ar4443 1478-6354 Research article <p>Induction of nerve growth factor expression and release by mechanical and inflammatory stimuli in chondrocytes: possible involvement in osteoarthritis pain</p> PecchiEmilieemiliepecchi@yahoo.fr PriamSabrinas.priam@gmail.com GossetMarjolainemarjolaine.gosset@parisdescartes.fr PigenetAudreyaudrey.pigenet@upmc.fr SudreLaurelaure.sudre@snv.jussieu.fr LaiguillonMarie-Charlottemarie-c.laiguillon@hotmail.fr BerenbaumFrancisfrancis.berenbaum@sat.aphp.fr HouardXavierxavier.houard@snv.jussieu.fr

INSERM UMRS938, UPMC, Univ Paris 06, 7 quai Saint-Bernard, Paris F-75252, Cedex 5, France

Department of Rheumatology, Inflammation-Immunopathology-Biotherapy Department (DHU i2B), AP-HP Saint-Antoine Hospital, 184, rue du Faubourg Saint-Antoine, F-75012 Paris, France

Département de Rhumatologie, Hôpital Saint-Antoine, 184, rue du Faubourg Saint-Antoine, F-75012 Paris, France

 Arthritis Research & Therapy 1478-6354 2014 16 1 R16 http://arthritis-research.com/content/16/1/R16 10.1186/ar444324438745 21520133120142012014 2014Pecchi et al.; licensee BioMed Central Ltd.This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Introduction

Nerve growth factor (NGF) level is increased in osteoarthritis (OA) joints and is involved in pain associated with OA. Stimuli responsible for NGF stimulation in chondrocytes are unknown. We investigated whether mechanical stress and proinflammatory cytokines may influence NGF synthesis by chondrocytes.

Methods

Primary cultures of human OA chondrocytes, newborn mouse articular chondrocytes or cartilage explants were stimulated by increasing amounts of IL-1β, prostaglandin E2 (PGE2), visfatin/nicotinamide phosphoribosyltransferase (NAMPT) or by cyclic mechanical compression (0.5 Hz, 1 MPa). Before stimulation, chondrocytes were pretreated with indomethacin, Apo866, a specific inhibitor of NAMPT enzymatic activity, or transfected by siRNA targeting visfatin/NAMPT. mRNA NGF levels were assessed by real-time quantitative PCR and NGF released into media was determined by ELISA.

Results

Unstimulated human and mouse articular chondrocytes expressed low levels of NGF (19.2 ± 8.7 pg/mL, 13.5 ± 1.0 pg/mL and 4.4 ± 0.8 pg/mL/mg tissue for human and mouse articular chondrocytes and costal explants, respectively). Mechanical stress induced NGF release in conditioned media. When stimulated by IL-1β or visfatin/NAMPT, a proinflammatory adipokine produced by chondocytes in response to IL-1β, a dose-dependent increase in NGF mRNA expression and NGF release in both human and mouse chondrocyte conditioned media was observed. Visfatin/NAMPT is also an intracellular enzyme acting as the rate-limiting enzyme of the generation of NAD. The expression of NGF induced by visfatin/NAMPT was inhibited by Apo866, whereas IL-1β-mediated NGF expression was not modified by siRNA targeting visfatin/NAMPT. Interestingly, PGE2, which is produced by chondrocytes in response to IL-1β and visfatin/NAMPT, did not stimulate NGF production. Consistently, indomethacin, a cyclooxygenase inhibitor, did not counteract IL-1β-induced NGF production.

Conclusions

These results show that mechanical stress, IL-1β and extracellular visfatin/NAMPT, all stimulated the expression and release of NGF by chondrocytes and thus suggest that the overexpression of visfatin/NAMPT and IL-1β in the OA joint and the increased mechanical loading of cartilage may mediate OA pain via the stimulation of NGF expression and release by chondrocytes.

Introduction

Osteoarthritis (OA) is a chronic and age-related joint disease leading to cartilage destruction. Whereas the mechanisms by which this degradation happens are more and more understood, the reasons why an OA joint is painful are quite mysterious. For a same degree of cartilage degradation, some patients have symptoms and others have not. Recently, novel pharmacological molecules, belonging to the anti-nerve growth factor (NGF) family, have shown a dramatic effect on OA symptoms, much more efficacious than non-steroidal anti-inflammatory drugs (NSAIDs), the usual treatment for symptomatic OA 1 2 3 4 . Unfortunately, all clinical trials were halted in 2011 due to an unexpected increase in the number of total joint prosthesis in the active compared to the control groups 5 . Reviewing all the cases, it has been shown that this increase was due to an accelerated OA process in a few patients, especially those co-treated with NSAIDs. Nevertheless, NGF displays proinflammatory effects, including the stimulation of cytokine and prostaglandin E2 (PGE2) synthesis, monocyte differentiation, mast cell proliferation and degranulation 6 . Moreover, the injection of NGF into the synovium of rats increased the density of mast cells 7 .

Since there is an unmet need for treating pain in OA patients, any explanations on the occurrence of such deleterious effects with anti-NGF drugs are welcome. It has been proposed that pain improvement allows increased joint activity leading to subsequent overuse 8 . More directly, NGF improves ligament healing 9 and decelerates chondrocyte differentiation in vitro 10 , which is suspected to play an important role in OA progression. In addition, NGF can also display anti-inflammatory effects 11 12 . Notably, NGF inhibited interleukin (IL)-1β-induced tumor necrosis factor-α (TNF-α) production in OA synovial fibroblasts 13 .

Inflammatory factors, including IL-1β, TNF-α and adipokines 14 are found in increased levels in OA joints and are proposed to play a role in OA progression, especially by switching chondrocytes towards a catabolic phenotype. IL-1β downregulates the expression of cartilage extracellular matrix components, while it stimulates matrix metalloproteinase (MMP), cytokine and PGE2 synthesis. Similarly, adipokines, including visfatin, stimulate the catabolic activity of chondrocytes 14 15 . NGF levels are increased in the synovial fluid of OA patients 16 . NGF and its two receptors, the high-affinity tyrosine kinase A receptor (trkA) and the low-affinity p75 receptor, are expressed by joint cells including chondrocytes 17 18 and are increased in OA cartilage 10 19 . Moreover, NGF synthesis is highly correlated with the degree of OA cartilage degradation in human 19 .

Interestingly, NGF expression is induced in an inflammatory context and mediators such as IL-1β and PGE2 can stimulate NGF synthesis 13 20 . For example, intra-articular injection of IL-1β induces an increase in NGF levels 21 . However, stimuli responsible for NGF expression and production in OA cartilage remain to be determined. In the present study, we investigated whether mechanical stress and proinflammatory factors, two main determinants of OA, may influence NGF synthesis by chondrocytes. For this purpose, primary cultures of human OA chondrocytes and newborn mouse articular chondrocytes or cartilage explants were stimulated by increasing amounts of IL-1β, PGE2, visfatin or by cyclic mechanical compression. NGF mRNA expression and protein released into media were determined.

