Inhibition of the protein kinase IKKepsilon attenuates neuropathic pain in mice
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)
- et al.
IKK-i/IKKe controls constitutive, cancer cell-associated NF-B activity via regulation of Ser-536 p65/RelA phosphorylation
J. Biol. Chem.
(2006) - et al.
Constitutive and interleukin-1-inducible phosphorylation of p65 NF-{kappa}B at serine 536 is mediated by multiple protein kinases including I{kappa}B kinase (IKK)-{alpha}, IKK{beta}, IKK{epsilon}, TRAF family member-associated (TANK)-binding kinase 1 (TBK1), and an unknown kinase and couples p65 to TATA-binding protein-associated factor II31-mediated interleukin-8 transcription
J. Biol. Chem.
(2004) - et al.
New insights of dimethyl sulphoxide effects (DMSO) on experimental in vivo models of nociception and inflammation
Pharmacol. Res.
(2008) - et al.
Spared nerve injury: an animal model of persistent peripheral neuropathic pain
Pain
(2000) - et al.
The role of neuroinflammation and neuroimmune activation in persistent pain
Pain
(2001) - et al.
Glia and pain: is chronic pain a gliopathy?
Pain
(2013) - et al.
Signaling via NF-kappaB in the nervous system
Biochim. Biophys. Acta
(2005) - et al.
Silencing of IRF3 alleviates chronic neuropathic pain following chronic constriction injury
Biomed. Pharmacother.
(2017) - et al.
Increased activation of nuclear factor kappa B in rat lumbar dorsal root ganglion neurons following partial sciatic nerve injuries
Brain Res.
(1998) - et al.
Inducible phosphorylation of NF-kappa B p65 at serine 468 by T cell costimulation is mediated by IKK epsilon
J. Biol. Chem.
(2006)
Akt is a downstream target of NF-kappa B
J. Biol. Chem.
Lentiviral-mediated targeted NF-kappaB blockade in dorsal spinal cord glia attenuates sciatic nerve injury-induced neuropathic pain in the rat
Mol. Ther.
Immune and inflammatory mechanisms in neuropathic pain
Brain Res. Rev.
A new family of IKK-related kinases may function as I kappa B kinase kinases
Biochim. Biophys. Acta
Embryonic lethality, liver degeneration, and impaired NF-kappa B activation in IKK-beta-deficient mice
Immunity
Activation of microglia and p38 mitogen-activated protein kinase in the dorsal column nucleus contributes to tactile allodynia following peripheral nerve injury
Neuroscience
Neuropathic pain in the general population: a systematic review of epidemiological studies
Pain
Phosphorylation of NF-kappaB and IkappaB proteins: implications in cancer and inflammation
Trends Biochem. Sci.
Protective effects of Garcinol against neuropathic pain - evidence from in vivo and in vitro studies
Neurosci. Lett.
IkappaB kinases: key regulators of the NF-kappaB pathway
Trends Biochem. Sci.
Silencing of FKBP51 alleviates the mechanical pain threshold, inhibits DRG inflammatory factors and pain mediators through the NF-kappaB signaling pathway
Gene
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
Neuropathic pain
Nat Rev Dis Primers
Neuropathic pain: a maladaptive response of the nervous system to damage
Annu. Rev. Neurosci.
Spinal microglia are required for long-term maintenance of neuropathic pain
Pain
Wallerian degeneration: gaining perspective on inflammatory events after peripheral nerve injury
J. Neuroinflammation
Immediate-early genes expression in spinal cord as related to acute noxious stimulus
Int. J. Clin. Pharmacol. Res.
Regulation and function of IKK and IKK-related kinases
Sci. STKE
Nuclear accumulation of cRel following C-terminal phosphorylation by TBK1/IKK epsilon
J. Immunol.
The roles of two IkappaB kinase-related kinases in lipopolysaccharide and double stranded RNA signaling and viral infection
J. Exp. Med.
Cited by (12)
The potential value of amlexanox in the treatment of cancer: Molecular targets and therapeutic perspectives
2022, Biochemical PharmacologyCitation Excerpt :AMX can be used also. Möser and coworkers have demonstrated that AMX administered orally (25 mg/kg) can reduce significantly mechanical hyperalgesia and cold allodynia in a spared nerve injury model of neuropathic pain in mice, through reduction of NFκB activation [47,92]. Moreover, the pharmacomodulation of TBK1 can reduce also inflammatory hyperalgesia in mice [93].
Inhibition of TANK-binding kinase1 attenuates the astrocyte-mediated neuroinflammatory response through YAP signaling after spinal cord injury
2023, CNS Neuroscience and TherapeuticsPromotion of Knee Cartilage Degradation by IκB Kinase ε in the Pathogenesis of Osteoarthritis in Human and Murine Models
2023, Arthritis and RheumatologyMitogen-activated protein kinases as a target for regulating connective tissue growth
2023, Advances in Health and Disease. Volume 67Intrathecal Administration of an Anti‐nociceptive Non-CpG Oligodeoxynucleotide Reduces Glial Activation and Central Sensitization
2021, Journal of Neuroimmune Pharmacology