New insights into protease-activated receptor 4 signaling pathways in the pathogenesis of inflammation and neuropathic pain: a literature review

Abstract

Pain is an unpleasant sensory and emotional experience that is commonly associated with actual or potential tissue damage. Despite decades of pain research, many patients continue to suffer from chronic pain that is refractory to current treatments. Accumulating evidence has indicated an important role of protease-activated receptor 4 (PAR4) in the pathogenesis of inflammation and neuropathic pain. Here we reviewed PAR4 expression and activation via intracellular signaling pathways and the role of PAR4 signaling pathways in the development and maintenance of pain. Understanding PAR4 and its corresponding signaling pathways will provide insight to further explore the molecular basis of pain, which will also help to identify new targets for pharmacological intervention for pain relief.

Introduction

Protease-activated receptors (PARs) comprise a subfamily of G-protein coupled receptors.^1-3 The study of PARs has been facilitated by the discovery that PARs can be selectively activated by synthetic peptides consisting of five to 6 amino acids presenting in the tethered ligand sequence, thereby negating the need for proteolytic cleavage.⁴ PARs have a unique mechanism of activation by which proteases cleave part of their extracellular N-terminal sequence, allowing the tethered ligand that is formed from the truncated portion of the N-terminus to bind to the receptor itself, leading to activation.⁵

Generally, several proteases can activate a single receptor, provided they cleave at a site that exposes the tethered ligand domain. For example, trypsin, tryptase, coagulation factors VIIa and Xa, and membrane-anchored thrombin and serine protease can all cleave and activate PAR2 with varying potencies.^6-8 PAR4 is the fourth identified PAR and the first to be described as an important thrombin receptor for the aggregation of both human and mouse platelets.⁹ It is also activated by trypsin, cathepsin G, activated factor X of the coagulation cascade, and trypsin IV.^10,11 Conversely, one protease usually activates several PARs with different efficiencies; for example, thrombin is released by blood clotting following blood vessel damage or tissue injury, and acts on PAR1, 3 and 4 expressed in primary sensory nerve terminals.^12-14

PARs are essential in pain response, specifically in nerve injury and inflammation.¹⁵ Particularly, PAR4 is considered to be an additional subclass of metabotropic G protein-coupled receptors (GPCRs), which is also involved in inflammation and pain responses,^16-20 and it could potentially represent a novel therapeutic target. The goal of this review is to summarize research on PAR4 signaling pathways in pain processes, in order to better understand the genesis of pain and to help to identify new targets for pharmacological intervention of pain relief.

Expression of PAR4 in Sensory Neurons

Increasing evidence has demonstrated that PARs contribute to peripheral nerve functions.²¹ It has been shown that PAR1 and PAR2 are expressed in primary spinal afferent neurons and modulate nociception.^2,22-24 PAR2 agonists (e.g. PAR2-activating peptides and trypsin) stimulate the release of nociception-related calcitonin gene-related protein (CGRP) and substance P (SP) from the peripheral and central projections of spinal afferent neurons in a calcium-dependent manner.²²

PAR4 expression has been demonstrated in various cell types including endothelial cells, neutrophils and sensory neurons.^16,19,20 Asfaha et al. have reported that the activation of PAR4 using the PAR4-activating peptide (PAR4-AP) AYPGKF-NH₂ in the rat hindpaw increased thermal and mechanical nociceptive thresholds and alleviated thermal and mechanical inflammatory hyperalgesia.^16,19 Moreover, PAR4 is expressed in peripheral nerves and cell bodies in the plexus, indicating its participation in neurogenic inflammation.²⁵ Furthermore, it is expressed in neurons, particularly in the sensory neurons isolated from the dorsal root ganglion (DRG) that express the sensory neuropeptides CGRP and SP. Accumulating evidence demonstrates that nociceptor activation and inflammation evoke a rapid change in the levels of CGRP mRNA and protein in DRG neurons,^26-28 and CGRP expression in these neurons contributes to the regulation of neurogenic inflammation and pain.^29-32 In a previous study, the PAR4 agonist did not induce a calcium signal in DRG neurons; it reduced the calcium signal of DRG neurons in response to KCl,¹⁶ suggesting that PAR4 activation could inhibit the nociceptive signal in DRG neurons. Furthermore, in an in vivo experiment, the PAR4 agonist was able to increase the nociceptive threshold to thermal and mechanical stimuli, and to reduce thermal and mechanical inflammatory hyperalgesia and allodynia. The intraplantar injection of 50 µg of the PAR4 agonist AYPGK-NH₂significantly increased the withdrawal latency from 45 min to 120 min and the nociceptive threshold from 30 min to 75 min; additionally, the agonist significantly reduced the nociceptive score in response to both noxious and non-noxious mechanical stimuli, thus inhibiting carrageenan-induced mechanical inflammatory hyperalgesia and allodynia.^16,33

