Elsevier

Neuropharmacology

Volume 146, 1 March 2019, Pages 198-211
Neuropharmacology

Inhibition of the protein kinase IKKepsilon attenuates neuropathic pain in mice

https://doi.org/10.1016/j.neuropharm.2018.12.004Get rights and content

Highlights

  • Expression of IKKε is regulated after nerve injury in the SNI model of neuropathic pain.

  • Inhibition of IKKε leads to a decrease of pain hypersensitivity.

  • IKKε knock-out and amlexanox-treated mice show decreased microglia activation after SNI.

  • IKKε knock-out mice show impaired p65 activation and MAPK activation after nerve injury.

  • IKKε knock-out and amlexanox-treated mice show reduced expression of several “pain-relevant” genes in the SNI model.

Abstract

Inhibitor-kappaB kinase epsilon (IKKε, Ikbke) constitutes an NF-κB activating kinase with high homology to the classical I-κB kinase subunits, IKKα and IKKβ. It is expressed in nociceptive neurons in the spinal cord and in dorsal root ganglia (DRG) and involved in inflammatory nociception. Under inflammatory conditions, IKKε deficient mice show significantly less nociceptive behavior in comparison to wild type mice associated with reduced activation of NF-κB and attenuated NF-κB-dependent gene expression. The role of IKKε in neuropathic pain has not been investigated so far. We applied the spared nerve injury (SNI) model of neuropathic pain in mice and found an increased expression of IKKε in the spinal cord, the DRGs and the sciatic nerve after induction of neuropathy. Genetic depletion of IKKε or pharmacological inhibition by amlexanox led to a significant reduction of mechanical hyperalgesia and cold allodynia in comparison to control mice. Transcription factor ELISA indicated that the effects are mediated by reduced activation of NF-κB. Furthermore, immunofluorescence staining, qPCR and Western Blot analyses revealed that the decreased pain-like behavior was associated with a reduced activation of microglia, diminished expression of c-fos as well as a decreased activation of MAP-Kinases. In summary, we conclude that IKKε modulates mechanisms of neuropathic pain by activating NF-κB. The administration of IKKε inhibitors might therefore constitute a new and promising approach for the therapy of neuropathic pain.

Introduction

Chronic neuropathic pain is associated with lesion or disease of the somatosensory system and associated with adaptive changes leading to pain hypersensitivity (hyperalgesia, allodynia) and spontaneous pain (Colloca et al., 2017; Costigan et al., 2009). This debilitating condition affects 7–10% people worldwide and it is not possible to treat neuropathic pain adequately yet (van Hecke et al., 2014). Therefore, novel therapeutic options targeting so far unknown mechanism of neuropathic pain are urgently needed. A number of reports already suggested that activation of the transcription factor NF-κB is involved in the induction of neuropathic pain (Ma and Bisby, 1998; Meunier et al., 2007; Niederberger and Geisslinger, 2008; Sakaue et al., 2001). NF-κB is expressed in neurons as well as astrocytes and microglia (Kaltschmidt et al., 2005). All these cell types contribute to the induction and maintenance of neuropathic pain and neuroinflammation (Moalem and Tracey, 2006; Watkins and Maier, 2002), which is accepted as a pathophysiological condition associated with nerve injury and neuropathic pain (DeLeo and Yezierski, 2001; Gaudet et al., 2011; Sacerdote et al., 2013; Xu et al., 2017). Inactive NF-κB is mostly localized in the cytoplasm of unstimulated cells as a dimer of different subunits trapped to the inhibitory subunit I-κB. NF-κB activation starts by phosphorylation of I-κB by I-κB kinases, followed by its ubiquitinylation and degradation by a proteasome complex. This allows the release of NF-κB from the trapping complex and its translocation into the nucleus where it binds to the promoter region of various genes (Karin et al., 2004; Sacerdote et al., 2013; Yamamoto and Gaynor, 2004). The most common NF-κB activation pathway, which is also relevant in neuropathic pain (Lim et al., 2017; Tegeder et al., 2004), is the so-called “classical” cascade. It depends on phosphorylation of NF-κB subunits by an inhibitor-κB kinase (IKK)-complex consisting of the regulatory subunit IKKγ (also known as NF-κB essential modulator (NEMO)) and the catalytical subunits IKKα and IKKβ (Hacker and Karin, 2006; Viatour et al., 2005; Yamamoto and Gaynor, 2004). Yet, another IKK complex of structurally similar IKKs has also been linked to induction and processing of pathophysiological pain (Moser et al., 2011). IKK epsilon (IKKε) and tank binding kinase 1 (TBK1) are activated by phorbol esters (PMA), lipopolysaccharide (LPS) and cytokines (Peters and Maniatis, 2001; Shimada et al., 1999). They contribute not only to NF-κB activation (Adli and Baldwin, 2006; Buss et al., 2004; Harris et al., 2006; Mattioli et al., 2006; Peters and Maniatis, 2001) but also to activation of type I interferon during viral infections by phosphorylation of interferon regulatory factors (IRF) 3 and 7 (McWhirter et al., 2004; Sharma et al., 2003). A recent study in our lab showed that IKKε is involved in inflammatory nociception. It is expressed in neurons, astrocytes and microglia and upregulated upon inflammatory stimulation. IKKε inhibition is associated with a reduced nociceptive response which is at least partially due to inhibition of NF-κB activation (Moser et al., 2011). Since neurons, microglia and astrocytes as well as IKKε-regulated genes have all been associated with the development of neuropathic pain, we hypothesized that IKKε might also play a role in neuropathy which has not been investigated so far.

