Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 Feb 21;9:36.
doi: 10.1186/1742-2094-9-36.

NOV/CCN3 Attenuates Inflammatory Pain Through Regulation of Matrix metalloproteinases-2 and -9

Affiliations
Free PMC article

NOV/CCN3 Attenuates Inflammatory Pain Through Regulation of Matrix metalloproteinases-2 and -9

Lara Kular et al. J Neuroinflammation. .
Free PMC article

Abstract

Background: Sustained neuroinflammation strongly contributes to the pathogenesis of pain. The clinical challenge of chronic pain relief led to the identification of molecules such as cytokines, chemokines and more recently matrix metalloproteinases (MMPs) as putative therapeutic targets. Evidence points to a founder member of the matricial CCN family, NOV/CCN3, as a modulator of these inflammatory mediators. We thus investigated the possible involvement of NOV in a preclinical model of persistent inflammatory pain.

Methods: We used the complete Freund's adjuvant (CFA)-induced model of persistent inflammatory pain and cultured primary sensory neurons for in vitro experiments. The mRNA expression of NOV and pro-inflammatory factors were measured with real-time quantitative PCR, CCL2 protein expression was assessed using ELISA, MMP-2 and -9 activities using zymography. The effect of drugs on tactile allodynia was evaluated by the von Frey test.

Results: NOV was expressed in neurons of both dorsal root ganglia (DRG) and dorsal horn of the spinal cord (DHSC). After intraplantar CFA injection, NOV levels were transiently and persistently down-regulated in the DRG and DHSC, respectively, occurring at the maintenance phase of pain (15 days). NOV-reduced expression was restored after treatment of CFA rats with dexamethasone. In vitro, results based on cultured DRG neurons showed that siRNA-mediated inhibition of NOV enhanced IL-1β- and TNF-α-induced MMP-2, MMP-9 and CCL2 expression whereas NOV addition inhibited TNF-α-induced MMP-9 expression through β1 integrin engagement. In vivo, the intrathecal delivery of MMP-9 inhibitor attenuated mechanical allodynia of CFA rats. Importantly, intrathecal administration of NOV siRNA specifically led to an up-regulation of MMP-9 in the DRG and MMP-2 in the DHSC concomitant with increased mechanical allodynia. Finally, NOV intrathecal treatment specifically abolished the induction of MMP-9 in the DRG and, MMP-9 and MMP-2 in the DHSC of CFA rats. This inhibitory effect on MMP is associated with reduced mechanical allodynia.

Conclusions: This study identifies NOV as a new actor against inflammatory pain through regulation of MMPs thus uncovering NOV as an attractive candidate for therapeutic improvement in pain relief.