Intracellular Signaling Pare Activated by PAR4

In human platelets, thrombin is a key factor in coagulation and inflammation, typically elicits cellular responses via activation of protease activated receptors (PARs).³⁴ Activation of human platelets by thrombin is mediated predominately through 2 proteinase-activated receptors (PARs), that is, PAR1 and PAR4.^35-37 Thrombin acts through the PAR4 receptor and causes activation of the Gq/phospholipase C (PLC) pathways.⁹ Gq-mediated PLC β₂activation leads to the generation of inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG), resulting in an increase in intracellular calcium and activation of protein kinase C (PKC).³⁸ In pheochromacytoma (PC12) cells, PKCδ promotes ERK phosphorylation and activation by epidermal growth factor (EGF)^39-41 (Fig. 2); however, endothelial cells influence leukocyte function directly by expressing and producing several regulatory factors, such as cytokines, chemokines, lipid mediators, NO, and adhesion molecules.⁴² Control of the inflammatory reaction also requires changes in vascular permeability, perfusion and coagulation.⁴² At sites of vascular damage, endothelial cells can be activated by thrombin⁴³ via the PARs. Cleavage of endothelial PAR1 and PAR4³⁷ results in morphological changes of endothelial cells and the release of vasoactive substances as well as cytokines.⁴³ Additionally, PAR4 is preferentially coupled to Gα_i/o and activates the PI3K-Akt pathway, thereby inducing NO production mostly in a calcium-independent manner.^44,45 Moreover, Sabri A.et al. provided evidence that thrombin-dependent activation of Src was mediated by PAR4 in cardiomyocytes.⁴⁶ AYPGKF, a modified PAR4 agonist with increased potency at PAR4, activates p38-mitogen-activated protein kinase but it is a weak activator of phospholipase C, extracellular signal-regulated kinase and cardiomyocyte hypertrophy.⁴⁶ Further studies implicate roles of Src and epidermal growth factor receptor (EGFR) kinase activities in the PAR4-dependent p38-mitogen-activated protein kinase signaling pathway.^46-48 Cardiomyocytes also express PAR4 which activates mechanisms that contribute to cardiac remodeling in areas of cardiac injury and/or inflammation.⁴⁶ Additionally, it is clear that PAR4 receptors are expressed in a population of peptide-expressing, IB4-negative nociceptive sensory neurons, where they couple to PKCε, causing sensitization of TRPV1 and promoting the heat-dependent release of the pro-inflammatory neuropeptide CGRP. These observations suggest a role for PAR4 receptors in promoting inflammation and pain following the release of thrombin;^30,33,49,50 however, evidence in the model of inflammation showed that the administration of a PAR4 activator peptide (PAR4-AP) caused the formation of edema neutrophils recruitment, PAR4-mediated edema is dependent on the recruitment of neutrophils and components of the kallikrein-kinin system.^18,19

Figure 1.

A diagram illustrating the mechanism of PAR activation by thrombin. Proteases cleave the N-terminal domain to release a new N-terminal tail (A), which acts as a tethered ligand that binds the receptor itself to induce an intracellular signal (B).

Figure 2.

A diagram illustrating the mechanisms of PAR4 signaling and the model depicting the mechanism and the function of tyrosine phosphorylation of PKC following PAR4 activation. Thrombin (indicated as cross) acts through PAR4 and causes activation of the Gq/PLC pathways. PLC activation leads to the generation of inositol trisphosphate (IP3), which mobilizes intracellular calcium and diacylglycerol (DAG) that activate protein kinase C (PKC). An increase in calcium directly or indirectly leads to an increase in Src activity and subsequently tyrosine phosphorylation of PKC. Additionally, PAR4 activates mitogen-activated protein kinase (MAPK) by transactivation of the epidermal growth factor receptor (EGFR), stimulating Raf kinase, which phosphorylates and activates mitogen and extracellular signal-regulated kinase kinase (MEK). Activated MEK in turn phosphorylates and activates ERK1/2, then sensitizes TRPV1. These activations result in an enhanced influx of calcium and elevated release of prostaglandin E (PGE), calcitonin gene-related peptide (CGRP), and substance P (SP) in the dorsal horn in response to mechanical and thermal stimuli. PGE, CGRP, and SP activate their receptors on spinal neurons [PGE, PGE receptor, SP, neurokinin 1 receptor (NK1R), CGRP, and CGRP1 receptor], resulting in enhanced transmission of nociceptive signals and mechanical hyperalgesia to the brain.

