significant at p < 0.05. B: Phosphorylation of ERK1/2 induced by NGF was inhibited by Ro 08-2750 treatment (0.64 ± 0.14-fold, p = 0.01; n = 4). *Significant at p < 0.05 com- pared with control. #Significant at p < 0.05 compared with NGF.

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Fig. 4. Human IVD samples stimulated ex vivo by NGF and analyzed by immunohistochemistry (n = 3). A: No stimula- tion. B: IVD tissue incubated with NGF. C: Human IVD tissue incubated with Ro 08-2750. D: Human IVD tissue incubated with NGF and Ro 08-2750. Clear staining of CHI3L1, LCN2, and MMP3 was found following NGF treatment for 12 hours that was inhibited by pretreatment of Ro 08-2750. Original magnification ×200.

CHI3L1 feeds back to control local tissue responses and progression of this condition that can lead to ankylosis that increased production is an attempt at tissue repair. and loss of motion at the segment. These therapies seek The CHI3L1 protein inhibits the degradation of type I to alter the balance of catabolic and anabolic factors to collagen by MMP1 and increases the rate of type I col- help preserve and regenerate tissue when possible. In vi- lagen fibril formation,21 a potentially beneficial response tro studies that lead to a reduction in catabolic proteinases to IVD damage. deserve consideration when formulating a clinical solu- Overexpression of MMPs is clearly associated with tion to degenerative disc disease. We believe that the data both osteoarthritis and IVD degeneration.28 Increased in this study support the further investigation of NGF as a expression of proteinases is presumably predominantly potential treatment target in this realm. through the activity of catabolic such as IL-1, Nerve growth factor can bind to both the p75 neu- but our observations suggest a role for NGF in the produc- rotrophin receptor (p75NTR) and trkA NGF receptors tion of MMP3 by AF cells. Indeed, NGF has been shown (NGFRs). In the presence of trkA NGFR, p75NTR par- to increase expression of MMPs in other tissues and cell ticipates in the formation of high-affinity binding sites, systems.23,33 Thus, because NGF may not only regulate resulting in enhanced NGF responsiveness.17 Signaling neuronal in-growth but also influence the production of through trkA NGFRs results in activation of a signal cas- MMPs and neoangiogenesis,32 it may be a prime target cade that includes the MAPK-Ras-Erk pathway, phospho- for intervention in the course of IVD degeneration to help lipase C, and phosphoinositide 3-kinase.30 In the present decrease pain and the rate of disease progression. study, we found that NGF activated ERK1/2 but not p38 Ongoing clinical trials for the treatment of degen- or NF-kB in AF cells, consistent with signaling through erative disc disease, involving intradiscal therapies with trkA NGFRs rather than p75NTR, by which NGF has genes, growth factors, and stem cells,10,44 aim to halt the been shown to stimulate NF-kB–dependent signaling.8