Materials and methods

Primary culture of human chondrocytes

Human cartilage samples were obtained from OA patients undergoing total knee arthroplasty at Saint-Antoine Hospital (Paris, France). Informed consent was obtained for each patient before surgery. The diagnosis of OA was based on clinical and radiographic evaluations according to the criteria of the American College of Rheumatology 22 . Experiments using human samples were approved by the local ethics committee (CPP Ile de France V, 2 May 2012).

Human articular chondrocytes were isolated by enzymatic digestion of cartilage, as previously described 15 . Cells were seeded in 12-well plates (500,000 cells/well) and allowed to grow to confluence in Dulbecco’s modified Eagle’s medium (DMEM) (4.5 mg/L glucose) supplemented with 10% fetal calf serum, 100 IU/mL penicillin, 100 μg/mL streptomycin and 4 mM L-glutamine (Sigma-Aldrich, Saint Quentin Fallavier, France). Cells were then starved in serum-free medium containing 0.3% bovine serum albumin for 24 hours before stimulation.

Primary culture of mouse chondrocytes

All experiments were performed according to the protocols approved by the French/European ethics committee. Immature murine articular chondrocytes were isolated by enzymatic digestion of articular cartilage from six-day-old newborn animals from one Swiss mouse litter (Janvier Labs, Le Genest Saint Isle, France), according to a previously described procedure 23 . Cells were seeded in 12-well plates (10,000 cells/cm2) and allowed to grow for six to seven days in DMEM (1 mg/L glucose) supplemented with 10% fetal calf serum, 100 IU/mL penicillin, 100 μg/mL streptomycin and 4 mM L-glutamine (Sigma-Aldrich). Cells were then starved in serum-free medium containing 0.1% bovine serum albumin for 24 hours before stimulation.

Cell stimulation

Human OA and murine articular chondrocytes were stimulated with IL-1β (PeproTech, from Tebu Bio, Le Perray-en-Yvelines, France; 0.1 to 10 ng/mL from 2 to 24 hours), PGE2 (Cayman Chemical, from SPI-Bio, Montigny-le-Bretonneux, France; 0.1 to 10 μM for 24 hours), or visfatin (Alexis Biochemical, Paris, France; 1 to 10 μg/mL for 24 hours) dissolved in starvation media. In additional experiments, chondrocytes were co-incubated with either IL-1β (1 ng/mL) and indomethacin (Sigma-Aldrich; 0.1 to 10 μM), an inhibitor of the cyclooxygenase activity, or IL-1β (1 ng/mL) and visfatin (5 μg/mL) or visfatin (5 μg/mL) and Apo866 (10 nM) (generously provided by Alexander So; Astellas Pharma, Munich, Germany), an inhibitor of the nicotinamide phosphoribosyltransferase activity of visfatin.

After stimulation, cells were disrupted in lysis buffer (RLT, from Qiagen, Courtaboeuf, France) and conditioned media were stored at -80°C until analysis.

Transfection of small interfering RNA (siRNA)

Small interfering RNA (siRNA) directed against mouse visfatin was designed and purchased from Ambion Cenix (Austin, TX, USA). The sequence specific for mouse visfatin was forward 5’-GGCACCACUAAUCAUCAGAtt-3’, reverse 5’-UCUGAUGAUUAGUGGUGCCtc-3’.

Mouse chondrocytes were cultured as described above. Confluent cells were removed with trypsin, and 6 × 105 chondrocytes were seeded in 6-cm tissue culture plates and grown for 24 hours, to 70 to 80% confluence. Normal growth medium containing 10% fetal bovine serum was changed prior to siRNA transfection. Transfections were performed as described for the RNAi Starter Kit (Qiagen). Cells were incubated for 18 hours with siRNA and transfection reagent, rinsed twice with phosphate-buffered saline (PBS), and placed in DMEM (1 mg/L glucose) supplemented with penicillin, streptomycin, and L-glutamine containing 1% BSA, with or without IL-1β (10 ng/ml) for 24 hours. Transfection of siRNA against MAPK-1, a ubiquitously produced mouse cell protein, was used as a positive control. A nonsilencing siRNA that has no homology with any known mammalian gene (RNAi Starter Kit) and scrambled siRNA (Ambion) were used as negative controls.

Mechanical compression

Mechanical compression was applied on costal cartilage, as previously described 24 . Briefly, ribs cages from six-day-old newborns from one Swiss mouse litter (Janvier Labs) were harvested and cartilage was separated from bone and soft tissues. Immediately after dissection, each sample, consisting of 50 mg of cartilage explants, were placed into Biopress culture plates (Flexercell International, Hillsborough, NC, USA) in DMEM (1 mg/L glucose) supplemented with 30 mM Hepes, 100 IU/mL penicillin, 100 μg/mL streptomycin and 4 mM L-glutamine (Sigma-Aldrich). During 4 to 24 hours, intermittent compression was applied by the Biopress system (Flexercell International) using a sinusoidal waveform at 0.5 Hz and 1.0 MPa of magnitude (Figure S1 in Additional file 1). Control explants were kept in unloaded condition.

<p>Additional file 1: Figure S1</p>

Compression system. (A) FX-4000C™ Flexercell™ Compression Plus™ System (Flexcell International Corp., Hillsborough, NC, USA). A positive pressure compresses samples between a piston and stationary platen on the BioPress™ culture. (B) Schematic diagram of the BioPress™ culture plate compression chamber in uncompressed or compressed position.

Click here for file

After compression, cartilage explants frizzed in liquid nitrogen and conditioned media were stored at -80°C until analysis.

RNA extraction, reverse transcription and quantitative real-time PCR

Total RNA was extracted from cultured cells with RNeasy Mini kit (Qiagen) according to the manufacturer’s instructions. Total RNA was reverse transcribed with Omniscript RT kit according to the manufacturer's instructions (Qiagen). NGF mRNA expression was analyzed by quantitative real-time PCR using the Light Cycler 480 (Roche Diagnostics, Meylan, France). The equivalent of 5 to 10 ng initial RNA was subjected to PCR amplification in a 12 μl final volume using specific primers at 10 μM and LC 480 SYBR Green I Master kit (Roche Diagnostics). PCR amplification conditions were: initial denaturation for 5 min at 95°C followed by 40 cycles consisting of 10 s at 95°C, 15 s at 60°C and 10 s at 72°C. Product formation was detected at 72°C in the fluorescein isothiocyanate channel. The generation of specific PCR products was confirmed by melting curve analysis. For each PCR, cDNAs were run in duplicate in parallel with serial dilutions of a cDNA mixture tested for each primer pair to generate a standard linear curve, which was used to estimate the amplification efficiency for NGF and HPRT. The relative mRNA expression of NGF normalized to that of HPRT, used as internal reference gene, was determined by the Efficiency method of the LightCycler 480 Software.