Figures

Figure 1
Figure 1
Neuronal expression of NOV in DRG. Immunolocalization using CT-Mu anti-mouse NOV antibody (red, b, e, h, k, n) and antibodies against neuronal markers NeuN (a), CGRP (g, m), IB4 (d) and NF200 (j). Overlay of the labeling (merge c, f, i, l, o). No primary antibody (control) and IgG depleted of NOV-specific IgG (flow through, FT) were used as negative controls. Scale: 100 μm.
Figure 2
Figure 2
Neuronal expression of NOV in the dorsal horn of the spinal cord. (A) NOV immunolabeling in dorsal spinal cord laminae and in the dorsolateral funiculus (a, asterisk). (b-d) Immunolocalization using neuronal NeuN (b) and NOV (c) antibodies, overlay (merge, d). No primary antibody (control) and IgG depleted of NOV-specific IgG (flow through, FT) were used as negative controls. Scale: 100 μm. (B) Co-immunostaining using antibodies against NOV (green, b, e, h, k), neuronal (CGRP, a; NF200, d), astrocytic (GFAP, g) and microglial (iba1, j) markers. Overlay of the labeling (merge c, f, i, l). Scale: 50 μm.
Figure 3
Figure 3
NOV expression in DRG and dorsal horn of spinal cord (DHSC) during CFA-induced pain. (A, B) NOV mRNA expression in DRG (A) and DHSC (B) ipsilateral to CFA injection in the hind paw. Transcript levels were quantified by RT-qPCR. Values were normalized to rat S26 mRNA levels. The corresponding control of the same time course (sham, sh) was set to 1. Data represent the mean value ± SEM of two independent experiments realized with four rats per condition. (**P < 0.01 CFA versus corresponding sham). (C) Expression of NOV protein in the DHSC. Representative western blot (left panel) and quantification of the protein levels normalized to GAPDH (right panel). Values are reported relative to the corresponding control (sham, sh) of the same time course (set to 1). Data represent the mean value ± SEM of two independent experiments realized with three rats per condition (*P < 0.05, CFA versus corresponding sham).
Figure 4
Figure 4
Effect of dexamethasone on NOV expression in DRG and dorsal spinal cord. mRNA expression of NOV (A, C) and the cytokines IL-6, CCL2, IL-1β and TNF-α (B, D) in the DRG (A, B) and DHSC (C, D) of rats treated with dexamethasone. Dexamethasone (DEXA, 2 mg/kg i.p) or vehicle (saline) were administered daily for 4 consecutive days to sham or CFA rats, starting from day 9 after CFA or saline injection into the paw. Transcript levels were quantified by RT-qPCR. Values were normalized to rat S26 mRNA levels and reported relative to the control group sham/saline set to 1 (dotted line in B and D). The data represent mean ± SEM of four rats per group of a representative experiment repeated twice (A, C: *P < 0.05, **P < 0.01 CFA/saline versus sham/saline or CFA-DEXA, B, D: *P < 0.05, **P < 0.01 CFA/saline versus sham/saline, †P < 0.05, ††P < 0.01 CFA/saline versus CFA/DEXA).
Figure 5
Figure 5
Effect of NOV knockdown on cytokine-stimulated MMP-2/-9, CCL2 and SP mRNA in DRG neuronal cultures. (A, B) NOV mRNA (A) and protein expression (B) 72 hours following transfection of primary sensory neurons with a control non-silencing siRNA (Ctr) or a specific siRNA against NOV (siNOV). NOV mRNA and secreted protein in the conditioned medium were quantified by RT-qPCR and western blot respectively. (C-F) Experiments were performed 48 hours following transfection with a control (control, Ctr) or a specific NOV siRNA (siNOV). MMP-9, CCL2, MMP-2 and substance P (SP) mRNA were evaluated by RT-qPCR after a 24 hour-exposure to cytokines TNF-α (1 ng/ml) or IL-1β (1 ng/ml). Data reported are fold changes versus the corresponding control of DRG cultures transfected with control siRNA exposed to normal medium (not treated, NT) and represent the mean of four values in a representative experiment performed at least two times with similar results (*P < 0.05 cytokine versus NT. †P < 0.05 siNOV versus Ctr).
Figure 6
Figure 6
Effect of NOV knockdown on cytokine-stimulated MMP-2/-9 activities and CCL2 concentration in DRG neuronal cultures. Experiments were performed 48 hours following transfection of primary sensory neurons with a control non-silencing siRNA (Ctr) or a specific NOV siRNA (siNOV). (A) Upper panel, representative gelatin zymograph showing MMP-9 and MMP-2 enzymatic activities after transfection and treatment for 24 hours with cytokines TNF-α (1 ng/ml) or IL-1β (1 ng/ml). Lower panel, quantification of MMP-9 and MMP-2 gelatinolytic bands. Data represent the mean of four values ± SEM (ns, not significant, *P < 0.05 cytokine versus not treated (NT), †P < 0.05 siNOV versus Ctr). (B, C) CCL2 protein levels produced in conditioned medium of cultured DRG after transfection and exposure to cytokines TNF-α (1 ng/ml) (B) or IL-1β (1 ng/ml) (C) for 24 hours (or no treatment, NT) determined by ELISA. Data represent the mean of four values in a representative experiment performed at least two times with similar results (* P < 0.05 IL-1β or TNF-α versus NT. † P < 0.05 siNOV versus Ctr).
Figure 7
Figure 7
Effect of NOV treatment on TNF-α-stimulated MMP-9 mRNA expression in DRG neuronal cultures. (A) DRG cultures were exposed to TNF-α (1 ng/ml) for 1 hour prior to NOV treatment (1 μg/ml). Cells were harvested 2, 5, 8 or 24 hours after NOV treatment. (B) DRG cultures were exposed to TNF-α (1 ng/ml) for 1 hour prior to NOV treatment (1 μg/ml) for 5 hours with recombinant human NOV protein produced using a baculovirus expression system in insect cells (bac) or with commercial human NOV protein produced in a murine cell line (mu). (C) Cells were infected with NOV adenovirus (Ad-NOV) or control virus for 48 hours and then exposed to TNF-α (1 ng/ml) for an additional 12 hours. MMP-9 mRNA expression was evaluated by RT-qPCR. Data reported are fold changes versus the corresponding control of DRG cultures exposed to normal medium (NT, not treated) and represent the mean of four values in a representative experiment out of two experiments giving similar results (*P < 0.05, **P < 0.01, ***P < 0.001 TNF-α + NOV versus TNF-α alone). (D) DRG cultures were exposed to TNF-α (1 ng/ml) for 1 hour prior to NOV treatment (1 μg/ml for 5 hours) 48 hours following transfection with a control non-silencing siRNA (Ctr) or a specific siRNA against integrin β1, β3 or β5. Values represent the percentage of inhibition ± SEM of MMP-9 by NOV upon TNF-α stimulation compared to TNF-α exposure of two independent experiments (ns: not significant, *P < 0.05, **P < 0.01, ***P < 0.001 TNF-α + NOV versus TNF-α).
Figure 8
Figure 8
MMP-2/-9 expression after CFA injection and the effect of MMP-9 inhibition on CFA-induced mechanical allodynia. (A, B) Levels of MMP-9 and MMP-2 mRNA in the ipsilateral DRG (A) and DHSC, (B) after CFA or vehicle (sham) injection (*P < 0.05, **P < 0.01 CFA versus corresponding sham, n = 4). (C, D) Dexamethasone (DEXA, 2 mg/kg i.p) or vehicle (saline) were daily administered for 4 consecutive days to sham or CFA rats, starting from day 9 after CFA or saline injection into the paw. Transcript levels were quantified by RT-qPCR. Values were normalized to rat S26 mRNA levels and reported to the control group sham/saline set at 1 (dotted line). The data represent the mean ± SEM of 4 rats per group (*P < 0.05, **P < 0.01 CFA/saline versus CFA/DEXA). (E) MMP-9 inhibitor (MMP-9i, 5 μg) or vehicle (10% DMSO) were intrathecally (i.t) injected daily for 4 consecutive days starting from day 9 after CFA injection. The paw withdrawal threshold (g) was evaluated using the von Frey test. Data represent the mean ± SEM of six rats per group (**P < 0.01 MMP-9i- versus vehicle-treated rats).
Figure 9
Figure 9
Effect of in vivo endogenous NOV inhibition on MMP-2/-9 expression and mechanical allodynia. In CFA rats, NOV-specific siRNA (2 μg) or control non-silencing siRNA (Ctr) were delivered intrathecally (i.t) daily for 3 consecutive days. (A) NOV protein levels in DHSC. Representative western blot (left panel) and quantification of protein levels normalized to GAPDH (right panel) (**P < 0.01, siNOV versus Ctr, n = 6) (B, C) Levels of MMP-9 and MMP-2 mRNA in DRG (B) and DHSC. (C) Transcript levels were quantified by RT-qPCR and values were normalized to rat S26 mRNA level. Data represent the mean value ± SEM of two independent experiments realized with three rats per condition (*P < 0.05 siNOV versus Ctr). (D) Representative gelatin zymograph showing MMP-9 and MMP-2 activities in DRG (left panel) and quantification of MMP-2 and MMP-9 gelatinolytic bands (right panel). Data represent the mean ± SEM of six rats per group (**P < 0.01 siNOV versus Ctr). (E) Paw withdrawal threshold (g) of CFA rats intrathecally injected with NOV-specific siRNA or control siRNA evaluated using the von Frey test. Data represent the mean ± SEM of eight rats per group (*P < 0.05 siNOV- versus Ctr-treated rats), BL: baseline.
Figure 10
Figure 10
Effect of NOV treatment of sham and CFA rats on MMP-2/-9 expression and mechanical allodynia. NOV (3 μg/rat) was administrated daily by a single intrathecal injection (i.t) for 4 consecutive days starting from the 9th day after CFA injection. (A, B) Levels of MMP-2 and MMP-9 mRNA in the DRG (A) and DHSC (B) following intrathecal (i.t) treatment with NOV protein or vehicle (*P < 0.05 NOV- versus saline-treated CFA rats, n = 4 to 5). (C) Representative gelatin zymograph showing MMP-9 and MMP-2 activities (left panels) and quantification of MMP-2 and MMP-9 gelatinolytic bands (right panels). Data represent the mean ± SEM of three to four rats per group (*P < 0.05 NOV- versus saline-treated CFA rats). (D) The paw withdrawal threshold (g) was evaluated using the von Frey test. Data represent the mean ± SEM of eight rats per group (*P < 0.05, **P < 0.01 CFA/NOV- versus CFA/saline-treated rats).