Therefore, we assessed the impact of IKKε in the spared nerve injury (SNI) model of neuropathic pain by performing behavioral analyses as well as molecular biological experiments using wild type and IKKε deficient mice as well as mice treated with the IKKε inhibitor amlexanox.

Section snippets

Animals

Homozygous IKKε−/--mice with a C57BL/6 background were purchased from The Jackson Laboratories, USA (B6.Cg-Ikbketm1Tman/J). In these mice, the exons 4–6 of the IKKε gene were replaced by a PGK-neo cassette resulting in an inactive protein. IKKε-/- mice are viable, fertile and healthy. Respective age-matched C57BL/6 wild type mice were purchased from Charles River, Germany as recommended by the supplier. C57BL/6 mice were also used for the experiments with amlexanox administration. Control

IKKε expression and regulation

IKKε mRNA and protein are constitutively expressed in the spinal cord and DRGs of C57BL/6 mice (Moser et al., 2011). Potential SNI-induced regulations of IKKε and TBK1 were analyzed in the spinal cord, the DRGs and the sciatic nerve 7 and 28d after surgery to assess effects of induction and maintenance of neuropathy, respectively. 28 days after SNI surgery, IKKε mRNA levels were significantly increased in the spinal cord, DRGs and the sciatic nerve of wild type mice. This increase was already

Discussion

IKKε is an NF-κB activating kinase involved in inflammatory nociception where its inhibition is associated with an attenuated nociceptive response and a reduction of NF-κB dependent inflammatory genes in the nervous system of mice (Moser et al., 2011). Inhibition of inflammatory responses can also alter the generation of neuropathic pain. Furthermore, IKKε is expressed in immune cells and neurons in the nervous system. For these reasons, we intended to investigate the role of IKKε in peripheral

Author contributions

CVM planned the experiments and performed animal, biochemical and molecular biological analyses and helped writing the manuscript, MM performed SNI surgery and was involved in the preparation of tissue samples and behavioral experiments, SCF was involved in establishing and performing LC-MS/MS analysis, DT was involved in establishing LC-MS/MS analysis, GG participated in the design of the study and editing of the manuscript. EN conceived and designed the study, co-ordinated subprojects,

Conflicts of interest

There is no conflict in financial interest.

Acknowledgement

The authors would like to thank Julia Häusler, Christine Manderscheid and Annett Wilken-Schmitz for excellent technical assistance.

References (61)

  • F. Meng et al.

    Akt is a downstream target of NF-kappa B

    J. Biol. Chem.

    (2002)
  • A. Meunier et al.

    Lentiviral-mediated targeted NF-kappaB blockade in dorsal spinal cord glia attenuates sciatic nerve injury-induced neuropathic pain in the rat

    Mol. Ther.

    (2007)
  • G. Moalem et al.

    Immune and inflammatory mechanisms in neuropathic pain

    Brain Res. Rev.

    (2006)
  • R.T. Peters et al.

    A new family of IKK-related kinases may function as I kappa B kinase kinases

    Biochim. Biophys. Acta

    (2001)
  • M. Tanaka et al.

    Embryonic lethality, liver degeneration, and impaired NF-kappa B activation in IKK-beta-deficient mice

    Immunity

    (1999)
  • R. Terayama et al.

    Activation of microglia and p38 mitogen-activated protein kinase in the dorsal column nucleus contributes to tactile allodynia following peripheral nerve injury

    Neuroscience

    (2008)
  • O. van Hecke et al.

    Neuropathic pain in the general population: a systematic review of epidemiological studies

    Pain

    (2014)
  • P. Viatour et al.

    Phosphorylation of NF-kappaB and IkappaB proteins: implications in cancer and inflammation

    Trends Biochem. Sci.

    (2005)
  • Y.W. Wang et al.

    Protective effects of Garcinol against neuropathic pain - evidence from in vivo and in vitro studies

    Neurosci. Lett.

    (2017)
  • Y. Yamamoto et al.

    IkappaB kinases: key regulators of the NF-kappaB pathway

    Trends Biochem. Sci.

    (2004)
  • H.M. Yu et al.

    Silencing of FKBP51 alleviates the mechanical pain threshold, inhibits DRG inflammatory factors and pain mediators through the NF-kappaB signaling pathway

    Gene

    (2017)
  • I. Bettoni et al.

    Glial TLR4 receptor as new target to treat neuropathic pain: efficacy of a new receptor antagonist in a model of peripheral nerve injury in mice

    Glia

    (2008)
  • L. Colloca et al.

    Neuropathic pain

    Nat Rev Dis Primers

    (2017)
  • M. Costigan et al.

    Neuropathic pain: a maladaptive response of the nervous system to damage

    Annu. Rev. Neurosci.

    (2009)
  • S. Echeverry et al.

    Spinal microglia are required for long-term maintenance of neuropathic pain

    Pain

    (2017)
  • A.D. Gaudet et al.

    Wallerian degeneration: gaining perspective on inflammatory events after peripheral nerve injury

    J. Neuroinflammation

    (2011)
  • L. Giorgi et al.

    Immediate-early genes expression in spinal cord as related to acute noxious stimulus

    Int. J. Clin. Pharmacol. Res.

    (1997)
  • H. Hacker et al.

    Regulation and function of IKK and IKK-related kinases

    Sci. STKE

    (2006)
  • J. Harris et al.

    Nuclear accumulation of cRel following C-terminal phosphorylation by TBK1/IKK epsilon

    J. Immunol.

    (2006)
  • H. Hemmi et al.

    The roles of two IkappaB kinase-related kinases in lipopolysaccharide and double stranded RNA signaling and viral infection

    J. Exp. Med.

    (2004)
  • Cited by (12)

    View all citing articles on Scopus
    View full text