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Histone acetylation and histone deacetylation in neuropathic pain: An unresolved puzzle?
European Journal of Pharmacology 795 (2017) 36–42
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Neuropharmacology and analgesia
Histone acetylation and histone deacetylation in neuropathic pain: An unresolved puzzle?
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Ravneet Kaur Khangura, Anjana Bali, Amteshwar Singh Jaggi , Nirmal Singh Department of Pharmaceutical Sciences and Drug Research, Punjabi University, Patiala 147002, India
A R T I C L E I N F O
A BS T RAC T
Keywords: Histone acetylase Histone deacetylase Epigenetic Cyclooxygenase-2 Glutamic acid decarboxylase 65 Tumor necrosis factor-ɑ
Chronic pain is broadly classified into somatic, visceral or neuropathic pain depending upon the location and extent of pain perception. Evidences from different animal studies suggest that inflammatory or neuropathic pain is associated with altered acetylation and deacetylation of histone proteins, which result in abnormal transcription of nociceptive processing genes. There have been a number of studies indicating that nerve injury up-regulates histone deacetylase enzymes, which leads to increased histone deacetylation and induce chronic pain. Treatment with histone deacetylase inhibitors relieves pain by normalizing nerve injury-induced down regulation of metabotropic glutamate receptors, glutamate transporters, glutamic acid decarboxylase 65, Neuron restrictive silencer factor and Serum and glucocorticoid inducible kinase 1. On the other hand, a few studies refer to increased expression of histone acetylase enzymes in response to nerve injury that promotes histone acetylation leading to pain induction. Treatment with histone acetyl transferase inhibitors have been reported to relieve chronic pain by blocking the up-regulation of chemokines and cyclooxygenase-2, the critical factors associated with histone acetylation-induced pain. The present review describes the dual role of histone acetylation/deacetylation in development or attenuation of neuropathic pain along with the underlying mechanisms.
1. Introduction Histone deacetylase enzymes, along with acetylpolyamine, amindohydrolases and the acetoin utilization proteins form a super family of histone deacetylase proteins (Leipe et al., 1997). There are 18 different histone deacetylases known till date and based on functions and structures, these are grouped into four classes, I, II, III and IV (Ruijter et al., 2003; Gregoretti et al., 2004). Class I includes histone deacetylase 1, 2, 3 and 8; class II includes histone deacetylase 4, 5, 6, 7, 9 and 10; class III (also termed as sirutins) includes sirutins 1–7 and class IV includes histone deacetylase 11. Class II is further classified into class IIa (includes 4,5,7 and 9) and class IIb (includes 6 and 10) (Xu et al., 2007; Wang et al., 2016). Histone deacetylases have known to play a crucial role in myriad of biological processes in the living organisms, including transcription, chromatin remodelling, cell cycle, signal transduction, and control of gene expression (Yang et al., 2007). The regulation of gene expression occurs through histone acetylation and deacetylation of transcription factors. Histone tails are positively charged due to the presence of amine groups on lysine and arginine amino acids, and are responsible for interaction between the negatively charged phosphate groups on the backbone of DNA. Histone acetyla-
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tion by histone acetylase transferases, counteracts the positive charge by converting amines to amides and decreases the electrostatic interaction of histones with DNA, which renders the chromosomes more accessible for transcription (Shahbazian et al., 2007). Histone deacetylases, on the other hand, removes the acetyl groups, thereby increasing the positive charge of histone tails and increasing the affinity for binding of DNA, leading to the formation of a compacted chromatin that restraints transcription. The opposite actions of two pivotal enzymes, histone acetyl transferases and histone deacetylases maintain the balance of the dynamic process of histone acetylation and deacetylation, which on disruption leads to many neurological disorders (Fig. 1). It has been shown that histone deacetylase enzymes are phosphoproteins (Cai et al., 2001) and their activity is influenced by phosphorylation or non-phosphorylation. It is suggested that Ser421 and Ser423 are constitutively phosphorylated and disruption of these sites reduces the enzymatic activity (Pflum et al., 2001). Studies have shown that these enzymes are critically involved in gene regulation and recent studies indicate a key role of epigenetic regulation in induction of pain which may be acute or chronic (Abel et al., 2008; Buchheit et al., 2012; Wang et al., 2016). There have been a number of experimental studies
Corresponding author. E-mail addresses: [email protected] (R.K. Khangura), [email protected] (A.S. Jaggi).
http://dx.doi.org/10.1016/j.ejphar.2016.12.001 Received 28 June 2016; Received in revised form 25 November 2016; Accepted 1 December 2016 Available online 02 December 2016 0014-2999/ © 2016 Elsevier B.V. All rights reserved.
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of histone deacetylase inhibitor in chronic constriction induced-induced neuropathic pain in rats. Sodium butyrate is a non-competitive inhibitor of histone deacetylase, which selectively inhibits class I and IIa (Khan et al., 2010) and its treatment (200 and 400 mg/kg) for 14 days ameliorated cold and mechanical allodynia, thermal hyperalgesia in a dose-dependent manner (Kukkar et al., 2014). Lu et al. (2010), demonstrated that treatment with histone deacetylase inhibitor, trichostatin A (0.5 mg/kg s.c.) for 4 weeks, suppresses the inflammatory immuno-reaction and manages the pain symptoms. Zammataro et al. (2014), reported the role of curcumin in regulation of pain through histone acetylation. Curcumin is a naturally occurring compound that contains a p300/CREB-binding protein with histone acetyl transferase inhibitory activity (Balasubramanyam et al., 2004). In a mice model of formalin (10 µl) induced pain, systemic administration of curcumin led to down-regulation of metabotropic glutamate receptors type 2 (mGlu2) in the spinal cord along with a marked hypo-acetylation of histones H3 and H4 in dorsal root ganglia. Furthermore, continuous treatment with curcumin (100 mg/kg i.p.) for 3 days suppressed the anti-nociceptive actions of glutamate agonist (LY379268 3 mg/kg s.c.). It suggests that decrease in histone acetylation may be an important mechanism in pain induction. However, pretreatment with histone deacetylase inhibitor, suberoylanilide hydroxamic acid, enhanced the analgesic activity of LY379268, again suggesting that histone acetylation may attenuate pain, while histone hypo-acetylation may potentiate pain. Hobo et al. (2011), demonstrated that administration of histone deacetylase inhibitor, valproic acid, for 3 weeks, produced anti-nociceptive effects, restored the expression of glutamate transporter-1 (GLT-1) and glutamate aspartate transporter (GLAST) in the spinal dorsal horn and enhanced the antinociceptive actions of riluzole in spinal nerve ligation model (Hobo et al., 2011). Previously, administration of histone deacetylase inhibitors, MS-275 and suberoylanilide hydroxamic acid is shown to reduce the nociceptive response in mice model of persistent inflammatory pain through increased expression of metabotropic glutamate receptor expression in dorsal root ganglia (Chiechio et al., 2009). Uchida et al. (2010) demonstrated that the increase in gene expression of neuron-restrictive silencer factor in dorsal root ganglia is the major factor in dysfunction of C-fibers. It was associated with an increase in H4 acetylation at neuron-restrictive silencer factor promoter II region and marked hypo-acetylation of H3 and H4 histones at neuron restrictive silencer element sequences of µ-opioid and Nav 1.8 genes, potential sites for neuron-restrictive silencer factor binding (Ballas et al., 2005). Furthermore, Matsushita et al. (2013), reported that treatment with histone deacetylase inhibitors restore injuryinduced C-fiber sensitivity and reduce thermal and mechanical hypersensitivity, thereby, supporting the anti-nociceptive effects in response to histone acetylation (Matsushita et al., 2013). In a mice model of partial sciatic nerve ligation, histone deacetylase inhibitors restored the expression of pain controlling genes and morphine's analgesic effects (Uchida et al., 2015), suggesting that the latter may act as adjuvant to morphine in pain management. Intrathecal treatment with histone deacetylase inhibitors (MS-275 30 nmol/d and MGCD 0103 60 nmol/ d), was shown to increase the histone acetylation (H3K9) in the spinal cord and reduce the mechanical and thermal hypersensitivity in partial sciatic nerve ligation and stavudine-induced neuropathy (Denk et al., 2013). Bai et al. (2010) demonstrated that histone deacetylase inhibitors specific to class II attenuate inflammatory pain in a more significant manner, as compared to class I deacetylase inhibitors. Furthermore, complete Freund's adjuvant led to selective up-regulation of histone deacetylase of class IIa (HDAC 4,5,7,9) in the spinal cord asserting that histone deacetylase inhibitors specific to class IIa may be sufficient in treating inflammatory pain. A very recent study of Kami et al. demonstrated the increase in the number of histone deacetylase 1 positive microglia and astrocytes in the dorsal horn along with the decrease in the nuclear expression of acetylated histones (H3K9). Interestingly, the authors demonstrated
Fig. 1. Histone acetylation decreases the binding affinity of histones to DNA and relaxes the chromatin to promote transcription. Histone deacetylation increases the binding affinity of histones to DNA and compacts the chromatin to restrain transcription.