The specific oligonucleotide primers used were as follows: human NGF, sense 5’GTCATCATCCCATCCCATCT3’ and anti-sense 5’AGTACTGTTTGAATACACTGTTGTTAAT3’; mouse NGF, sense 5’CACCCACCCAGTCTTCC3’ and anti-sense 5’CTCGGCACTTGGTCTCAAA3’; human GAPDH, sense 5’CCATCACCATCTTCCA3’ and anti-sense 5’CCTTCTCCATGGTGGT3’; mouse HPRT, sense, 5’GCTGGTGAAAAGGACCTCT3’ and anti-sense, 5’CACAGGACTAGAACACCTGC3’.

NGF assay

NGF concentrations in the medium were measured using an enzyme-linked immunosorbent assay (ELISA) kit from Promega (Charbonnières, France) according to the manufacturer’s instructions. The detection limit of NGF was 7.8 pg/mL and the intra-assay coefficients of variance was 4.2%. The NGF concentrations were analyzed in duplicate at serial dilution and were read against a standard curve.

Statistical analysis

Results are expressed as means ± standard deviation (SD). Results were compared using one-way factorial analysis (analysis of variance) followed by the Scheff´s F test (Statview software, version 4.57; Abacus Concepts Inc., Berkeley, CA, USA). Statistical significance was accepted for P <0.05.

Results

Induction of NGF expression and release by costal cartilage explants in response to mechanical stress

Since mechanical overload is considered as a strong inducer of cartilage damage and pain, the influence of in vitro mechanical compression on NGF release by costal cartilage explants was investigated. For this purpose, murine costal cartilages were subjected to intermittent compression for 4 and 24 hours. Costal cartilage explants constitutively released low levels of NGF that accumulated within conditioned media between 4 and 24 hours (Figure  1A). Compression for 4 hours did not stimulate NGF release. In contrast, accumulation of NGF into tissue-conditioned media was significantly increased after compression for 24 hours (4.7-fold increase versus control, P = 0.04) (Figure  1).

<p>Figure 1</p>

Stimulation of NGF release by cartilage compression

Stimulation of NGF release by cartilage compression. Explants of costal cartilages from Swiss mice were compressed (C) or not (NC) for 4 and 24 hours (n = 4). NGF protein levels were measured in conditioned media. *P <0.05. NGF, nerve growth factor.

Induction of NGF expression and release by articular chondrocytes in response to visfatin

Visfatin is an adipokine found in OA synovial fluids 25 , which displays catabolic activity on chondrocytes 15 . We thus determined whether NGF expression and release could be modulated by visfatin. Human OA and mouse articular chondrocytes were stimulated with increasing concentrations of visfatin (1, 2.5, 5 and 10 μg/mL) for 24 hours and NGF mRNA expression and release into conditioned media was measured. Both human and mouse articular chondrocytes spontaneously synthesized NGF mRNA and protein (Figure  2). Visfatin induced a dose-dependent increase in NGF mRNA expression in human OA chondrocytes (Figure  2A). The stimulation of NGF mRNA expression was observed with 1 μg/mL visfatin and became statistically significant with 2.5 μg/mL visfatin (14-fold increase as compared to control, P = 0.02). Consequently, an accumulation of NGF into human chondrocyte-conditioned media was measured after visfatin stimulation (Figure  2B). Similarly, a dose-dependent increased accumulation of NGF was observed in conditioned media of mouse articular chondrocytes (Figure  2C). A significant increase, as compared to unstimulated chondrocytes, was obtained with 2.5 μg/mL visfatin (2.6-fold versus control, P = 0.03) and the highest concentration of visfatin (10 μg/mL) induced the highest accumulation of NGF into cell supernatant (4.9-fold versus control, P = 0.0003) (Figure  2C). Visfatin-mediated release of NGF was associated with an increase in NGF mRNA expression (2-fold versus control, P = 0.045) (Figure  2C, inset).

<p>Figure 2</p>

Visfatin induces NGF expression and release by chondrocytes

Visfatin induces NGF expression and release by chondrocytes. Human OA chondrocytes were stimulated for 24 hours with increasing concentrations of visfatin (0, 1, 2.5 and 5 μg/mL) and NGF mRNA expression (A) and release into cell supernatant (B) were measured by quantitative RT-PCR and ELISA, respectively (n = 3) Control cells released 20.4 ± 12.2 pg/mL NGF. (C) Primary cultures of mouse articular chondrocytes were stimulated for 24 hours with increasing concentration of visfatin (0, 1, 2.5, 5 and 10 μg/mL) and NGF levels were measured in conditioned media (n = 3). Inset: NGF mRNA expression was determined by quantitative RT-PCR in control cells and chondrocytes stimulated with visfatin (5 μg/mL) for 24 hours. The amount of NGF mRNA was normalized against the amount of HPRT mRNA measured in the same cDNA. (D) Mouse articular chondrocytes were incubated with or without Apo866 (10 nM) for 4 hours and then treated or not with visfatin (5 μg/mL) for 24 hours (n = 3). NGF release was determined into cell-conditioned media. *P <0.05, **P <0.01, ***P <0.001. ELISA, enzyme-linked immunosorbent assay; NGF, nerve growth factor; OA, osteoarthritis.

In addition to its cytokine-like activity, visfatin displays a nicotinamide phosphoribosyltransferase (NAMPT) enzymatic activity responsible for the synthesis of nicotinamide adenine dinucleotide (NAD). Several lines of evidence support the involvement of this enzymatic activity in the cellular effects of visfatin/NAMPT 26 . To investigate the involvement of visfatin/NAMPT enzymatic activity in NGF stimulation, chondrocytes were pretreated with 10 nM Apo866, a specific inhibitor of NAMPT 27 , for 4 hours before the addition of exogenous visfatin/NAMPT (5 μg/mL). Apo866 treatment led to a 56% and 68% drop in NAD concentration in control and visfatin/NAMPT-treated chondrocytes (P = 0.0003 and P <0.0001, respectively) (not shown). The inhibition of visfatin/NAMPT enzymatic activity by Apo866 prevented the increase in NGF synthesis (P = 0.02 versus visfatin/NAMPT-treated cells without Apo866) (Figure  2D). In contrast, no modification of spontaneous release of NGF by chondrocytes was observed in the presence of Apo866.

