How then is this exaggerated response to PGE₂, and other mediators like serotonin or A₂ adenosine receptor agonists (Aley et al. 2000), which can also precipitate hyperalgesia in primed animals, generated? Extensive studies have demonstrated that this priming effect is dependent on switches in signaling mechanisms in nociceptors. In naïve animals, the hyperalgesic effects of PGE₂ injection are mediated by adenylyl cyclase (AC) activation downstream of PGE₂ receptors causing protein kinase A (PKA) activation (Fig. 1b) (Aley and Levine 1999). This effect can be attenuated via injection of PKA antagonists (Aley and Levine 1999). Although the second messenger pathway underlying the early phase of PGE₂-induced hyperalgesia is PKA mediated, even in primed animals, the same cannot be said for the pathway responsible for the long-lasting hyperalgesia that is uniquely present in primed rodents. While PGE₂-induced hyperalgesia in primed animals is still cyclic AMP (cAMP) dependent, it now bypasses PKA to activate exchange proteins directly activated by cAMP (Epac) which can activate protein kinase Cε (PKCε, Fig. 1b, Hucho et al. 2005). Importantly, inflammatory stimulation of nociceptors leads to a decrease in G-protein receptor kinase 2 (GRK2) which results in enhanced Epac activity (Eijkelkamp et al. 2010; Wang et al. 2013). These changes occur in IB₄-positive nociceptors, and decreases in GRK2 and increases in Epac expression are correlated with the persistence of priming (Wang et al. 2013). Moreover, in primed animals, PGE₂ results in an activation of pertussis toxin-sensitive G-protein α_i subunits (Dina et al. 2009) and phospholipase Cβ (PLCβ) leading to a downstream engagement of PKCε (Joseph et al. 2007); hence, multiple pathways for PKCε engagement may exist in primed nociceptors (Fig. 1b). Critically, in primed animals, the long-lasting hyperalgesia arising from exposure to compounds that can precipitate priming is blocked by selective inhibition of PKCε (Aley et al. 2000) and by intrathecal delivery of antisense oligonucleotides knocking down PKCε expression (Parada et al. 2003). Additionally, injection of a PKCε agonist alone results in a prolonged hyperalgesic state and hyperalgesic priming, pointing to a key role for nociceptor PKCε in hyperalgesic priming (Reichling and Levine 2009). Importantly, this PKCε-dependent primed state does not require an initial bout of hyperalgesia as subthreshold doses of PKCε activators (Parada et al. 2003), previous exposure to unpredictable sound stress (Khasar et al. 2008), and even repeated administration of opioid agonists into the paw (Joseph et al. 2010) are capable of causing an emergence of a primed state (Fig. 1a).

Is there a distinct subset of nociceptors required for PKCε-dependent priming? To elucidate the population of nociceptors involved in hyperalgesic priming, Joseph and colleagues lesioned IB₄(+) nociceptors via intrathecal administration of IB₄-saporin (Joseph and Levine 2010). In these animals, the PKCε activator, ψεRACK, produces an initial hyperalgesia; however, the PGE₂-induced prolonged hyperalgesia is absent in animals treated with toxin suggesting that priming is localized to IB₄(+) neurons. Furthermore, select agents known to act on peptidergic vs. IB₄(+) cells stimulate an initial hyperalgesic state in IB₄-lesioned animals but fail to establish priming indicating that a switch in signaling to PKCε selectively occurs in IB₄(+) nociceptors (Joseph and Levine 2010). As mentioned above, this may be linked to a pronounced loss of GRK2 expression and an increase in Epac activity in IB₄(+) nociceptors of primed animals (Wang et al. 2013).

Collectively these findings support a key role for PKCε in the initiation and maintenance of nociceptor priming therefore making this PKC isoform a key therapeutic target. However, PKCε-dependent forms of priming show a marked sexual dimorphism in rats with female rats failing to show priming, an effect that is apparently due to a protective effect of estrogen (Joseph et al. 2003). Importantly, this does not appear to be the case with mice where both carrageenan and PKCε-activating peptides cause robust priming to subsequent PGE₂ injection in females (Wang et al. 2013). At this point, it remains unclear as to whether there is sexual dimorphism in PKCε-mediated effects in humans. Despite these potential discrepancies, it is clear that one mechanism of priming is shared across species and sexes, a requirement for changes in gene expression at the level of protein synthesis.