documenting that nerve injury up-regulates the levels of histone deacetylase and resulting decreased histone acetylation has been correlated with induction of pain. Accordingly, histone deacetylase inhibitors are documented to attenuate neuropathic and inflammatory pain by increasing histone acetylation (Uchida et al., 2015; Kukkar et al., 2014; Denk et al., 2013; Bai et al., 2010). On the contrary, there have been some studies suggesting that histone acetylation may promote pain and histone acetyl transferase inhibitors attenuate nerve injury-induced pain by decreasing histone acetylation (Kiguchi et al., 2012; Zhu et al., 2012). The present review describes the role of histone acetylation and deacetylation in neuropathic and inflammatory pain along with possible mechanisms. 2. Histone deacetylation promotes pain There have been number of studies documenting that nerve injury or inflammatory conditions increase the expression of histone deacetylase enzymes and induce deacetylation of histone proteins, which may eventually result in pain induction. Accordingly, histone deacetylase inhibitors have been shown to attenuate pain in experimental models of neuropathic and inflammatory pain (Cherng et al., 2014; Zhang et al., 2011). 2.1. Evidences Lin et al. (2015) reported an increase in expression of histone deacetylase 4 in spinal nerve ligation-induced nociceptive hypersensitivity model in rats. Moreover, there was an increase in 14-3-3β, a ubiquitous phosphor-binding protein (Gannon-Murakami and Murakami, 2002), accompanied by its coupling with histone deacetylase 4, leading to retention of deacetylase in the cytoplasm. The selective inhibition of phosphorylation of histone deacetylase 4, using LMK 235, effectively prevented nerve ligation-induced allodynia. Furthermore, knockdown of spinal 14-3-3β prevented coupling of histone deacetylase and 14-3-3β, and behavioural allodynia. However, it failed to prevent phosphorylation of deacetylase, therefore, the authors concluded that phosphorylation of histone deacetylase 4 is upstream to its coupling with 14-3-3β. A recent experiment conducted in the spinal nerve ligation model of neuropathic pain in rats evaluated the ameliorative effect of baicalin, an anti-inflammatory flavonoid (Cherng et al., 2014). Baicalin has been shown to possess an anti-nociceptive effect in carrageenan-induced thermal hyperalgesia (Gao et al., 1999), hence, referring to its potential in treating neuropathic pain. Observations showed that nerve injury resulted in over-expression of histone deacetylase 1 and decrease in histone (H3) acetylation along with development of allodynia and hyperalgesia. Treatment with 10 µg of baicalin was found to significantly decrease deacetylase 1 expression, increase the spinal histone acetylation, reverse pain sensitivity and increase the anti-nociceptive effect of morphine (15 µg). Our own study, explored the beneficial role 37
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systemic inflammation. Many studies conducted on pain models have revealed the predominant role of TNF-ɑ in central sensitization and neuropathic pain (Jaggi and Singh, 2011; Leung et al., 2010). Our study demonstrated that administration of histone deacetylase inhibitor attenuated CCI-induced increase in TNF-ɑ levels, which may be responsible for anti-nociceptive effects (Kukkar et al., 2014). Other studies have also reported that inhibition of histone deacetylation leads to reduced production of cytokines molecules (Leoni et al., 2005, 2002). Lu et al. (2010), showed that treatment with histone deacetylase inhibitor for 4 weeks attenuated endometriosis-induced hyperalgesia and decreased the levels of pain mediators like TNF-ɑ (Lin et al., 2006). Therefore, it may be asserted that inhibition of histone deacetylase leads to decreased production of pro-inflammatory molecules that eventually leads to attenuation of pain symptoms.
that histone deacetylase enzymes were not expressed in the neurons of dorsal horn. Based on these, it was suggested that increase in histone deacetylase 1 activity in the astrocytes may contribute in inducing pain (Kami et al., 2016). A study of Mariaru et al. explored the changes in the expression of histone deacetylase 2 following spinal nerve injuryinduced pain. The authors demonstrated that in the normal state this enzyme is expressed in neurons and astrocytes (more in neurons) with no expression n microglia. However, spinal nerve injury led to increase in expression of histone deacetylase 2 specifically in astrocytes, with no significant change in neurons of dorsal horn (Maiarù et al., 2016). Apart from the studies showing changes in the spinal cord, studies have also shown the changes in histone deacetylases in brainstem raphe magnus. The study of Zhang et al. showed that spinal nerve ligation induces hypo-acetylation of H3 and H4 in nucleus raphe magnus and histone deacetylase inhibitors attenuate nociception (Zhang et al., 2011). A study of Tao et al. reported that histone deacetylase inhibitors attenuate inflammatory pain by altering the gene expression in the raphe nucleus (Tao et al., 2015).
2.2.3. Metabotropic Glutamate receptors type 2 (mGlu2) and glutamate transporters Metabotropic Glutamate receptors type 2 (mGlu2) are located in the glial cells as well as in neurons in the central nervous system (CNS) (Teichberg et al., 1991). Chiechio et al. (2009), demonstrated that treatment with histone deacetylase inhibitors for 5 days, significantly reduced the nociception in formalin-induced inflammatory pain, along with increase in the expression of mGlu2 receptors in the dorsal root ganglia and spinal cord. The mGlu2/3 receptor antagonist (LY341495) blocked the anti-nociceptive actions signifying that the increased expression of mGlu2 receptors in dorsal root ganglia is one of the mechanisms involved in anti-nociceptive actions of histone deacetylase inhibitors. Activation of metabotropic glutamate receptors checks the neurotransmitter release from primary afferent neurons in the dorsal horn (Gerber et al., 2000), and their activation abrogates pain in the models of inflammatory and neuropathic pain (Simmons et al., 2002; Jones at al., 2005). It is proposed that histone deacetylase inhibitors induce the over-expression of mGlu2 receptors via activation of the nuclear factor-ĸB (NF-ĸB) pathway secondary to increased acetylation of p65 subunit at lysine 310 (K310) (Chiechio et al., 2009). Zammataro et al. (2014) reported that curcumin, a compound with histone acetyl transferase inhibitory activity (Balasubramanyam et al., 2004), blocks the acetylation of H3 and H4 histone in the dorsal root ganglia to down regulate mGlu2 receptors in the spinal cord and attenuate the antinociceptive effects of mGlu2 agonist. Studies have explored the effects of repeated administration of histone deacetylase inhibitors on the expression of Glutamate-aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1) in the spinal dorsal horn in spinal nerve ligation model (Hobo et al., 2011). Both GLAST and GLT-1 are present in astrocytes, and GLT-1 plays a more predominant role in the regulation of extra-cellular glutamate in the spinal cord and brain (Beart et al., 2007). Evidences from various studies indicate that deficiency or down-regulation of GLAST and GLT1 in the spinal dorsal horn lead to spontaneous nociceptive behaviours resulting in development of neuropathic pain (Sung et al., 2003; Binns
2.2. Mechanisms The following mechanisms have been described in histone deacetylation (histone hypo-acetylation)-induced pain (Table 1). 2.2.1. Serum and Glucocorticoid-inducible Kinase 1 (SGK 1) These are the subfamily of genes that encodes serine/threonine protein kinase and play a vital role in cellular stress responses. Lin et al. (2015), demonstrated that spinal nerve ligation activates serum and glucocorticoid-inducible kinase 1 that in turn leads to phosphorylation of histone deacetylase 4, followed by its coupling with 14-3-3β, resulting in the retention of deacetylase 4 in the cytoplasm of dorsal horn neurons. Furthermore, treatment with GSK-650394 (serum and glucocorticoid-inducible kinase 1 inhibitor) prevented nerve injuryinduced co-localization of phosphorylated kinase and phosphorylated histone deacetylase 4. Administration of LMK 235 (histone deacetylase 4 inhibitor) prevented allodynia, without modulating phosphorylation of kinase and deacetylase or expression of 14-3-3β expression, suggesting that histone deacetylase 4 is a downstream mediator of serum and glucocorticoid-inducible kinase 1. Furthermore, the observation that LMK 235 inhibits pain without altering histone deacetylase phosphorylation indicates that the steps subsequent to histone deacetylase phosphorylation are more critical in pain induction. Accordingly, it is suggested that 14-3-3β interacts with phosphorylated histone deacetylase 4 and prevents its translocation in the nucleus by cytoplasmic retention of 14-3-3β- histone deacetylase 4 complex, which in turn is critical for neuropathic pain. 2.2.2. Tumor necrosis factor- ɑ (TNF- ɑ) TNF-ɑ is a cytokine cell signaling molecule that is primarily released by macrophages and other immune cell types, triggering the Table 1 Mechanisms involved in histone deacetylation -induced pain. S. No.