Induction of NGF expression and release by articular chondrocytes in response to IL-1β

We next investigated the action of IL-1β on NGF mRNA expression and release by human OA and murine articular chondrocytes. IL-1β dose-dependently increased NGF mRNA expression in human OA articular chondrocytes (Figure  3A). A 9.3-fold induction was observed with 0.1 ng/mL IL-1β and became statistically significant with 1 ng/mL (P = 0.004). This was associated with increased levels of NGF measured in conditioned media of chondrocytes stimulated with IL-1β (Figure  3B). IL-1β also stimulated NGF mRNA expression in mouse articular chondrocytes since a 1.8-fold induction was observed with 0.1 ng/mL IL-1β and became statistically significant with 1 ng/mL (P = 0.01). NGF mRNA expression further increased using 10 ng/mL of IL-1β (5.7-fold compared to control, P = 0.004). IL-1β-induced NGF mRNA expression paralleled the release of NGF protein into cell-conditioned media (Figure  3C and D). NGF protein release was slightly increased at 0.1 ng/mL and this increase became significant at 1 ng/mL IL-1β (P = 0.024). In the time-course experiment, IL-1β-induced NGF release was observed after a 4-hour stimulation (4.2-fold increase versus control, P = 0.049) and further increased up to 24 hours (9.2-fold increase versus control, P = 0.0005) (Figure  3E).

<p>Figure 3</p>

Stimulation of articular chondrocyte expression and release of NGF by IL-1β

Stimulation of articular chondrocyte expression and release of NGF by IL-1β. Human OA chondrocytes were stimulated for 24 hours with increasing concentrations of IL-1β (0, 0.1, 1 and 10 ng/mL) and NGF mRNA expression (A) and release into cell supernatant (B) were measured by quantitative RT-PCR and ELISA, respectively (n = 4). Control cells released 19.2 ± 8.7 pg/mL NGF. (C-E) Primary cultures of mouse articular chondrocytes were stimulated either with increasing concentrations of IL-1β (0, 0.1, 1 and 10 ng/mL) for 24 hours (n = 3) (C and D) or with IL-1β (10 ng/mL) for 2, 4, 6, 8 and 24 hours (n = 3) (E). As a control (ctrl), chondrocytes were incubated without IL-1β for 24 hours (E). NGF mRNA expression was determined by quantitative RT-PCR (C). The amount of NGF mRNA was normalized against the amount of HPRT mRNA measured in the same cDNA. NGF protein levels were measured in conditioned media by ELISA (D and E). *P <0.05, **P <0.01, ***P <0.001. ELISA, enzyme-linked immunosorbent assay; IL-1β, interleukin 1β; NGF, nerve growth factor; OA, osteoarthritis.

IL-1β is a strong inducer of PGE2 synthesis by cartilage and chondrocytes. However, PGE2 was not involved in IL-1β-mediated NGF stimulation. Indeed, PGE2 (0.01 to 10 μM) did not stimulate NGF release by articular chondrocytes (Figure  4A). Furthermore, indomethacin (0.1 to 10 μM), a specific inhibitor of cyclooxygenase enzymatic activity, was unable to prevent the release of NGF induced by IL-1β (1 ng/mL) (Figure  4B), although it efficiently inhibited the IL-1β-mediated production of PGE2 by chondrocytes (not shown).

<p>Figure 4</p>

PGE2 and endogenous visaftin/NAMPT are not involved in IL-1β-mediated stimulation of NGF release by articular chondrocytes

PGE2 and endogenous visaftin/NAMPT are not involved in IL-1β-mediated stimulation of NGF release by articular chondrocytes. (A and B) Primary cultures of mouse articular chondrocytes were stimulated for 24 hours either with increasing concentrations of PGE2 (0, 0.01, 0.01, 1 and 10 ng/mL) (n = 3) (A) or with IL-1β (1 ng/mL) in the presence of increasing concentrations of indomethacin (0, 0.1, 1, 10 μM) (n = 3) (B). As a control (ctrl), chondrocytes were incubated without IL-1β for 24 hours (B). NGF protein levels were measured in conditioned media by ELISA. (C) Primary culture of mouse articular chondrocytes were transfected by siRNA targeting visfatin/NAMPT before stimulation with IL-1β (10 ng/mL) for 24 hours (n = 2). Each experiment was performed in triplicate. Results are expressed as NGF release as compared to IL-1β-stimulated chondrocytes. *P <0.05, ***P <0.001. ELISA, enzyme-linked immunosorbent assay; IL-1β, interleukin 1β; NAMPT, nicotinamide phosphoribosyltransferase activity. Supress activity; NGF, nerve growth factor; PGE2, prostaglandin E2; siRNA, small interfering RNA.

Since some of the effects of IL-1β on chondrocytes involve visfatin/NAMPT 15 , consequences of visfatin/NAMPT knockdown on IL-1β-mediated NGF stimulation were assessed (Figure  4C). No effect of visfatin/NAMPT siRNA on the constitutive release of NGF was observed. Similarly, the potent stimulation of NGF secretion induced by IL-1β (10 ng/mL) (5-fold as compared to control, P >0.0002) was not blocked by visfatin/NAMPT siRNA, suggesting that endogenous visfatin/NAMPT is not involved in the IL-1β-stimulated release of NGF by chondrocytes (Figure  4C).

<p>Figure 5</p>

Potentiation of IL-1β-stimulated NGF expression and release by visfatin/NAMPT

Potentiation of IL-1β-stimulated NGF expression and release by visfatin/NAMPT. Primary cultures of mouse articular chondrocytes were stimulated or not for 24 hours either with IL-1β (1 ng/mL) or visfatin/NAMPT (5 μg/mL) or with IL-1β (1 ng/mL) and visfatin/NAMPT (5 μg/mL) (n = 3). The mRNA expression of NGF (A) and the NGF protein levels in chondrocyte conditioned media were determined (B). *P <0.05, ***P <0.001. IL-1β, interleukin 1β; NAMPT, nicotinamide phosphoribosyltransferase activity. Supress activity; NGF, nerve growth factor.

Potentiation of IL-1β-stimulated NGF expression and release by visfatin/NAMPT

To determine whether the stimulation of NGF mRNA expression and release by IL-1β and visfatin/NAMPT could be additive, murine articular chondrocytes were co-stimulated with IL-1β (1 ng/mL) and visfatin/NAMPT (5 μg/mL). A slight potentiation of IL-1β-mediated stimulation of NGF was observed both at mRNA (1.4-fold) and protein levels (1.3-fold) when chondrocytes were co-stimulated with visfatin/NAMPT (Figure  5). This potentiation was similar to the stimulatory effect of visfatin/NAPMT since visfatin/NAMPT induced a 1.5-fold and 1.4-fold stimulation of NGF mRNA expression and release, respectively (Figure  5). Compression did not further increase the release of NGF from cartilage induced by either IL-1β or visfatin/NAMPT or IL-1β and visfatin/NAMPT (not shown).