Due to the continuous turnover of cellular proteins, a simple switch in G-protein coupling is likely not sufficient to explain such a long-lasting maladaptive change in peripheral nociceptors (Bogen et al. 2012). It is likely that this effect is coupled to changes in gene expression and a possible solution to this problem is a localized change occurring at the level of protein translation. Translation can be controlled by extracellular factors signaling via kinase cascades offering a rapid, locally mediated control of gene expression. Two important kinases for translation control are the mechanistic target of rapamycin complex 1 (mTORC1) and extracellular signal-regulated kinase (ERK) (Fig. 2a). Both of these kinases signal to proteins that bind to the 5′ cap structure of mRNAs. mTORC1 phosphorylates 4E-binding proteins (4EBPs) leading to a release of inhibition on eukaryotic initiation factor (eIF) proteins allowing for the eIF4F complex (composed of eIF4E, the 5′ cap-binding protein; eIF4G, a scaffolding protein; and eIF4A, a helicase that unwinds secondary structure in mRNAs) to form (Fig. 2a) (Sonenberg and Hinnebusch 2009). ERK, on the other hand, activates the mitogen-activated kinase-interacting kinases (MNKs), which then phosphorylate eIF4E (Fig. 2a) (Wang et al. 1998). Phosphorylation of eIF4E, which is mediated specifically by MNK1/2, is thought to enhance eIF4F complex formation leading to an increase in translation (Ueda et al. 2010; Herdy et al. 2012). In sensory neurons, nerve growth factor (NGF) and interleukin 6 (IL6), two factors known to induce priming, induce an increase in ERK and mTORC1 signaling leading to a local, axonal increase in protein synthesis (Melemedjian et al. 2010, 2014). Blockade of these kinases, or blockade of eIF4F complex formation with the eIF4F inhibitor compound 4EGI1 (Fig. 2a), inhibits mechanical hypersensitivity induced by these factors and abrogates priming to subsequent PGE₂ exposure (Melemedjian et al. 2010; Asiedu et al. 2011). Hence, local translation is required for the induction of priming downstream of NGF and IL6 signaling.

An alternative mechanism to decrease ERK and mTORC1 signaling is via stimulation of adenosine monophosphate-activated protein kinase (AMPK) (Fig. 2b). AMPK is a ubiquitously expressed energy sensing kinase well known to inhibit mTORC1 signaling through multiple phosphorylation events (Carling et al. 2012). AMPK also abrogates ERK signaling in many cell types, and this effect has recently been linked to negative regulation of the upstream ERK activator BRaf (Fig. 2b) (Shen et al. 2013). In sensory neurons, AMPK activation with pharmacological stimulators (for review see Price and Dussor 2013) leads to decreased ERK and mTORC1 activity (Melemedjian et al. 2011; Tillu et al. 2012), decreased eIF4F complex formation (Melemedjian et al. 2011; Tillu et al. 2012), and inhibition of axonal protein synthesis as measured by enhanced processing body (P body) formation (Melemedjian et al. 2014). AMPK activators decrease peripheral nerve injury- and inflammation-induced mechanical hyperalgesia (Melemedjian et al. 2011; Russe et al. 2013) suggesting an important role for this kinase in pain plasticity. In the context of hyperalgesic priming, AMPK activation with the natural product resveratrol (Fig. 2b) decreases mechanical hypersensitivity caused by incision and completely blocks the development of priming when given locally around the time of incision (Tillu et al. 2012). These findings further suggest a role for local translation in the initiation of plasticity leading to priming of nociceptors.

Epac signaling, which was described above as an important mediator of priming induced by inflammatory mediators (Wang et al. 2013), may also play an important role in regulating translation in sensory neurons. Decreased GRK2 leads to enhanced activation of Epac causing an increase in ERK activity (Eijkelkamp et al. 2010). While this signaling event has not been linked to translation control, based on findings involving IL6-induced priming and its dependence of ERK/eIF4E-mediated changes in translation control, it is conceivable that this is an important mediator for priming where ERK activity is increased. Furthermore, Epac activation causes an increase in mTORC1 activity in some transformed cells (Misra and Pizzo 2009) suggesting that enhanced Epac signaling in primed nociceptors may lead to convergent signaling onto the eIF4F complex in a manner analogous to that observed with NGF and IL6 (Melemedjian et al. 2010). It remains to be seen if these Epac-mediated events occur in sensory neurons and their axons, but this may be a fruitful area for further work and therapeutic intervention.