Target
Effect
HDAC inhibitor/ HAT inhibitor
References
1.
Serum glucocorticoid-inducible kinase-I (SGK) Tumor necrosis factor-ɑ
Cytoplasmic retention of 14-3-3β- histone deacetylase 4 complex ↑TNF-ɑ and other inflammatory mediators
Lin et al., 2015
3.
Metabotropic Glutamate receptors-2 and glutamate transporters
4.
Neuron restrictive silencer factor
5.
Glutamic acid decarboxylase 65
Increase in the expression of mGlu2 receptors; glutamateaspartate transporter (GLAST) and glutamate transporter-1 (GLT-1) ↓Transcription of Nav 1.8 genes on C-fibers; reduction in mu-opioid receptor expression ↓GABAergic transmission
GSK-650395 (SGK inhibitor), LMK-235 (HDAC-4 inhibitor) Sodium butyrate (non selective HDAC inhibitors) mGlu2/3 receptor antagonist (LY341495; sodium valproate
2.
38
Non selective HDAC inhibitors Non selective HDAC inhibitors
Kukkar et al., 2014; Lin et al., 2006 Chiechio et al., 2009; Hobo et al., 2011 Matsushita et al., 2013;Uchida et al., 2010 Zhang et al., 2011; Knabl, 2008
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nucleus raphe magnus was blocked by TrB receptor antagonist (K252a), in inflammatory pain model of rats, suggesting a role of brain derived nerve factor-TrB signaling in glutamic acid decarboxylase 65 dependent GABAergic function (Tao et al., 2015). Moreover, the ameliorative effects of histone deacetylase inhibitors on pain behaviour were shown to be suppressed by TrB receptor antagonist (K252a) infusion. Based on this, it may be proposed that brain derived nerve factor-TrB signaling along with epigenetic actions of histone deacetylase inhibitors is of great importance for the inducing anti-nociceptive effects.
et al., 2005). Hobo et al. (2011) demonstrated that peripheral nerve injury led to down-regulation of these spinal glutamate transporters, which resulted in increase in behavioural sensitivity in rats. However, repeated treatment with sodium valproate for ten days restored the expression of GLT-1 and GLAST in the spinal dorsal horn and reduced pain hypersensitivity. Moreover, the authors demonstrated that riluzole and valproate may work synergistically to produce analgesia, as valproate may restore the spinal expression of glutamate transporters and riluzole may activate these transporters to produce analgesia. Riluzole is known to activate the glutamate transporters and enhance glutamate uptake (Frizzo et al., 2004). Hence, it can be asserted that histone acetylation may up-regulate mGlu2 receptors and glutamate transporters to attenuate pain, while decrease in histone acetylation may promote pain by down regulating mGlu2 receptors.
3. Histone acetylation promotes pain In contrast to above described studies, there have been few studies documenting that histone acetylation may actually induce pain. Accordingly, histone acetyl transferase inhibitors (which decrease histone acetylation) are reported to attenuate pain. On the other hand histone deacetylase inhibitors (which promote histone acetylation) are suggested to induce pain in neuropathic models (Kiguchi et al., 2012; Sun et al., 2013).
2.2.4. Neuron restrictive silencer factor (NRSF) It is a transcriptional repressor gene in humans and is responsible for the repressed transcription of neuron-restrictive silencer element (NRSE)-containing genes Nav1.8, Kav4.3 and µ-opioid receptor genes by recruiting histone deacetylase enzymes (Schoenherr et al., 1995; Chong et al., 1995; Ooi et al., 2007). Nav 1.8 channels are expressed on unmyelinated neurons known as C-fibers, specifically confined to dorsal root ganglia (Akopian et al., 1996; Rabert et al., 1998) and play an important role in pain perception through nociceptive pathway (Akopian et al., 1999). Matsushita et al. (2013), demonstrated that inhibitors of histone deacetylase restore the electrophysiological sensitivity of C-fibers along with recovery of Nav 1.8 sodium channel transcription in a mice model of partial sciatic nerve ligation. It was suggested that during nerve injury, extensive histone deacetylation of histone 3 and histone 4 bound to Nav 1.8 sodium channel gene around the neuron-restrictive silencing elements (NRSE) region, resulted in more condensed hetero-chromatin structure, leading to inaccessibility of RNA polymerases and transcriptional repression of Nav 1.8 sodium channel genes. However, impediment of histone deacetylation led to increased accessibility of RNA polymerase leading to reversal of down regulated genes including Nav1.8 sodium channels and reversal of injury-induced insensitivity of C-fibers (Vanhaecke et al., 2004). Uchida et al. (2015), further studied the involvement of histone deacetylase inhibitors in injury-induced down-regulation of mu-opioid receptor gene expression and attenuation of morphine resistance in neuropathic pain. Histone deacetylase inhibitor was shown to reverse nerve injury-induced reduction in mu-opioid receptor expression and restore the anti-nociceptive actions of morphine. Previous studies of same authors showed that histone hypoacetylation-mediated silencing of NRSE-containing mu-opioid receptor and Nav 1.8 genes may contribute in morphine resistance and hypoesthesia (Uchida et al., 2010).
3.1. Evidences Kiguchi et al. (2012), reported a critical role of histone acetylation and chemokines in the augmentation of neuropathic pain. In a partial sciatic nerve ligation model of mice, it was demonstrated that the upregulation of certain chemokines after nerve injury led to accumulation of macrophages and neutrophils in the injured sciatic nerve to induce pain. The elevated chemokine level was found to be a consequence of increased acetylation of histone H3 [lysine (Lys9)-acetylated histone H3 (AcK9-H3)] on the chemokine specific promoter regions in the nuclei of neutrophils and macrophages surrounding the epineurium, which resulted in severe neuroinflammation. Administration of histone acetyl transferase inhibitor attenuated pain signifying that nerve injury may promote histone acetylation and histone acetyl transferase inhibitors may reduce nerve injury-induced inflammation and pain. Another study reveals the opposite effects of histone acetyl transferase inhibitor and histone deacetylase inhibitor in hind paw incision model of pain in mice (Sun et al., 2013). Following the incision, increased histone acetylation and mechanical sensitization was found to be suppressed by histone acetyl transferase inhibitor. However, treatment with histone deacetylase inhibitor aggravated the mechanical sensitization and increased the acetylation of H3 histone at lysine residue 9 (H3K9) in the spinal dorsal horn. The attenuation of nerve pain, by virtue of decreased histone acetylation and aggravation of pain by increase in histone acetylation, emphasizes that histone acetylation and deacetylation is a critical step in nerve injury pain. Zhu et al. (2012), demonstrated that the levels of p300, a histone acetyl transferase, were increased in the spinal cord and inhibition of p300, using small interfering RNA or a p300 inhibitor (C646) attenuated CCIinduced pain again suggesting that histone acetyl transferase inhibitors attenuate pain.