Discussion

Several lines of evidence highlight NGF as an attractive therapeutic target to control OA pain. NGF is a key factor in inflammation-associated hyperalgesia, whose protein is detected in OA synovial fluid 16 and mRNA expression is enhanced in OA chondrocytes 19 . NGF may also mediate joint damage 6 . Preclinical studies on experimental models of joint pain 1 3 and clinical trials on OA patients 2 4 thus evidenced the efficiency of NGF targeting in the reduction of joint pain. However, the increased number of total joint prosthesis due to acceleration in lesion progression in some patients decided the FDA to stop all clinical trials. A better understanding of the involvement of NGF in OA is therefore required. In this study, we investigated whether proinflammatory factors and mechanical compression, the main determinants of OA, may lead to the expression and the release of NGF by chondrocytes.

We first show that human OA chondrocytes as well as mouse cartilage and articular chondrocytes constitutively expressed NGF mRNA and released NGF protein into conditioned media, as already reported by Iannone et al. 19 in human normal articular chondrocytes. We also observed an increase in NGF expression and release in response to OA stimuli, consistently with the enhanced expression of NGF by OA chondrocytes 19 . In this context, we show for the first time that dynamic compression induced the release of NGF by cartilage, highlighting NGF as a mechanosensitive gene in chondrocytes. This result is in accordance with data showing that mechanical stretch can modulate NGF expression with an induction observed in smooth muscle cells 28 and sympathetic neurocytes 29 , whereas a downregulation was obtained in rat cardiomyocytes 30 .

We also show that IL-1β, which is considered as the main inflammatory mediator involved in cartilage degradation in OA, induced in a time- and dose-dependent manner an increase in NGF expression and release by both human and mouse articular chondrocytes. Similarly, IL-1β has been reported to stimulate the expression of NGF in several cell types, including human nucleus pulposus cells isolated from patients with intervertebral disc (IVD) degeneration 31 32 33 . The intra-articular injection of IL-1β induced an increase in NGF levels 21 and a positive correlation between IL-1β and NGF immunostaining in human IVD tissues was observed 34 .

In addition to its well-known pro-degradative property on cartilage, IL-1β plays also a role in joint pain. Indeed, IL-1β levels in OA synovial fluids are associated with pain and hyperalgesia of the temporomandibular joint 35 and the synovial mRNA expression of IL-1β is correlated with the degree of pain in rotator cuff diseases 36 . OA patients showing a 2-fold higher expression of IL-1β by peripheral blood leukocytes compared to non-OA controls have higher pain scores and risks of radiographic OA progression than patients displaying similar peripheral blood leukocyte IL-1β expression than non-OA controls 37 . Furthermore, a reduced pain was obtained following the treatment of patients with chronic active gouty arthritis with rilonacept, an inhibitor of IL-1β 38 . Similarly, the subcutaneous injection of canakinumab, an anti-human IL-1β antibody, to patients with cryopyrin-associated periodic syndrome provoked a sustained remission of symptoms, including pain 39 . All these data suggest that IL-1β-mediated stimulation of NGF expression by chondrocytes may account for the pain associated with IL-1β levels in OA joints.

Adipokines have recently been proposed as molecular mediators probably involved in OA 40 . Among them, visfatin/NAMPT was characterized as a visceral fat cytokine, a 52 kDa protein with insulin mimetic activity 41 . Visfatin/NAMPT is released by all joint tissues in OA patients 25 and its concentration in synovial fluids is linked to cartilage degradation biomarkers 42 . Visfatin/NAMPT stimulates chondrocyte catabolic activity measured by PGE2 and protease synthesis 15 and induces the expression of several proinflammatory cytokines by chondrocytes (Laiguillon, submitted). Our results show that visfatin also stimulated both NGF expression and release by human OA and mouse articular chondrocytes, suggesting that it may favor OA pain in addition to mediate cartilage damage.

In addition to its cytokine-like activity, visfatin displays a NAMPT enzymatic activity responsible for the synthesis of NAD. NAD biosynthetic activity of visfatin/NAMPT is involved in some cellular effects of extracellular visfatin/NAMPT 43 44 45 . Interestingly, the pharmacological inhibition of visfatin/NAMPT by Apo866, a specific inhibitor of NAMPT activity, prevents arthritis progression 46 47 . In a previous study, we showed that blocking NAMPT enzymatic activity inhibited the catabolic response of chondrocytes to exogenous visfatin/NAMPT 26 . Similarly, we show here that NAMPT enzymatic activity was also crucial for the visfatin/NAMPT-mediated NGF stimulation in chondrocytes, suggesting the involvement of NAD in the signaling pathway of visfatin/NAMPT leading to NGF expression. NAD-consuming proteins, including sirtuins and poly(ADP-ribose) polymerases (PARPs), are known actors in the expression of many genes 48 . However, nothing is known about a putative role of such proteins in the expression of NGF. In addition, NAD consumption generates second messengers, which contribute to intracellular calcium signaling 49 . Intracellular calcium can regulate the expression of NGF 28 30 .

IL-1β-mediated NGF expression involves nuclear factor kappa B (NF-κB) activity 50 . Interestingly, the proinflammatory effect of visfatin/NAMPT requires NF-κB activity in a NAMPT enzymatic activity-dependent manner. Some of the effects of IL-1β involve endogenous visfatin/NAMPT, as demonstrated by siRNA strategy 15 . However, we did not obtain any inhibition of IL-1β-mediated NGF secretion by using siRNA targeting endogenous visfatin/NAMPT, showing that endogenous visfatin/NAMPT is not involved in the stimulation of NGF induced by IL-1β. As NAD-derived second messengers activate intracellular calcium signaling, these results could explain why intracellular calcium signaling is not involved in IL-1β-mediated NGF expression 51 . Taken together, our results support that different signaling pathways, requiring endogenous visfatin/NAMPT or not, can be activated by IL-1β. Our results also show that the stimulation of NGF expression and release in response to IL-1β and visfatin/NAMPT involves different pathways, thus explaining the potentiation of IL-1β-mediated stimulation of NGF expression and release by visfatin/NAMPT.