The regulation of translation via 5′ cap-binding proteins and their upstream kinases clearly comprise an important mechanism for the priming of nociceptors. However, translation is likewise regulated by RNA-binding proteins that bind to either 5′ or 3′ untranslated regions (UTRs). The fragile X mental retardation protein (FMRP) is a key RNA-binding protein regulating plasticity in the PNS and CNS (Bassell and Warren 2008). Mice lacking FMRP fail to sensitize in several preclinical pain models (Price et al. 2007; Price and Melemedjian 2012), and these mice also have deficits in priming induced by NGF and IL6 (Asiedu et al. 2011). Another important RNA-binding protein in hyperalgesic priming is the cytoplasmic polyadenylation element-binding protein (CPEB). CPEB binds preferentially to mRNAs containing a CPE sequence in their 3′ UTR near the polyadenylation sequence. These mRNAs contain short poly A tails, and CPEB, upon phosphorylation, can enhance the poly A tail length leading to enhanced translation efficiency (Fig. 2a) (Richter 2007). This process is linked to long-term potentiation (LTP) in the CNS (Udagawa et al. 2012) and has recently been shown to play an important role in nociceptor priming (Bogen et al. 2012; Ferrari et al. 2012, 2013a). CPEB is phosphorylated by the aurora family kinases (Mendez et al. 2000) and, likely more importantly for neurons, by Ca²⁺/calmodulin-activated protein kinase IIα (Fig. 2a) (CaMKIIα, Atkins et al. 2005). In the PNS, CPEB is primarily expressed by IB₄(+) nociceptors (Bogen et al. 2012). Knockdown of CPEB with intrathecally injected antisense oligonucleotides leads to inhibition of the initiation of priming induced by PKCε activators linking translation events to PKCε-dependent priming (Bogen et al. 2012). Importantly, in priming induced by peripheral inflammation, CPEB may act downstream of PKCε and CaMKIIα to initiate and maintain a primed state (Ferrari et al. 2013a). Since CPEB is thought to have prion-like properties that are crucial for its role in memory maintenance (Si et al. 2003a, b), these findings point to a potential role for CPEB in creating a permanently primed state in peripheral nociceptors.

While it is clear that translation regulation is required to initiate a primed state in the periphery, a crucial question is whether disruption of local translation is capable of interrupting priming once it has been fully established. This is an important question because it gives insight into devising therapeutic strategies to reverse priming in the clinical state. While there are certain situations (e.g., surgery) where inhibition of priming mechanisms at the time of injury is a viable strategy, the majority of clinical situations are likely to require intervention following the full establishment of priming. An experimental paradigm to test this translation dependency is to induce priming with a locally administered stimulus and then allow the initial hyperalgesia to resolve. Then, prior to injection of the stimulus to precipitate hyperalgesia in primed animals, translation inhibitors can be administered locally to test whether continuous translation is required to maintain a primed state (Asiedu et al. 2011; Ferrari et al. 2013a). In this regard, following injection of IL6 and resolution of mechanical hyperalgesia in mice, injection of anisomycin or rapamycin (at doses that block the initiation of priming) fail to reverse a primed state (Asiedu et al. 2011). In contrast, in rats, injection of carrageenan causes priming that is disrupted both at the time of carrageenan injection and during the maintenance phase by either the mTORC1 inhibitor rapamycin or the polyadenylation inhibitor cordycepin (Ferrari et al. 2013b). Interestingly, in addition to cordycepin action on polyadenylation, this compound has recently been identified as an AMPK activator (Wu et al. 2014). Similar effects with rapamycin and cordycepin are observed in rats primed with paw injection of CaMKIIα. Since CaMKIIα phosphorylates CPEB and CPEB regulates CaMKIIα translation, this raises the intriguing possibility that CaMKIIα/CPEB signaling could represent a positive feedback mechanism to maintain pain memory in the peripheral nociceptor (Ferrari et al. 2013a). Importantly, in rats where PKCε-induced priming is sexually dimorphic, priming that is dependent on local translation occurs both in male and female rats (Ferrari et al. 2013a). Hence, while there are conflicting results in different models, it is formally possible that brief disruption of local translation in primed nociceptors is capable of resolving a primed state. Further work is clearly needed to further interrogate mechanisms involved in these effects; however, they point to the tantalizing possibility that therapeutics targeting translation regulation might have disease-modifying effects in chronic pain conditions. This hypothesis is consistent with the observation that AMPK activators, which have a strong effect on translation regulation pathways, have disease-modifying effects in neuropathic pain models (Melemedjian et al. 2011, 2013b).