2.2.5. Glutamic acid decarboxylase 65 (GAD 65) It is an enzyme essential for the synthesis of gamma amino butyric acid (GABA) at the synaptic terminals (Tian et al., 1999) and the loss of glutamic acid decarboxylase 65 activity in the spinal dorsal horn after peripheral nerve injury has been indicated in induction of pain behaviour (Lorenzo et al., 2014). In spinal nerve ligation model, a restrained GABAergic synaptic inhibition due to decrease in glutamic acid decarboxylase 65 gene expression and hypo-acetylation of H3 and H4 in nucleus raphe magnus was associated with persistent pain sensitization in rats (Zhang et al., 2011). Similarly, the results from glutamic acid decarboxylase 65 knockout mice support that the impaired GABA activity in the neurons is responsible for increased pain perception. However, treatment with histone deacetylase inhibitors, leading to histone hyper-acetylation, promotes glutamic acid decarboxylase 65 expression and improves GABA activity, thereby, reducing the sensitized pain behaviour (Knabl, 2008). Recently, it was observed that histone deacetylase inhibitors-induced increase in glutamic acid decarboxylase 65 expression in pre-synaptic terminals of
3.2. Mechanisms Nerve injury induced histone acetylation may promote pain perception through following underlying mechanisms (Fig. 2). 3.2.1. Chemokines An increase in the level of inflammatory molecules like cytokines and chemokines are known key regulators of neuropathic pain (Marchand et al., 2005; Gao et al., 2010). The acetylation of histone proteins specifically promotes the up-regulation of cytokine molecules (Schmeck et al., 2008; Yu et al., 2002), and accordingly, histone acetylation may promote the development of pain through increase in the levels of cytokines (Doehring et al., 2011). Kiguchi et al. (2012), 39
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nerve injury-induced or inflammatory pain (Matsushita et al., 2013; Bai et al., 2010). The different mechanisms involved in decrease in histone acetylation-induced pain include decrease in mGlu2 receptors in the DRG and spinal cord (Zammataro et al., 2014); up-regulation of TNF-ɑ levels (Kukkar et al., 2014); down-regulation of glutamate transporters such as GLAST and GLT-1 (Hobo et al., 2011); downregulation of mu-opioid receptors (Uchida et al., 2015) and decrease in glutamic acid decarboxylase 65 expression, leading to decrease in synaptic GABAergic inhibition (Zhang et al., 2011). In contrast, there are studies that acknowledge histone acetylation as a prominent cause for pain perception. In chronic constriction injury model, it has been shown that levels of histone acetyl transferase are elevated along with cyclooxygenase-2, which resulted in release of prostaglandins leading to spinal hyper-excitability. Treatment with histone acetyl transferase inhibitor or p300 inhibitor significantly attenuated pain symptoms, thereby suggesting a major role of histone acetylation in induction of pain (Zhu et al., 2012; Kiguchi et al., 2012; Sun et al., 2013). Accordingly, increased histone acetylation may have ameliorative effects under one chronic pain condition (Zhang et al., 2011; Bai et al., 2010), while it could trigger nociception under different chronic pain conditions (Sun et al., 2013). A number of critical issues remain unanswered, which require an addressing in the view of histone acetylation and deacetylation resulting in epigenetic regulation. Firstly, there is a lack of consistency in these pain studies, which makes the interpretation more difficult. The studies employing changes in histone acetylation and histone deacetylase have not taken into account the global change of histone acetylation or deacetylation at the relevant sites. A single histone contains multiple lysine residues that are sensitive to acetylation (Tan et al., 2011). Most of the studies consider only a few of lysine residues (H3K9 and H3K18) and had no view of remaining lysine residues. Furthermore, histone deacetylase enzymes are of different types, with different distribution pattern in the central nervous system, different acetylation pattern and hence, functional significance. Secondly, there has been lack of specificity of histone deacetylase inhibitors employed in different studies (Chang et al., 2012). For example, sodium valproate has been very commonly employed histone deacetylase inhibitors in pain studies. However, the other properties of sodium valproate (for which it is clinically employed) may also influence the pain sensitivity, which makes the interpretation more complex. Lastly, there is not much evidence to compare the potential effects of histone deacetylase inhibitors in neuropathic pain due to employment of different techniques of inducing pain, different species and sex in these models of pain. A study conducted on stress associated hypersensitivity revealed positive effects of histone acetyl transferase inhibitors on female offspring, when compared to male offspring (Winston et al., 2014). Nevertheless, the more critical analysis of these reports suggest that apart from the study of Sun et al., 2013, all other studies have shown the pain attenuating actions of histone deacetylase inhibitors. The contradictory results of histone deacetylase inhibitor in this study may be possibly due to employment of non-neuropathic pain model i.e., hind paw incision pain model, which is distinctly different from nerve injury models (Sun et al., 2013). Furthermore, studies have also shown that histone acetyl transferase inhibitors reduce pain in different pain models (Kiguchi et al., 2012; Zhu et al., 2012; Sun et al., 2013). Therefore, it is possible to suggest that perhaps both histone deacetylase and histone acetyl transferase inhibitors are useful in pain management. The possibility of activation of both histone deacetylase and histone acetyl transferase in response to nerve injury may not be ruled out. During nerve injury, the activation of histone deacetylase may possibly inhibit certain transcriptional processes, while histone acetyl transferase may possibly activate other transcriptional processes. It is well known that neuropathic pain conditions are associated with increase as well as decrease in transcriptional factors (Jaggi and Singh, 2011, 2012). Accordingly, further studies may be designed to identify the relative activation of both enzymes (deacetylase and acetyl trans-
Fig. 2. Histone acetylation promotes pain through different pathways including: chemokines and cyclooxygenase-2 pathway.
demonstrated that the expression of CXC chemokine ligand type 2 [macrophage inflammatory protein 2 (MIP-2)] and CXC chemokine receptor type 2 (CXCR2) was up-regulated in the neutrophils and macrophages of the injured nerves, that was reversed by perineural injection of MIP-2-neutralizing antibody (anti-MIP-2) or the CXCR2 antagonist N-(2-bromophenyl)-N-(2-hydroxy-4-nitrophenyl) urea (SB225002). Furthermore, there was increase in acetylation of histone H3 on the promoter region of MIP-2 and CXCR2 that was suppressed by histone acetyl transferase inhibitor, anacardic acid. Furthermore, histone acetyl transferase inhibitor also blocked the accumulation of neutrophils in injured sciatic nerve. Hence, it can be implied that augmentation of the MIP-2/CXCR2 axis by hyper-acetylation of histone H3 on the promoter region of MIP-2 and CXCR2 may induce chronic neuroinflammation that results in neuropathic pain. Sun et al. (2013) asserted that histone deacetylase inhibitor aggravates the levels of two major CXCR2 ligands: KC (CXCL1) and MIP-2 (CXCL2) in response to hind paw incision, without any relevant change in expressions of MIP-2 suggesting that histone acetylation may lead to up-regulation of chemokines. There have been number of studies indicating the vital role of chemokines in nociceptive sensitization (Manjavachi et al., 2010). Based on the observations, it can be proposed that nerve injury may induce histone acetylation, which inturn may induce neuro-inflammation by up-regulating chemokine receptors to produce neuropathic pain. 3.2.2. Cyclooxygenase-2 (COX-2) Many studies have revealed the over-expression of COX-2 in the injured nerve in different models of neuropathic pain (Ma, 2003; Jean et al., 2009). A study conducted by Zhu et al. (2012), correlated the increased expression of COX-2 with histone acetylation in a neuropathy model. The authors observed that increase in the expression of p300, a histone acetyl transferase, in the spinal cord along with up-regulation of COX-2 levels and inhibition of histone acetyl transferase activity by p300 inhibitor lowered nerve injury-induced increase in p300 expression and COX-2 levels. Therefore, it has been proposed that overexpression of histone acetyl transferase, p300, may produce neuropathic pain through up-regulation of COX-2 expression. 4. Summary and discussion Most of the studies have described that during nerve injury or inflammatory conditions, there is an increased expression or activity of histone deacetylases, which tends to deacetylase histone proteins. The hypo-acetylation of histone in-turn activates the different pathways to induce pain and, accordingly, histone deacetylase inhibitors attenuate 40
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Contents lists available at ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Neuropharmacology and analgesia
Histone acetylation and histone deacetylation in neuropathic pain: An unresolved puzzle?