Interestingly, mechanical loading, IL-1β and visfatin all induce PGE2 production by cartilage and chondrocytes 15 52 53 . PGE2 was reported to induce the expression of NGF in mouse astrocyte cultures 54 55 . Blocking the generation of prostaglandins by celecoxib (Celebrex) prevented the increased secretion of NGF by tooth perfusates in rats after an inflammatory stress 56 . PGE2 may be also involved in IL-1β-mediated pain 57 . PGE2 may thus represent a common way for NGF expression in chondrocytes in response to OA stimuli. However, our results show that PGE2, whatever the concentration used, was unable to induce NGF expression and release in chondrocytes. In addition, indomethacin, an inhibitor of cyclooxygenase enzymatic activity, did not prevent the IL-1β-mediated increase in NGF expression in chondrocytes. These results suggest that the increased chondrocyte expression of NGF in response to several OA stimuli did not involve PGE2. In contrast, other studies showed that PGE2 modulates the expression and synthesis of NGF. Friedman et al. reported that indomethacin partly blocked the increase in NGF production in response to IL-1β in embryonic rat hippocampal cultures 20 . PGE2 stimulates the secretion of NGF in astrocytes 54 55 , whereas in 3T3-L1 adipocytes it decreases both NGF expression and synthesis 58 . Thus, the influence of PGE2 on NGF synthesis appears to be cell-type-dependent and may be related to the differential expression pattern of PGE2 receptor in these cells. High amounts of E-type prostanoid (EP)3 receptor are indeed expressed in mature adipocytes 59 , whereas chondrocytes express low levels of EP3 60 61 .

Conclusions

OA is a painful disease, in which NGF plays a crucial role. Indeed, pivotal clinical trials have demonstrated that counteracting NGF leads to a dramatic reduction of OA pain 2 . Unfortunately, all the NGF antibodies in development have induced an acceleration of the OA process in a subgroup of patients. It is thus mandatory to accumulate more information on the mechanism of action of NGF in order to enhance the benefit-risk ratio of this new family of effective symptomatic drugs. Our results show that two main OA determinants, mechanical stress and proinflammatory factors, including IL-1β and extracellular visfatin/NAMPT, stimulated the expression and the release of NGF by articular chondrocytes. These results suggest that OA pain may involve the release of NGF by chondrocytes in response to the increased levels of IL-1β and extracellular visfatin/NAMPT, and to the increased mechanical loading of cartilage.

Abbreviations

DMEM: Dulbecco’s modified Eagle’s medium; ELISA: enzyme-linked immunosorbent assay; IL-1β: interleukin 1β; IVD: intervetebral disc; MMP: matrix metalloproteinase; NAMPT: nicotinamide phosphoribosyltransferase activity. Supress activity; NGF: nerve growth factor; NSAID: non-steroidal anti-inflammatory drug; OA: osteoarthritis; PGE2: prostaglandin E2; siRNA: small interfering RNA; TNF-α: tumor necrosis factor-α; trkA: tyrosine kinase A receptor.

Competing interests

All authors state that there is no conflict of interests.

Author’s contributions

EP participated in the conception and design of the study, data collection and analysis, the manuscript writing and final approval of the manuscript. SP carried out data collection and analysis, and participated in the critical revision and final approval of the manuscript. MG carried out data collection and analysis, and participated in the critical revision and final approval of the manuscript. AP contributed to data collection and analysis and the final approval of the manuscript. LS contributed to data collection and analysis and the final approval of the manuscript. MCL contributed to data collection and analysis and the final approval of the manuscript. FB participated in the conception and design of the study, provided financial support, and contributed to manuscript writing and the final approval of the manuscript. XH participated in the conception and design of the study, data collection and analysis, the manuscript writing, and final approval of the manuscript. All authors read and approved the final manuscript.

Acknowledgments

The authors thank Colette Salvat for her excellent technical help. This work was supported by Labex Transimmunom, managed by the ANR within the Investissements d'Avenir programme under reference ANR-11-IDEX-0004-02.