Specific Signaling Pathways of PAR4 in Neurons

PAR4-dependent ERK/MAPK activation

Mitogen-activated protein kinases (MAPKs) are a family of evolutionally conserved molecules that are essential in cell signaling and gene expression. The MAPK family includes 3 major members: extracellular signal-regulated kinase (ERK), p38-mitogen-activated protein kinase (p38), and c-Jun N-terminal kinase (JNK), representing 3 different signaling cascades. MAPKs are activated by phosphorylation and transduce a broad range of extracellular stimuli into diverse intracellular responses via both transcriptional and non-transcriptional regulation.^47,48,51

ERKs are activated by diverse extracellular stimuli, including several hormones and growth factors that activate GPCRs or receptor tyrosine kinases, leading to stimulation of Raf kinases that phosphorylate and activate mitogen and extracellular signal-regulated kinase kinase (MEK). Activated MEK in turn phosphorylates and activates ERK1/2.⁵² Protein kinase A (PKA) and cAMP can promote ERK activation via a Rap1-dependent pathway in neural cells, such as PC12 cells, which use B-Raf as the major Raf isoform.^53-56 Initiation of the ERK/MAPK cascade involves activation of kinases: Ras→Raf→MEK→ERK/MAPK (Fig. 2), which mediate several cellular responses to mitogenic and differentiation signals.⁵⁷ ERK/MAPK activation has been shown to contribute to nociceptive responses in the dorsal horn and DRG following inflammation and/or nerve injury.^58,59 Following nerve injury, p-ERK levels are sequentially increased in neurons, microglia, and astrocytes of the dorsal horn. Moreover, nerve injury-induced p-ERK activation occurs early and lasts a long time.⁶⁰

In microglia, the PAR4 signaling pathway is featured by a prolonged increase in calcium and ERK/MAPK activation.⁶¹ And in primary afferent neurons, PAR4 can induce strong activation of a non-receptor Src tyrosine kinase-p38- MAPK cascade which has been described as being proalgesic;⁶² however, in visceral pain, according to the analgesic effects of PAR4 agonists, Auge et al. speculated that P38-MAPK was not involved in PAR4 signaling in sensory neurons.⁴⁹ Therefore, this would have to be demonstrated by extensive signaling studies in neurons; however, to date, no studies have been reported that c-Jun is associated with PAR4 activation in neurons.

Sensitization of TRPV1 in PAR4 Signaling Pathway

Transient receptor potential vanilloid 1 (TRPV1) is an important molecular integrator of pain-producing stimuli that is highly expressed in nociceptive C and Aδ fibers. TRPV1 functions as a non-selective cation channel activated by noxious heat, acidic pH, or lipid-derived endovanilloids, which enables nociceptors to respond to a range of noxious stimuli with an electrical discharge.^63-65 TRPV1 has been linked to a number of chronic pain conditions associated with inflammation, including arthritic pain, cancer pain, visceral pain and migraine.^65-68

TRPV1 is widely distributed in primary nociceptive afferent neurons, and its expression extensively overlaps with PAR4 immunoreactivity in cultured primary sensory neurons.^31,33,69 PAR4 is expressed in both large neurons with myelinated fiber and small nociceptors of the peptidergic subclass, which is able to potentiate TRPV1 activity and promotes the heat-dependent release of the proinflammatory neuropeptide CGRP.³¹ Capsaicin-activated TRPV1 is involved in noxious responses and induction of TRPV1 and CGRP mRNA and protein expression in DRG neurons.²⁸ Thrombin or PAR4-AP causes activation of the PLCβ/PKC pathway: intracellular calcium release, sensitization of TRPV1, and translocation of the epsilon isoform of PKC (PKCε) to the neuronal cell membrane.³³