crossmark
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Ravneet Kaur Khangura, Anjana Bali, Amteshwar Singh Jaggi , Nirmal Singh Department of Pharmaceutical Sciences and Drug Research, Punjabi University, Patiala 147002, India
A R T I C L E I N F O
A BS T RAC T
Keywords: Histone acetylase Histone deacetylase Epigenetic Cyclooxygenase-2 Glutamic acid decarboxylase 65 Tumor necrosis factor-ɑ
Chronic pain is broadly classified into somatic, visceral or neuropathic pain depending upon the location and extent of pain perception. Evidences from different animal studies suggest that inflammatory or neuropathic pain is associated with altered acetylation and deacetylation of histone proteins, which result in abnormal transcription of nociceptive processing genes. There have been a number of studies indicating that nerve injury up-regulates histone deacetylase enzymes, which leads to increased histone deacetylation and induce chronic pain. Treatment with histone deacetylase inhibitors relieves pain by normalizing nerve injury-induced down regulation of metabotropic glutamate receptors, glutamate transporters, glutamic acid decarboxylase 65, Neuron restrictive silencer factor and Serum and glucocorticoid inducible kinase 1. On the other hand, a few studies refer to increased expression of histone acetylase enzymes in response to nerve injury that promotes histone acetylation leading to pain induction. Treatment with histone acetyl transferase inhibitors have been reported to relieve chronic pain by blocking the up-regulation of chemokines and cyclooxygenase-2, the critical factors associated with histone acetylation-induced pain. The present review describes the dual role of histone acetylation/deacetylation in development or attenuation of neuropathic pain along with the underlying mechanisms.
1. Introduction Histone deacetylase enzymes, along with acetylpolyamine, amindohydrolases and the acetoin utilization proteins form a super family of histone deacetylase proteins (Leipe et al., 1997). There are 18 different histone deacetylases known till date and based on functions and structures, these are grouped into four classes, I, II, III and IV (Ruijter et al., 2003; Gregoretti et al., 2004). Class I includes histone deacetylase 1, 2, 3 and 8; class II includes histone deacetylase 4, 5, 6, 7, 9 and 10; class III (also termed as sirutins) includes sirutins 1–7 and class IV includes histone deacetylase 11. Class II is further classified into class IIa (includes 4,5,7 and 9) and class IIb (includes 6 and 10) (Xu et al., 2007; Wang et al., 2016). Histone deacetylases have known to play a crucial role in myriad of biological processes in the living organisms, including transcription, chromatin remodelling, cell cycle, signal transduction, and control of gene expression (Yang et al., 2007). The regulation of gene expression occurs through histone acetylation and deacetylation of transcription factors. Histone tails are positively charged due to the presence of amine groups on lysine and arginine amino acids, and are responsible for interaction between the negatively charged phosphate groups on the backbone of DNA. Histone acetyla-
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tion by histone acetylase transferases, counteracts the positive charge by converting amines to amides and decreases the electrostatic interaction of histones with DNA, which renders the chromosomes more accessible for transcription (Shahbazian et al., 2007). Histone deacetylases, on the other hand, removes the acetyl groups, thereby increasing the positive charge of histone tails and increasing the affinity for binding of DNA, leading to the formation of a compacted chromatin that restraints transcription. The opposite actions of two pivotal enzymes, histone acetyl transferases and histone deacetylases maintain the balance of the dynamic process of histone acetylation and deacetylation, which on disruption leads to many neurological disorders (Fig. 1). It has been shown that histone deacetylase enzymes are phosphoproteins (Cai et al., 2001) and their activity is influenced by phosphorylation or non-phosphorylation. It is suggested that Ser421 and Ser423 are constitutively phosphorylated and disruption of these sites reduces the enzymatic activity (Pflum et al., 2001). Studies have shown that these enzymes are critically involved in gene regulation and recent studies indicate a key role of epigenetic regulation in induction of pain which may be acute or chronic (Abel et al., 2008; Buchheit et al., 2012; Wang et al., 2016). There have been a number of experimental studies
Corresponding author. E-mail addresses: [email protected] (R.K. Khangura), [email protected] (A.S. Jaggi).
http://dx.doi.org/10.1016/j.ejphar.2016.12.001 Received 28 June 2016; Received in revised form 25 November 2016; Accepted 1 December 2016 Available online 02 December 2016 0014-2999/ © 2016 Elsevier B.V. All rights reserved.
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of histone deacetylase inhibitor in chronic constriction induced-induced neuropathic pain in rats. Sodium butyrate is a non-competitive inhibitor of histone deacetylase, which selectively inhibits class I and IIa (Khan et al., 2010) and its treatment (200 and 400 mg/kg) for 14 days ameliorated cold and mechanical allodynia, thermal hyperalgesia in a dose-dependent manner (Kukkar et al., 2014). Lu et al. (2010), demonstrated that treatment with histone deacetylase inhibitor, trichostatin A (0.5 mg/kg s.c.) for 4 weeks, suppresses the inflammatory immuno-reaction and manages the pain symptoms. Zammataro et al. (2014), reported the role of curcumin in regulation of pain through histone acetylation. Curcumin is a naturally occurring compound that contains a p300/CREB-binding protein with histone acetyl transferase inhibitory activity (Balasubramanyam et al., 2004). In a mice model of formalin (10 µl) induced pain, systemic administration of curcumin led to down-regulation of metabotropic glutamate receptors type 2 (mGlu2) in the spinal cord along with a marked hypo-acetylation of histones H3 and H4 in dorsal root ganglia. Furthermore, continuous treatment with curcumin (100 mg/kg i.p.) for 3 days suppressed the anti-nociceptive actions of glutamate agonist (LY379268 3 mg/kg s.c.). It suggests that decrease in histone acetylation may be an important mechanism in pain induction. However, pretreatment with histone deacetylase inhibitor, suberoylanilide hydroxamic acid, enhanced the analgesic activity of LY379268, again suggesting that histone acetylation may attenuate pain, while histone hypo-acetylation may potentiate pain. Hobo et al. (2011), demonstrated that administration of histone deacetylase inhibitor, valproic acid, for 3 weeks, produced anti-nociceptive effects, restored the expression of glutamate transporter-1 (GLT-1) and glutamate aspartate transporter (GLAST) in the spinal dorsal horn and enhanced the antinociceptive actions of riluzole in spinal nerve ligation model (Hobo et al., 2011). Previously, administration of histone deacetylase inhibitors, MS-275 and suberoylanilide hydroxamic acid is shown to reduce the nociceptive response in mice model of persistent inflammatory pain through increased expression of metabotropic glutamate receptor expression in dorsal root ganglia (Chiechio et al., 2009). Uchida et al. (2010) demonstrated that the increase in gene expression of neuron-restrictive silencer factor in dorsal root ganglia is the major factor in dysfunction of C-fibers. It was associated with an increase in H4 acetylation at neuron-restrictive silencer factor promoter II region and marked hypo-acetylation of H3 and H4 histones at neuron restrictive silencer element sequences of µ-opioid and Nav 1.8 genes, potential sites for neuron-restrictive silencer factor binding (Ballas et al., 2005). Furthermore, Matsushita et al. (2013), reported that treatment with histone deacetylase inhibitors restore injuryinduced C-fiber sensitivity and reduce thermal and mechanical hypersensitivity, thereby, supporting the anti-nociceptive effects in response to histone acetylation (Matsushita et al., 2013). In a mice model of partial sciatic nerve ligation, histone deacetylase inhibitors restored the expression of pain controlling genes and morphine's analgesic effects (Uchida et al., 2015), suggesting that the latter may act as adjuvant to morphine in pain management. Intrathecal treatment with histone deacetylase inhibitors (MS-275 30 nmol/d and MGCD 0103 60 nmol/ d), was shown to increase the histone acetylation (H3K9) in the spinal cord and reduce the mechanical and thermal hypersensitivity in partial sciatic nerve ligation and stavudine-induced neuropathy (Denk et al., 2013). Bai et al. (2010) demonstrated that histone deacetylase inhibitors specific to class II attenuate inflammatory pain in a more significant manner, as compared to class I deacetylase inhibitors. Furthermore, complete Freund's adjuvant led to selective up-regulation of histone deacetylase of class IIa (HDAC 4,5,7,9) in the spinal cord asserting that histone deacetylase inhibitors specific to class IIa may be sufficient in treating inflammatory pain. A very recent study of Kami et al. demonstrated the increase in the number of histone deacetylase 1 positive microglia and astrocytes in the dorsal horn along with the decrease in the nuclear expression of acetylated histones (H3K9). Interestingly, the authors demonstrated
Fig. 1. Histone acetylation decreases the binding affinity of histones to DNA and relaxes the chromatin to promote transcription. Histone deacetylation increases the binding affinity of histones to DNA and compacts the chromatin to restrain transcription.