 <p>Role of low-affinity nerve growth factor receptor inhibitory antibody in reducing pain behavior and calcitonin gene-related Peptide expression in a rat model of wrist joint inflammatory pain</p>IwakuraNOhtoriSOritaSYamashitaMTakahashiKKuniyoshiKJ Hand Surg Am20103526727310.1016/j.jhsa.2009.10.03020060234<p>Tanezumab for the treatment of pain from osteoarthritis of the knee</p>LaneNESchnitzerTJBirbaraCAMokhtaraniMSheltonDLSmithMDBrownMTN Engl J Med20103631521153110.1056/NEJMoa090151020942668<p>Treatment of murine osteoarthritis with TrkAd5 reveals a pivotal role for nerve growth factor in non-inflammatory joint pain</p>McNameeKEBurleighAGompelsLLFeldmannMAllenSJWilliamsRODawbarnDVincentTLInglisJJPain201014938639210.1016/j.pain.2010.03.00220350782<p>Tanezumab reduces osteoarthritic hip pain: results of a randomized, double-blind, placebo-controlled phase 3 trial</p>BrownMTMurphyFTRadinDMDavignonISmithMDWestCRArthritis Rheum2013651795180323553790<p>Adjudication of reported serious adverse joint events in the Tanezumab clinical development program</p>HochbergMCAbramsonSBHungerford DSEMVignonEPSmithMCTiveLVerburgKMWestCRArthritis Rheum201264S113<p>Nerve growth factor in rheumatic diseases</p>SeidelMFHerguijuelaMForkertROttenUSemin Arthritis Rheum20104010912610.1016/j.semarthrit.2009.03.00219481238<p>Nerve growth factor and distribution of mast cells in the synovium of adult rats</p>AloeLTuveriMALevi-MontalciniRClin Exp Rheumatol1992102032041505118<p>New treatments for osteoarthritis</p>SmelterEHochbergMCCurr Opin Rheumatol20132531031610.1097/BOR.0b013e32835f69b423425965<p>Nerve growth factor improves ligament healing</p>MammotoTSeerattanRAPaulsonKDLeonardCABrayRCSaloPTJ Orthop Res20082695796410.1002/jor.2061518302239<p>Nerve growth factor induced after temporomandibular joint inflammation decelerates chondrocyte differentiation</p>HuangHShankGMaLTallentsRKyrkanidesSOral Dis20131960461010.1111/odi.1204523231346<p>Nerve growth factor inhibits some acute experimental inflammations</p>Amico-RoxasMCarusoALeoneMGScifoRVanellaAScapagniniUArch Int Pharmacodyn Ther19892992692852774768<p>The neurotrophin receptor p75NTR mediates early anti-inflammatory effects of estrogen in the forebrain of young adult rats</p>NordellVLLewisDKBakeSSohrabjiFBMC Neurosci200565810.1186/1471-2202-6-58123991816156894<p>Nerve growth factor release by human synovial fibroblasts prior to and following exposure to tumor necrosis factor-alpha, interleukin-1 beta and cholecystokinin-8: the possible role of NGF in the inflammatory response</p>ManniLLundebergTFioritoSBoniniSVignetiEAloeLClin Exp Rheumatol20032161762414611111<p>What's new in our understanding of the role of adipokines in rheumatic diseases?</p>GomezRCondeJScoteceMGomez-ReinoJJLagoFGualilloONat Rev Rheumatol2011752853610.1038/nrrheum.2011.10721808287<p>Crucial role of visfatin/pre-B cell colony-enhancing factor in matrix degradation and prostaglandin E2 synthesis in chondrocytes: possible influence on osteoarthritis</p>GossetMBerenbaumFSalvatCSautetAPigenetATahiriKJacquesCArthritis Rheum2008581399140910.1002/art.2343118438860<p>Nerve growth factor in the synovial fluid of patients with chronic arthritis</p>AloeLTuveriMACarcassiULevi-MontalciniRArthritis Rheum19923535135510.1002/art.17803503151536673<p>Expression of NGF, Trka and p75 in human cartilage</p>GiganteABevilacquaCPagnottaAManzottiSToescaAGrecoFEur J Histochem20034733934414706929<p>Expression patterns of neurotrophins and neurotrophin receptors in articular chondrocytes and inflammatory infiltrates in knee joint arthritis</p>GrimsholmOGuoYNyTForsgrenSCells Tissues Organs200818829930910.1159/00012143218349525<p>Increased expression of nerve growth factor (NGF) and high affinity NGF receptor (p140 TrkA) in human osteoarthritic chondrocytes</p>IannoneFDe BariCDell'AccioFCovelliMPatellaVLo BiancoGLapadulaGRheumatology (Oxford)2002411413141810.1093/rheumatology/41.12.141312468822<p>Regulation of beta-nerve growth factor expression by inflammatory mediators in hippocampal cultures</p>FriedmanWJLarkforsLAyer-LeLievreCEbendalTOlsonLPerssonHJ Neurosci Res19902737438210.1002/jnr.4902703162129046<p>Role of IL-1 beta and TNF-alpha in the regulation of NGF in experimentally induced arthritis in mice</p>ManniLAloeLRheumatol Int1998189710210.1007/s0029600500659833249<p>Development of criteria for the classification and reporting of osteoarthritis, Classification of osteoarthritis of the knee. Diagnostic and Therapeutic Criteria Committee of the American Rheumatism Association</p>AltmanRAschEBlochDBoleGBorensteinDBrandtKChristyWCookeTDGreenwaldRHochbergMHowellDKaplanDKoopmanWLongleySIIIMankinHMcShaneDJMedsgerTJrMeenanRMikkelsenWMoskowitzRMurphyWRotschildBSegalMSokoloffLWolfeFArthritis Rheum1986291039104910.1002/art.17802908163741515<p>Immature murine articular chondrocytes in primary culture: a new tool for investigating cartilage</p>SalvatCPigenetAHumbertLBerenbaumFThirionSOsteoarthritis Cartilage20051324324910.1016/j.joca.2004.11.00815727891<p>Prostaglandin E2 synthesis in cartilage explants under compression: mPGES-1 is a mechanosensitive gene</p>GossetMBerenbaumFLevyAPigenetAThirionSSaffarJLJacquesCArthritis Res Ther20068R13510.1186/ar2024177939216872525<p>Increased serum concentrations of visfatin and its production by different joint tissues in patients with osteoarthritis</p>ChenWPBaoJPFengJHuPFShiZLWuLDClin Chem Lab Med2010481141114520482384<p>Proinflammatory actions of visfatin/nicotinamide phosphoribosyltransferase (Nampt) involve regulation of insulin signaling pathway and Nampt enzymatic activity</p>JacquesCHolzenbergerMMladenovicZSalvatCPecchiEBerenbaumFGossetMJ Biol Chem2012287151001510810.1074/jbc.M112.350215334021722399297<p>FK866, a highly specific noncompetitive inhibitor of nicotinamide phosphoribosyltransferase, represents a novel mechanism for induction of tumor cell apoptosis</p>HasmannMSchemaindaICancer Res2003637436744214612543<p>Stretch-activated signaling of nerve growth factor secretion in bladder and vascular smooth muscle cells from hypertensive and hyperactive rats</p>ClemowDBSteersWDTuttleJBJ Cell Physiol200018328930010.1002/(SICI)1097-4652(200006)183:3<289::AID-JCP1>3.0.CO;2-610797303<p>Mechanical stretch induces nerve sprouting in rat sympathetic neurocytes</p>RanaORSchauertePHommesDSchwingerRHSchroderJWHoffmannRSaygiliEAuton Neurosci2010155253210.1016/j.autneu.2010.01.00320122881<p>Regulation of nerve growth factor in the heart: the role of the calcineurin-NFAT pathway</p>RanaORSaygiliEMeyerCGemeinCKruttgenAAndrzejewskiMGLudwigASchottenUSchwingerRHWeberCWeisJMischkeKRassafTKelmMSchauertePJ Mol Cell Cardiol20094656857810.1016/j.yjmcc.2008.12.00619150448<p>Proinflammatory cytokines stimulate the expression of nerve growth factor by human intervertebral disc cells</p>AbeYAkedaKAnHSAokiYPichikaRMuehlemanCKimuraTMasudaKSpine (Phila Pa 1976)20073263564210.1097/01.brs.0000257556.90850.5317413467<p>Expression and