Apart from PKC, PKA is essential for mediating phenyl glycidyl ether 2 (PGE2)-induced modulation of TRPV1. The cAMP/PKA pathway is a major signaling cascade that mediates TRPV1 modulation by PGE2 in DRG neurons.⁷⁰ Additionally, PKA in primary sensory neurons is involved in the modulation of hypersensitivity in response to noxious stimuli after TRPV1 activation.^32,70-72 TRPV1 contains a phosphorylation site in its amino acid sequence (at Ser116) for PKA.^73-75 TRPV1 activation induces Ca²⁺ influx into neurons, leading to activation of Ca²⁺-mediated signal transduction, including activation of Ca²⁺/calmodulin-dependent protein kinase (CaMK)⁷⁶ and PKC isoenzymes.⁷⁷ CaMK activates the transcription factor cAMP response element-binding protein (CREB) via phosphorylation of Ser-133.⁷⁸ Activated CREB induces target gene expression and regulates various neuronal functions.⁷⁹ It has been shown that the inflammation caused by complete Freund's adjuvant increases phosphorylation of CREB in DRG neurons.⁸⁰ Taken together, proton-activated TRPV1 upregulates CGRP expression through the activation of CREB, leading to the induction of pain,^81,82 and phosphorylation-dependent modulation of TRPV1 by inflammatory mediators is an important mechanism underlying the sensitization of nociceptors.^83,84

Role of TRPV4 in visceral pain

transient receptor potential vanilloid 4 (TRPV4) is a widely expressed cation channel of the TRP superfamily, the mammalian homolog of the Caenorhabditis elegans gene Osm-9, and it is a potential mediator of mechanical hyperalgesia.⁸⁵ TRPV4 is gated by altered tonicity and temperatures >27°C.^86,87 Hypo-osmotic stimuli cause cell swelling, phospholipase A (PLA)-2 activation, and production of arachidonic acid (AA).⁸⁸ A cytochrome P450 product of AA, 5′,6′-epoxyeicosatrienoic acid, activates TRPV4 and is a potential endogenous agonist.⁸⁹ TRPV4 is expressed by neurosensory structures, including circumventricular organs, inner ear hair cells, Merkel cells and sensory neurons. Trpv4^−/− mice showed abnormal osmotic regulation and decreased nociceptive responses to pressure,^90,91 and knockdown or deletion of TRPV4 reduces nociceptive responses to hypotonic and mildly hypertonic stimuli.^92,93 Its activation by hypotonic stimuli suggests that it detects osmotic and mechanical stimuli.⁹⁴ One recent study has demonstrated that PAR4 is present in sensory neurons projecting from the colon, and it is co-expressed with PAR2 and TRPV4. PAR4 agonist exposure alone has no effect on free intracellular calcium mobilization in sensory neurons in vitro; however, it inhibits calcium mobilization induced by PAR2 and TRPV4 agonists in all sizes of isolated neurons,⁴⁹ suggesting that the analgesic properties of PAR4 are not specific for one type of pronociceptive signal. Thus, PAR4 activation can be mediated by different painful stimuli (i.e., PAR2 agonists, TRPV4 agonists, or others).

TRPV4 has been shown to function in visceral nociception and hypersensitivity symptoms.^95-97 While a synthetic TRPV4 agonist, the phorbol ester 4a-phorbol 12,13-didecanoate (4aPDD),⁹⁸ has been shown to promote the release of SP and CGRP from the central projections of primary afferents in the spinal cord, suggesting a role of TRPV4 in nociception.⁹⁶ SP activates the NK1 receptor on endothelial cells of postcapillary venules, causing plasma extravasation and granulocyte recruitment. Additionally, CGRP induces arteriolar vasodilatation and hyperemia. The combined effects of SP-stimulated plasma extravasation and CGRP-stimulated vasodilatation may contribute to the measured increase in paw diameter, and SP-stimulated granulocyte recruitment may account for the increased myeloperoxidase (MPO) activity in tissues.^94,96,99 This response was Ca²⁺-dependent; and thus, it is dependent on neurosecretion and is prevented by pretreatment with a desensitizing concentration of capsaicin, indicating that CGRP and SP originate from peripheral terminals of capsaicin-sensitive nociceptive fibers.⁹⁴

This mechanism may mediate the generation and maintenance of inflammation, suggesting that TRPV4 blockade could be a novel therapy for the treatment of neurogenic inflammation.