documenting that nerve injury up-regulates the levels of histone deacetylase and resulting decreased histone acetylation has been correlated with induction of pain. Accordingly, histone deacetylase inhibitors are documented to attenuate neuropathic and inflammatory pain by increasing histone acetylation (Uchida et al., 2015; Kukkar et al., 2014; Denk et al., 2013; Bai et al., 2010). On the contrary, there have been some studies suggesting that histone acetylation may promote pain and histone acetyl transferase inhibitors attenuate nerve injury-induced pain by decreasing histone acetylation (Kiguchi et al., 2012; Zhu et al., 2012). The present review describes the role of histone acetylation and deacetylation in neuropathic and inflammatory pain along with possible mechanisms. 2. Histone deacetylation promotes pain There have been number of studies documenting that nerve injury or inflammatory conditions increase the expression of histone deacetylase enzymes and induce deacetylation of histone proteins, which may eventually result in pain induction. Accordingly, histone deacetylase inhibitors have been shown to attenuate pain in experimental models of neuropathic and inflammatory pain (Cherng et al., 2014; Zhang et al., 2011). 2.1. Evidences Lin et al. (2015) reported an increase in expression of histone deacetylase 4 in spinal nerve ligation-induced nociceptive hypersensitivity model in rats. Moreover, there was an increase in 14-3-3β, a ubiquitous phosphor-binding protein (Gannon-Murakami and Murakami, 2002), accompanied by its coupling with histone deacetylase 4, leading to retention of deacetylase in the cytoplasm. The selective inhibition of phosphorylation of histone deacetylase 4, using LMK 235, effectively prevented nerve ligation-induced allodynia. Furthermore, knockdown of spinal 14-3-3β prevented coupling of histone deacetylase and 14-3-3β, and behavioural allodynia. However, it failed to prevent phosphorylation of deacetylase, therefore, the authors concluded that phosphorylation of histone deacetylase 4 is upstream to its coupling with 14-3-3β. A recent experiment conducted in the spinal nerve ligation model of neuropathic pain in rats evaluated the ameliorative effect of baicalin, an anti-inflammatory flavonoid (Cherng et al., 2014). Baicalin has been shown to possess an anti-nociceptive effect in carrageenan-induced thermal hyperalgesia (Gao et al., 1999), hence, referring to its potential in treating neuropathic pain. Observations showed that nerve injury resulted in over-expression of histone deacetylase 1 and decrease in histone (H3) acetylation along with development of allodynia and hyperalgesia. Treatment with 10 µg of baicalin was found to significantly decrease deacetylase 1 expression, increase the spinal histone acetylation, reverse pain sensitivity and increase the anti-nociceptive effect of morphine (15 µg). Our own study, explored the beneficial role 37
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systemic inflammation. Many studies conducted on pain models have revealed the predominant role of TNF-ɑ in central sensitization and neuropathic pain (Jaggi and Singh, 2011; Leung et al., 2010). Our study demonstrated that administration of histone deacetylase inhibitor attenuated CCI-induced increase in TNF-ɑ levels, which may be responsible for anti-nociceptive effects (Kukkar et al., 2014). Other studies have also reported that inhibition of histone deacetylation leads to reduced production of cytokines molecules (Leoni et al., 2005, 2002). Lu et al. (2010), showed that treatment with histone deacetylase inhibitor for 4 weeks attenuated endometriosis-induced hyperalgesia and decreased the levels of pain mediators like TNF-ɑ (Lin et al., 2006). Therefore, it may be asserted that inhibition of histone deacetylase leads to decreased production of pro-inflammatory molecules that eventually leads to attenuation of pain symptoms.
that histone deacetylase enzymes were not expressed in the neurons of dorsal horn. Based on these, it was suggested that increase in histone deacetylase 1 activity in the astrocytes may contribute in inducing pain (Kami et al., 2016). A study of Mariaru et al. explored the changes in the expression of histone deacetylase 2 following spinal nerve injuryinduced pain. The authors demonstrated that in the normal state this enzyme is expressed in neurons and astrocytes (more in neurons) with no expression n microglia. However, spinal nerve injury led to increase in expression of histone deacetylase 2 specifically in astrocytes, with no significant change in neurons of dorsal horn (Maiarù et al., 2016). Apart from the studies showing changes in the spinal cord, studies have also shown the changes in histone deacetylases in brainstem raphe magnus. The study of Zhang et al. showed that spinal nerve ligation induces hypo-acetylation of H3 and H4 in nucleus raphe magnus and histone deacetylase inhibitors attenuate nociception (Zhang et al., 2011). A study of Tao et al. reported that histone deacetylase inhibitors attenuate inflammatory pain by altering the gene expression in the raphe nucleus (Tao et al., 2015).
2.2.3. Metabotropic Glutamate receptors type 2 (mGlu2) and glutamate transporters Metabotropic Glutamate receptors type 2 (mGlu2) are located in the glial cells as well as in neurons in the central nervous system (CNS) (Teichberg et al., 1991). Chiechio et al. (2009), demonstrated that treatment with histone deacetylase inhibitors for 5 days, significantly reduced the nociception in formalin-induced inflammatory pain, along with increase in the expression of mGlu2 receptors in the dorsal root ganglia and spinal cord. The mGlu2/3 receptor antagonist (LY341495) blocked the anti-nociceptive actions signifying that the increased expression of mGlu2 receptors in dorsal root ganglia is one of the mechanisms involved in anti-nociceptive actions of histone deacetylase inhibitors. Activation of metabotropic glutamate receptors checks the neurotransmitter release from primary afferent neurons in the dorsal horn (Gerber et al., 2000), and their activation abrogates pain in the models of inflammatory and neuropathic pain (Simmons et al., 2002; Jones at al., 2005). It is proposed that histone deacetylase inhibitors induce the over-expression of mGlu2 receptors via activation of the nuclear factor-ĸB (NF-ĸB) pathway secondary to increased acetylation of p65 subunit at lysine 310 (K310) (Chiechio et al., 2009). Zammataro et al. (2014) reported that curcumin, a compound with histone acetyl transferase inhibitory activity (Balasubramanyam et al., 2004), blocks the acetylation of H3 and H4 histone in the dorsal root ganglia to down regulate mGlu2 receptors in the spinal cord and attenuate the antinociceptive effects of mGlu2 agonist. Studies have explored the effects of repeated administration of histone deacetylase inhibitors on the expression of Glutamate-aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1) in the spinal dorsal horn in spinal nerve ligation model (Hobo et al., 2011). Both GLAST and GLT-1 are present in astrocytes, and GLT-1 plays a more predominant role in the regulation of extra-cellular glutamate in the spinal cord and brain (Beart et al., 2007). Evidences from various studies indicate that deficiency or down-regulation of GLAST and GLT1 in the spinal dorsal horn lead to spontaneous nociceptive behaviours resulting in development of neuropathic pain (Sung et al., 2003; Binns
2.2. Mechanisms The following mechanisms have been described in histone deacetylation (histone hypo-acetylation)-induced pain (Table 1). 2.2.1. Serum and Glucocorticoid-inducible Kinase 1 (SGK 1) These are the subfamily of genes that encodes serine/threonine protein kinase and play a vital role in cellular stress responses. Lin et al. (2015), demonstrated that spinal nerve ligation activates serum and glucocorticoid-inducible kinase 1 that in turn leads to phosphorylation of histone deacetylase 4, followed by its coupling with 14-3-3β, resulting in the retention of deacetylase 4 in the cytoplasm of dorsal horn neurons. Furthermore, treatment with GSK-650394 (serum and glucocorticoid-inducible kinase 1 inhibitor) prevented nerve injuryinduced co-localization of phosphorylated kinase and phosphorylated histone deacetylase 4. Administration of LMK 235 (histone deacetylase 4 inhibitor) prevented allodynia, without modulating phosphorylation of kinase and deacetylase or expression of 14-3-3β expression, suggesting that histone deacetylase 4 is a downstream mediator of serum and glucocorticoid-inducible kinase 1. Furthermore, the observation that LMK 235 inhibits pain without altering histone deacetylase phosphorylation indicates that the steps subsequent to histone deacetylase phosphorylation are more critical in pain induction. Accordingly, it is suggested that 14-3-3β interacts with phosphorylated histone deacetylase 4 and prevents its translocation in the nucleus by cytoplasmic retention of 14-3-3β- histone deacetylase 4 complex, which in turn is critical for neuropathic pain. 2.2.2. Tumor necrosis factor- ɑ (TNF- ɑ) TNF-ɑ is a cytokine cell signaling molecule that is primarily released by macrophages and other immune cell types, triggering the Table 1 Mechanisms involved in histone deacetylation -induced pain. S. No.