regulation of neurotrophins in the nondegenerate and degenerate human intervertebral disc</p>PurmessurDFreemontAJHoylandJAArthritis Res Ther200810R9910.1186/ar2487257561318727839<p>Cytokine mRNA induction by interleukin-1beta or tumor necrosis factor alpha in vitro and in vivo</p>TaishiPChurchillLDeAObalFJrKruegerJMBrain Res200812268998264247818620339<p>Interleukin-1beta induces angiogenesis and innervation in human intervertebral disc degeneration</p>LeeJMSongJYBaekMJungHYKangHHanIBKwonYDShinDEJ Orthop Res20112926526910.1002/jor.2121020690185<p>Interleukin-1beta in synovial fluid from the arthritic temporomandibular joint and its relation to pain, mobility, and anterior open bite</p>AlstergrenPErnbergMKvarnstromMKoppSJ Oral Maxillofac Surg19985610591065discussion 106610.1016/S0278-2391(98)90256-79734768<p>Interleukin-1-induced subacromial synovitis and shoulder pain in rotator cuff diseases</p>GotohMHamadaKYamakawaHYanagisawaKNakamuraMYamazakiHUeyamaYTamaokiNInoueAFukudaHRheumatology (Oxford)200140995100110.1093/rheumatology/40.9.99511561109<p>Increased interleukin-1beta gene expression in peripheral blood leukocytes is associated with increased pain and predicts risk for progression of symptomatic knee osteoarthritis</p>AtturMBelitskaya-LevyIOhCKrasnokutskySGreenbergJSamuelsJSmilesSLeeSPatelJAl-MussawirHMcDanielGKrausVBAbramsonSBArthritis Rheum2011631908191710.1002/art.30360312842921717421<p>The interleukin 1 inhibitor rilonacept in treatment of chronic gouty arthritis: results of a placebo-controlled, monosequence crossover, non-randomised, single-blind pilot study</p>TerkeltaubRSundyJSSchumacherHRMurphyFBookbinderSBiedermannSWuRMellisSRadinAAnn Rheum Dis2009681613161710.1136/ard.2009.108936273289819635719<p>Sustained remission of symptoms and improved health-related quality of life in patients with cryopyrin-associated periodic syndrome treated with canakinumab: results of a double-blind placebo-controlled randomized withdrawal study</p>Kone-PautILachmannHJKuemmerle-DeschnerJBHachullaELeslieKSMouyRFerreiraALheritierKPatelNPreissRHawkinsPNArthritis Res Ther201113R20210.1186/ar3535333465522152723<p>Osteoarthritis, inflammation and obesity</p>BerenbaumFEymardFHouardXCurr Opin Rheumatol20132511411810.1097/BOR.0b013e32835a941423090672<p>Visfatin: a protein secreted by visceral fat that mimics the effects of insulin</p>FukuharaAMatsudaMNishizawaMSegawaKTanakaMKishimotoKMatsukiYMurakamiMIchisakaTMurakamiHIchisakaTMurakamiHWatanabeETakagiTAkiyoshiMOhtsuboTKiharaSYamashitaSMakishimaMFunahashiTYamanakaSHiramatsuRMatsuzawaYShimomuraIScience200530742643010.1126/science.109724315604363<p>Increased synovial fluid visfatin is positively linked to cartilage degradation biomarkers in osteoarthritis</p>DuanYHaoDLiMWuZLiDYangXQiuGRheumatol Int20123214331437<p>The adipokine visfatin induces tissue factor expression in human coronary artery endothelial cells: another piece in the adipokines puzzle</p>CirilloPDi PalmaVMarescaFPacificoFZivielloFBevilacquaMTrimarcoBLeonardiAChiarielloMThromb Res201213040340810.1016/j.thromres.2012.06.00722726553<p>Visfatin/PBEF/Nampt induces EMMPRIN and MMP-9 production in macrophages via the NAMPT-MAPK (p38, ERK1/2)-NF-kappaB signaling pathway</p>FanYMengSWangYCaoJWangCInt J Mol Med20112760761521327328<p>Extracellular PBEF/NAMPT/visfatin activates pro-inflammatory signalling in human vascular smooth muscle cells through nicotinamide phosphoribosyltransferase activity</p>RomachoTAzcutiaVVazquez-BellaMMatesanzNCercasENevadoJCarraroRRodriguez-ManasLSanchez-FerrerCFPeiroCDiabetologia2009522455246310.1007/s00125-009-1509-219727662<p>Pharmacological inhibition of nicotinamide phosphoribosyltransferase/visfatin enzymatic activity identifies a new inflammatory pathway linked to NAD</p>BussoNKarababaMNobileMRolazAVan GoolFGalliMLeoOSoADe SmedtTPLoS One20083e226710.1371/journal.pone.0002267237733618493620<p>Selective inhibition of PBEF/Visfatin/NAMPT suppresses leukocyte infiltration and cartilage degradation</p>EvansLWilliamsASHayesAJJonesSANowellMArthritis Rheum2011631866187710.1002/art.3033821400478<p>Review: NAD +: a modulator of immune functions</p>GrahnertAGrahnertAKleinCSchillingEWehrhahnJHauschildtSInnate Immun20101721223320388721<p>Compartmentation of NAD + -dependent signalling</p>Koch-NolteFFischerSHaagFZieglerMFEBS Lett20115851651165610.1016/j.febslet.2011.03.04521443875<p>The role of nerve growth factor in hyperosmolar stress induced apoptosis</p>ChangEJImYSKayEPKimJYLeeJELeeHKJ Cell Physiol2008216697710.1002/jcp.2137718300262<p>Mechanisms of nerve growth factor mRNA regulation by interleukin-1 beta in hippocampal cultures: role of second messengers</p>FriedmanWJAltiokNFredholmBBPerssonHJ Neurosci Res199233374610.1002/jnr.4903301061333537<p>Mechanical stress and prostaglandin E2 synthesis in cartilage</p>GossetMBerenbaumFLevyAPigenetAThirionSCavadiasSJacquesCBiorheology20084530132018836232<p>Synergistic effect of interleukin-1 beta and tumor necrosis factor alpha on PGE2 production by articular chondrocytes does not involve PLA2 stimulation</p>BerenbaumFJacquesCThomasGCorvolMTBereziatGMasliahJExp Cell Res199622237938410.1006/excr.1996.00478598226<p>Prostaglandins are powerful inducers of NGF and BDNF production in mouse astrocyte cultures</p>ToyomotoMOhtaMOkumuraKYanoHMatsumotoKInoueSHayashiKIkedaKFEBS Lett200456221121510.1016/S0014-5793(04)00246-715044028<p>Beta adrenergic and prostaglandin receptor activation increases nerve growth factor mRNA content in C6-2B rat astrocytoma cells</p>Dal TosoRDe BernardiMABrookerGCostaEMocchettiIJ Pharmacol Exp Ther1988246119011932458446<p>Inflammatory mediators released following application of irritants on the rat injured incisors, The effect of treatment with anti-inflammatory drugs</p>ChidiacJJAl-AsmarBRifaiKJabburSJSaadeNECytokine20094619420010.1016/j.cyto.2009.01.00819261487<p>Interleukin-1beta-mediated induction of Cox-2 in the CNS contributes to inflammatory pain hypersensitivity</p>SamadTAMooreKASapirsteinABilletSAllchorneAPooleSBonventreJVWoolfCJNature200141047147510.1038/3506856611260714<p>Stimulation of NGF expression and secretion in 3 T3-L1 adipocytes by prostaglandins PGD2, PGJ2, and Delta12-PGJ2</p>BulloMPeeraullyMRTrayhurnPAm J Physiol Endocrinol Metab2005289E62E6710.1152/ajpendo.00008.200515713689<p>Differential expression of prostaglandin receptor mRNAs during adipose cell differentiation</p>BorglumJDPedersenSBAilhaudGNegrelRRichelsenBProstaglandins Other Lipid Mediat19995730531710.1016/S0090-6980(98)00082-310480485<p>Differential regulation of EP receptor isoforms during chondrogenesis and chondrocyte maturation</p>ClarkCASchwarzEMZhangXZiranNMDrissiHO'KeefeRJZuscikMJBiochem Biophys Res Commun200532876477610.1016/j.bbrc.2004.11.07415694412<p>Characterization of the PGE2 receptor subtype in bovine chondrocytes in culture</p>de Brum-FernandesAJMorissetSBkailyGPatryCBr J Pharmacol19961181597160410.1111/j.1476-5381.1996.tb15580.x19098468842420