Similar articles

See all similar articles

Cited by 13 articles

See all "Cited by" articles

References

    1. Uceyler N, Schafers M, Sommer C. Mode of action of cytokines on nociceptive neurons. Exp Brain Res. 2009;196:67–78. doi: 10.1007/s00221-009-1755-z. - DOI - PubMed
    1. Miller RJ, Jung H, Bhangoo SK, White FA. Cytokine and chemokine regulation of sensory neuron function. Handb Exp Pharmacol. 2009;194:417–449. doi: 10.1007/978-3-540-79090-7_12. - DOI - PMC - PubMed
    1. Abbadie C, Bhangoo S, De Koninck Y, Malcangio M, Melik-Parsadaniantz S, White FA. Chemokines and pain mechanisms. Brain Res Rev. 2009;60:125–134. doi: 10.1016/j.brainresrev.2008.12.002. - DOI - PMC - PubMed
    1. Kawasaki Y, Zhang L, Cheng JK, Ji RR. Cytokine mechanisms of central sensitization: distinct and overlapping role of interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha in regulating synaptic and neuronal activity in the superficial spinal cord. J Neurosci. 2008;28:5189–5194. doi: 10.1523/JNEUROSCI.3338-07.2008. - DOI - PMC - PubMed
    1. Van Steenwinckel J, Reaux-Le Goazigo A, Pommier B, Mauborgne A, Dansereau MA, Kitabgi P, Sarret P, Pohl M, Melik Parsadaniantz S. CCL2 released from neuronal synaptic vesicles in the spinal cord is a major mediator of local inflammation and pain after peripheral nerve injury. J Neurosci. 2011;31:5865–5875. doi: 10.1523/JNEUROSCI.5986-10.2011. - DOI - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources

Feedback