Target
Effect
HDAC inhibitor/ HAT inhibitor
References
1.
Serum glucocorticoid-inducible kinase-I (SGK) Tumor necrosis factor-ɑ
Cytoplasmic retention of 14-3-3β- histone deacetylase 4 complex ↑TNF-ɑ and other inflammatory mediators
Lin et al., 2015
3.
Metabotropic Glutamate receptors-2 and glutamate transporters
4.
Neuron restrictive silencer factor
5.
Glutamic acid decarboxylase 65
Increase in the expression of mGlu2 receptors; glutamateaspartate transporter (GLAST) and glutamate transporter-1 (GLT-1) ↓Transcription of Nav 1.8 genes on C-fibers; reduction in mu-opioid receptor expression ↓GABAergic transmission
GSK-650395 (SGK inhibitor), LMK-235 (HDAC-4 inhibitor) Sodium butyrate (non selective HDAC inhibitors) mGlu2/3 receptor antagonist (LY341495; sodium valproate
2.
38
Non selective HDAC inhibitors Non selective HDAC inhibitors
Kukkar et al., 2014; Lin et al., 2006 Chiechio et al., 2009; Hobo et al., 2011 Matsushita et al., 2013;Uchida et al., 2010 Zhang et al., 2011; Knabl, 2008
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nucleus raphe magnus was blocked by TrB receptor antagonist (K252a), in inflammatory pain model of rats, suggesting a role of brain derived nerve factor-TrB signaling in glutamic acid decarboxylase 65 dependent GABAergic function (Tao et al., 2015). Moreover, the ameliorative effects of histone deacetylase inhibitors on pain behaviour were shown to be suppressed by TrB receptor antagonist (K252a) infusion. Based on this, it may be proposed that brain derived nerve factor-TrB signaling along with epigenetic actions of histone deacetylase inhibitors is of great importance for the inducing anti-nociceptive effects.
et al., 2005). Hobo et al. (2011) demonstrated that peripheral nerve injury led to down-regulation of these spinal glutamate transporters, which resulted in increase in behavioural sensitivity in rats. However, repeated treatment with sodium valproate for ten days restored the expression of GLT-1 and GLAST in the spinal dorsal horn and reduced pain hypersensitivity. Moreover, the authors demonstrated that riluzole and valproate may work synergistically to produce analgesia, as valproate may restore the spinal expression of glutamate transporters and riluzole may activate these transporters to produce analgesia. Riluzole is known to activate the glutamate transporters and enhance glutamate uptake (Frizzo et al., 2004). Hence, it can be asserted that histone acetylation may up-regulate mGlu2 receptors and glutamate transporters to attenuate pain, while decrease in histone acetylation may promote pain by down regulating mGlu2 receptors.
3. Histone acetylation promotes pain In contrast to above described studies, there have been few studies documenting that histone acetylation may actually induce pain. Accordingly, histone acetyl transferase inhibitors (which decrease histone acetylation) are reported to attenuate pain. On the other hand histone deacetylase inhibitors (which promote histone acetylation) are suggested to induce pain in neuropathic models (Kiguchi et al., 2012; Sun et al., 2013).
2.2.4. Neuron restrictive silencer factor (NRSF) It is a transcriptional repressor gene in humans and is responsible for the repressed transcription of neuron-restrictive silencer element (NRSE)-containing genes Nav1.8, Kav4.3 and µ-opioid receptor genes by recruiting histone deacetylase enzymes (Schoenherr et al., 1995; Chong et al., 1995; Ooi et al., 2007). Nav 1.8 channels are expressed on unmyelinated neurons known as C-fibers, specifically confined to dorsal root ganglia (Akopian et al., 1996; Rabert et al., 1998) and play an important role in pain perception through nociceptive pathway (Akopian et al., 1999). Matsushita et al. (2013), demonstrated that inhibitors of histone deacetylase restore the electrophysiological sensitivity of C-fibers along with recovery of Nav 1.8 sodium channel transcription in a mice model of partial sciatic nerve ligation. It was suggested that during nerve injury, extensive histone deacetylation of histone 3 and histone 4 bound to Nav 1.8 sodium channel gene around the neuron-restrictive silencing elements (NRSE) region, resulted in more condensed hetero-chromatin structure, leading to inaccessibility of RNA polymerases and transcriptional repression of Nav 1.8 sodium channel genes. However, impediment of histone deacetylation led to increased accessibility of RNA polymerase leading to reversal of down regulated genes including Nav1.8 sodium channels and reversal of injury-induced insensitivity of C-fibers (Vanhaecke et al., 2004). Uchida et al. (2015), further studied the involvement of histone deacetylase inhibitors in injury-induced down-regulation of mu-opioid receptor gene expression and attenuation of morphine resistance in neuropathic pain. Histone deacetylase inhibitor was shown to reverse nerve injury-induced reduction in mu-opioid receptor expression and restore the anti-nociceptive actions of morphine. Previous studies of same authors showed that histone hypoacetylation-mediated silencing of NRSE-containing mu-opioid receptor and Nav 1.8 genes may contribute in morphine resistance and hypoesthesia (Uchida et al., 2010).
3.1. Evidences Kiguchi et al. (2012), reported a critical role of histone acetylation and chemokines in the augmentation of neuropathic pain. In a partial sciatic nerve ligation model of mice, it was demonstrated that the upregulation of certain chemokines after nerve injury led to accumulation of macrophages and neutrophils in the injured sciatic nerve to induce pain. The elevated chemokine level was found to be a consequence of increased acetylation of histone H3 [lysine (Lys9)-acetylated histone H3 (AcK9-H3)] on the chemokine specific promoter regions in the nuclei of neutrophils and macrophages surrounding the epineurium, which resulted in severe neuroinflammation. Administration of histone acetyl transferase inhibitor attenuated pain signifying that nerve injury may promote histone acetylation and histone acetyl transferase inhibitors may reduce nerve injury-induced inflammation and pain. Another study reveals the opposite effects of histone acetyl transferase inhibitor and histone deacetylase inhibitor in hind paw incision model of pain in mice (Sun et al., 2013). Following the incision, increased histone acetylation and mechanical sensitization was found to be suppressed by histone acetyl transferase inhibitor. However, treatment with histone deacetylase inhibitor aggravated the mechanical sensitization and increased the acetylation of H3 histone at lysine residue 9 (H3K9) in the spinal dorsal horn. The attenuation of nerve pain, by virtue of decreased histone acetylation and aggravation of pain by increase in histone acetylation, emphasizes that histone acetylation and deacetylation is a critical step in nerve injury pain. Zhu et al. (2012), demonstrated that the levels of p300, a histone acetyl transferase, were increased in the spinal cord and inhibition of p300, using small interfering RNA or a p300 inhibitor (C646) attenuated CCIinduced pain again suggesting that histone acetyl transferase inhibitors attenuate pain.