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The observation that Ro 08-2750, a nonpeptide inhibitor tice guidelines in the management of chronic spinal pain. of NGF that binds the NGF dimer and inhibits binding Pain Physician 10:7–111, 2007 to both p75NTR and trkA, prevented both activation of 6. Burke JG, Watson RW, McCormack D, Dowling FE, Walsh MG, Fitzpatrick JM: Intervertebral discs which cause low ERK1/2 and increased expression of Chi3l1 and Lcn2 in back pain secrete high levels of proinflammatory mediators. J NGF-treated AF cells supports a direct role for NGF in Bone Joint Surg Br 84:196–201, 2002 regulating AF function that may be amenable to thera- 7. Carragee EJ: Clinical practice. Persistent low back pain. N peutic intervention. Engl J Med 352:1891–1898, 2005 8. Chacón PJ, Arévalo MA, Tébar AR: NGF-activated protein tyrosine phosphatase 1B mediates the phosphorylation and Conclusions degradation of I-κ-Bα coupled to NF-κ-B activation, thereby controlling dendrite morphology. Mol Cell Neurosci 43:384– We have shown, for the first time, that NGF increas- 393, 2010 es expression of a range of genes in AF cells, includ- 9. Coppes MH, Marani E, Thomeer RT, Groen GJ: Innervation ing Chi3l1, Lcn2, and Mmp3, promoting a procatabolic of “painful” lumbar discs. Spine (Phila Pa 1976) 22:2342– phenotype that, when replicated in vivo, would lead to 2350, 1997 IVD degeneration. The effects appear to be via the high- 10. Coric D, Pettine K, Sumich A, Boltes MO: Prospective study affinity trkA NGFR. Anti-NGF therapies are currently of disc repair with allogeneic chondrocytes. Clinical article. J tested in clinical trials for chronic LBP and pain from Neurosurg Spine 18:85–95, 2013 osteoarthritis of hip or knee joints, with good preliminary 11. Davis TT, Delamarter RB, Sra P, Goldstein TB: The IDET 24,27,39 procedure for chronic discogenic low back pain. Spine (Phila results. Our study shows that NGF potentially influ- Pa 1976) 29:752–756, 2004 ences the process of IVD matrix breakdown, in addition 12. Deyo RA, Weinstein JN: Low back pain. N Engl J Med to contributing to pain pathways, and that these activities 344:363–370, 2001 may be blocked by specific NGF inhibitors. Novel treat- 13. Freemont AJ, Peacock TE, Goupille P, Hoyland JA, O’Brien J, ments targeting NGF/NGFRs have the potential to influ- Jayson MI: Nerve ingrowth into diseased intervertebral disc ence a range of adverse biological responses, including in chronic back pain. Lancet 350:178–181, 1997 neurite and vascular infiltration and tissue degeneration, 14. Freemont AJ, Watkins A, Le Maitre C, Baird P, Jeziorska M, that result in the clinical symptoms and pathology of IVD Knight MT, et al: Nerve growth factor expression and inner- vation of the painful intervertebral disc. J Pathol 197:286– disease. Adding outcome measurements that assess the 292, 2002 extent of IVD degradation in clinical trials of anti-NGF 15. Gupta K, Shukla M, Cowland JB, Malemud CJ, Haqqi TM: therapies is indicated to establish whether these treat- Neutrophil gelatinase-associated lipocalin is expressed in os- ments have effects on disease progression. teoarthritis and forms a complex with matrix metalloprotein- ase 9. Arthritis Rheum 56:3326–3335, 2007 Disclosure 16. Hakala BE, White C, Recklies AD: Human cartilage gp-39, a major secretory product of articular chondrocytes and sy- The study was supported by grants from the National Science novial cells, is a mammalian member of a chitinase protein Council and National Defense Medical Center, Tri-Service General family. J Biol Chem 268:25803–25810, 1993 Hospital, Taiwan (NSC100-2320-B-016-006-MY3, MAB101-29, 17. Hempstead BL: The many faces of p75NTR. Curr Opin Neu- TSGH-C101-075), and Taichung Veterans General Hospital, Tai- robiol 12:260–267, 2002 wan (TCVGH-964904B). The authors report no conflict of interest 18. Horii M, Orita S, Nagata M, Takaso M, Yamauchi K, Yamashita concerning the materials or methods used in this study or the find- M, et al: Direct application of the tumor necrosis factor-α in- ings specified in this paper. hibitor, etanercept, into a punctured intervertebral disc decreas- Author contributions to the study and manuscript preparation es gene-related peptide expression in rat dorsal root include the following. Conception and design: Lee, Kao. Acquisition ganglion . Spine (Phila Pa 1976) 36:E80–E85, 2011 of data: Kao. Analysis and interpretation of data: Kao. Drafting the 19. Huang K, Wu LD: YKL-40: a potential biomarker for osteoar- article: Kao. Critically revising the article: all authors. Reviewed thritis. J Int Med Res 37:18–24, 2009 submitted version of manuscript: all authors. Approved the final 20. Imai S, Matsusue Y: Neuronal regulation of bone metabolism version of the manuscript on behalf of all authors: Lee. Statistical and anabolism: calcitonin gene-related peptide-, substance P-, analysis: Kao. Administrative/technical/material support: Peng, and tyrosine hydroxylase-containing nerves and the bone. Mi- Tsou. Study supervision: Salter. crosc Res Tech 58:61–69, 2002 21. Iwata T, Kuwajima M, Sukeno A, Ishimaru N, Hayashi Y, References Wabitsch M, et al: YKL-40 secreted from adipose tissue in- hibits degradation of type I collagen. Biochem Biophys Res 1. Andersson GB: Epidemiological features of chronic low-back Commun 388:511–516, 2009 pain. Lancet 354:581–585, 1999 22. Kallewaard JW, Terheggen MA, Groen GJ, Sluijter ME, Der- 2. Ashton IK, Roberts S, Jaffray DC, Polak JM, Eisenstein SM: by R, Kapural L, et al: 15. Discogenic low back pain. Pain Neuropeptides in the human intervertebral disc. J Orthop Pract 10:560–579, 2010 Res 12:186–192, 1994 23. 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