2.2.5. Glutamic acid decarboxylase 65 (GAD 65) It is an enzyme essential for the synthesis of gamma amino butyric acid (GABA) at the synaptic terminals (Tian et al., 1999) and the loss of glutamic acid decarboxylase 65 activity in the spinal dorsal horn after peripheral nerve injury has been indicated in induction of pain behaviour (Lorenzo et al., 2014). In spinal nerve ligation model, a restrained GABAergic synaptic inhibition due to decrease in glutamic acid decarboxylase 65 gene expression and hypo-acetylation of H3 and H4 in nucleus raphe magnus was associated with persistent pain sensitization in rats (Zhang et al., 2011). Similarly, the results from glutamic acid decarboxylase 65 knockout mice support that the impaired GABA activity in the neurons is responsible for increased pain perception. However, treatment with histone deacetylase inhibitors, leading to histone hyper-acetylation, promotes glutamic acid decarboxylase 65 expression and improves GABA activity, thereby, reducing the sensitized pain behaviour (Knabl, 2008). Recently, it was observed that histone deacetylase inhibitors-induced increase in glutamic acid decarboxylase 65 expression in pre-synaptic terminals of
3.2. Mechanisms Nerve injury induced histone acetylation may promote pain perception through following underlying mechanisms (Fig. 2). 3.2.1. Chemokines An increase in the level of inflammatory molecules like cytokines and chemokines are known key regulators of neuropathic pain (Marchand et al., 2005; Gao et al., 2010). The acetylation of histone proteins specifically promotes the up-regulation of cytokine molecules (Schmeck et al., 2008; Yu et al., 2002), and accordingly, histone acetylation may promote the development of pain through increase in the levels of cytokines (Doehring et al., 2011). Kiguchi et al. (2012), 39
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nerve injury-induced or inflammatory pain (Matsushita et al., 2013; Bai et al., 2010). The different mechanisms involved in decrease in histone acetylation-induced pain include decrease in mGlu2 receptors in the DRG and spinal cord (Zammataro et al., 2014); up-regulation of TNF-ɑ levels (Kukkar et al., 2014); down-regulation of glutamate transporters such as GLAST and GLT-1 (Hobo et al., 2011); downregulation of mu-opioid receptors (Uchida et al., 2015) and decrease in glutamic acid decarboxylase 65 expression, leading to decrease in synaptic GABAergic inhibition (Zhang et al., 2011). In contrast, there are studies that acknowledge histone acetylation as a prominent cause for pain perception. In chronic constriction injury model, it has been shown that levels of histone acetyl transferase are elevated along with cyclooxygenase-2, which resulted in release of prostaglandins leading to spinal hyper-excitability. Treatment with histone acetyl transferase inhibitor or p300 inhibitor significantly attenuated pain symptoms, thereby suggesting a major role of histone acetylation in induction of pain (Zhu et al., 2012; Kiguchi et al., 2012; Sun et al., 2013). Accordingly, increased histone acetylation may have ameliorative effects under one chronic pain condition (Zhang et al., 2011; Bai et al., 2010), while it could trigger nociception under different chronic pain conditions (Sun et al., 2013). A number of critical issues remain unanswered, which require an addressing in the view of histone acetylation and deacetylation resulting in epigenetic regulation. Firstly, there is a lack of consistency in these pain studies, which makes the interpretation more difficult. The studies employing changes in histone acetylation and histone deacetylase have not taken into account the global change of histone acetylation or deacetylation at the relevant sites. A single histone contains multiple lysine residues that are sensitive to acetylation (Tan et al., 2011). Most of the studies consider only a few of lysine residues (H3K9 and H3K18) and had no view of remaining lysine residues. Furthermore, histone deacetylase enzymes are of different types, with different distribution pattern in the central nervous system, different acetylation pattern and hence, functional significance. Secondly, there has been lack of specificity of histone deacetylase inhibitors employed in different studies (Chang et al., 2012). For example, sodium valproate has been very commonly employed histone deacetylase inhibitors in pain studies. However, the other properties of sodium valproate (for which it is clinically employed) may also influence the pain sensitivity, which makes the interpretation more complex. Lastly, there is not much evidence to compare the potential effects of histone deacetylase inhibitors in neuropathic pain due to employment of different techniques of inducing pain, different species and sex in these models of pain. A study conducted on stress associated hypersensitivity revealed positive effects of histone acetyl transferase inhibitors on female offspring, when compared to male offspring (Winston et al., 2014). Nevertheless, the more critical analysis of these reports suggest that apart from the study of Sun et al., 2013, all other studies have shown the pain attenuating actions of histone deacetylase inhibitors. The contradictory results of histone deacetylase inhibitor in this study may be possibly due to employment of non-neuropathic pain model i.e., hind paw incision pain model, which is distinctly different from nerve injury models (Sun et al., 2013). Furthermore, studies have also shown that histone acetyl transferase inhibitors reduce pain in different pain models (Kiguchi et al., 2012; Zhu et al., 2012; Sun et al., 2013). Therefore, it is possible to suggest that perhaps both histone deacetylase and histone acetyl transferase inhibitors are useful in pain management. The possibility of activation of both histone deacetylase and histone acetyl transferase in response to nerve injury may not be ruled out. During nerve injury, the activation of histone deacetylase may possibly inhibit certain transcriptional processes, while histone acetyl transferase may possibly activate other transcriptional processes. It is well known that neuropathic pain conditions are associated with increase as well as decrease in transcriptional factors (Jaggi and Singh, 2011, 2012). Accordingly, further studies may be designed to identify the relative activation of both enzymes (deacetylase and acetyl trans-
Fig. 2. Histone acetylation promotes pain through different pathways including: chemokines and cyclooxygenase-2 pathway.
demonstrated that the expression of CXC chemokine ligand type 2 [macrophage inflammatory protein 2 (MIP-2)] and CXC chemokine receptor type 2 (CXCR2) was up-regulated in the neutrophils and macrophages of the injured nerves, that was reversed by perineural injection of MIP-2-neutralizing antibody (anti-MIP-2) or the CXCR2 antagonist N-(2-bromophenyl)-N-(2-hydroxy-4-nitrophenyl) urea (SB225002). Furthermore, there was increase in acetylation of histone H3 on the promoter region of MIP-2 and CXCR2 that was suppressed by histone acetyl transferase inhibitor, anacardic acid. Furthermore, histone acetyl transferase inhibitor also blocked the accumulation of neutrophils in injured sciatic nerve. Hence, it can be implied that augmentation of the MIP-2/CXCR2 axis by hyper-acetylation of histone H3 on the promoter region of MIP-2 and CXCR2 may induce chronic neuroinflammation that results in neuropathic pain. Sun et al. (2013) asserted that histone deacetylase inhibitor aggravates the levels of two major CXCR2 ligands: KC (CXCL1) and MIP-2 (CXCL2) in response to hind paw incision, without any relevant change in expressions of MIP-2 suggesting that histone acetylation may lead to up-regulation of chemokines. There have been number of studies indicating the vital role of chemokines in nociceptive sensitization (Manjavachi et al., 2010). Based on the observations, it can be proposed that nerve injury may induce histone acetylation, which inturn may induce neuro-inflammation by up-regulating chemokine receptors to produce neuropathic pain. 3.2.2. Cyclooxygenase-2 (COX-2) Many studies have revealed the over-expression of COX-2 in the injured nerve in different models of neuropathic pain (Ma, 2003; Jean et al., 2009). A study conducted by Zhu et al. (2012), correlated the increased expression of COX-2 with histone acetylation in a neuropathy model. The authors observed that increase in the expression of p300, a histone acetyl transferase, in the spinal cord along with up-regulation of COX-2 levels and inhibition of histone acetyl transferase activity by p300 inhibitor lowered nerve injury-induced increase in p300 expression and COX-2 levels. Therefore, it has been proposed that overexpression of histone acetyl transferase, p300, may produce neuropathic pain through up-regulation of COX-2 expression. 4. Summary and discussion Most of the studies have described that during nerve injury or inflammatory conditions, there is an increased expression or activity of histone deacetylases, which tends to deacetylase histone proteins. The hypo-acetylation of histone in-turn activates the different pathways to induce pain and, accordingly, histone deacetylase inhibitors attenuate 40
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