PAR4 activation increases the release of CGRP

CGRP is a member of the calcitonin family of peptides, which is produced in both peripheral and central neurons.¹⁰⁰ It is a potent peptide vasodilator and functions in pain transmission.^101,102 PAR4 activation induced by the PAR4-AP AYPGKF-NH₂ is associated with an increase in CGRP expression in DRG neurons and indicates that PAR4 activation evokes a remarkable increase in the number of CGRP-labeled neurons and CGRP mRNA and protein levels in DRG neurons.^31,32 There is a close correlation between CGRP mRNA and protein levels; therefore, it seems more likely that the effect of PAR4 on CGRP expression results from a change in transcription.³¹ Several studies have shown that these rapid changes in CGRP mRNA and protein levels were evoked by nociceptor activation and inflammation.^26,28 PAR4-AP induces the upregulation of CGRP expression in primary sensory neurons via activation of the ERK1/2 signaling pathway. Notably, this upregulation was significantly inhibited after inhibition of ERK1/2 phosphorylation,³² intraplantar injection of PAR4-AP, or treatment of cultured DRG neurons with PAR4-AP evoked a significant increase in the number of DRG cells expressing CGRP and cytoplasmic and nuclear staining for phospho-ERK1/2 (p-ERK1/2). The percentages of total DRG neurons expressing both CGRP and PAR4 or p-ERK1/2 also increased significantly at 2h after PAR4-AP treatment.^32,103,104 Moreover, previous studies have reported that noxious stimulation immediately increases p-ERK1/2 expression in the cytoplasm and nucleus of DRG neurons, all of which are features of small- and medium-sized nociceptive neuronal cells.^16,33,105 Furthermore, membrane depolarization and calcium influx evoked by noxious stimulation lead to ERK1/2 activation,¹⁰⁶ which triggers CREB-induced transcription and upregulation of CGRP mRNA levels in DRG neurons.^82,107-111

The role of PAR4 signaling pathways in various types of pain conditions

PAR4 activation in the modulation of visceral pain

Recent studies have focused on the role of PAR2 in visceral pain and hypersensitivity.^112,113 As opposed to PAR2, the activation of PAR4 not only decreases the nociceptive response to colorectal distension in basal conditions but also significantly inhibits colonic hypersensitivity. For the first time, Auge et al. have described the expression of PAR4 in visceral primary afferent neurons and its role in modulating colonic nociceptive responses, colonic hypersensitivity and primary afferent.⁴⁹ They showed that intra-colonic administration of a sub-inflammatory dose of a PAR4 agonist peptide in mice reduced the visceromotor response to colorectal distension (CRD) and inhibited the exacerbated visceromotor response to CRD. In contrast, a higher dose of the PAR4 agonist increased the visceral sensitivity to CRD and induced a small increase in macroscopic damage scores and myeloperoxidase activity, indicating the presence of inflammation.⁴⁹

PAR4 as a central and common mediator has been shown to inhibit colonic hypersensitivity associated with inflammatory bowel syndrome.⁹⁵ Importantly, it exerts an endogenous role in modulating visceral pain, as PAR4-deficient mice experienced significantly more pain in response to mustard oil compared with wild-type littermates.⁴⁹ Additionally, low doses of intraplantar injection of PAR4 agonist exert analgesic properties. In contrast, PAR4 activation could exert an opposite effect as higher doses of PAR4 agonists caused inflammation in both the paw and colon.¹⁶ It is believed that a range of inflammatory mediators are released by infiltrated cells after proinflammatory doses of PAR4 agonists are administered, and they exert proalgesic effects that contribute to modulation of nociceptor sensitization and inflammatory pain.^49,114 It is noteworthy that in the setting of PAR4-induced inflammation, the direct anti-nociceptive effects of PAR4 activation on sensory neurons could be masked by the pronociceptive effects of other inflammatory mediators. Hollenberg et al. have shown that PAR4-induced paw inflammation is not mediated by a neurogenic mechanism¹⁷ but by a mechanism involving the kinin/kallikrein system activated by endothelial cells and leukocytes.¹⁸ These findings indicate that the effect of PAR4 agonists on the development of inflammation and hyperalgesia occurs in a dose-dependent manner in an inflammatory situation. Furthermore, Vellani et al. found that PAR4 receptors are expressed in 2 distinct populations of sensory neurons, suggesting a possible basis for this dual effect. Activation of large-diameter myelinated afferents is well known to have an anti-nociceptive effect, and the activation of PAR4 in these afferents could therefore have an analgesic action.³³

PAR4 activation in the modulation of inflammatory pain

PAR4 expression was observed in the rat endothelium from mensenteric venules and isolated leukocytes, and the selective PAR4-AP could reproduce thrombin-induced leukocyte rolling and adhesion.²⁰ Administration of the PAR4-AP into the rat hindpaw caused acute edema, which was mast cell-independent and unaffected by pretreatment with capsaicin.¹⁷ Thus, unlike PAR1 and PAR2, PAR4 does not appear to mediate any of its inflammatory effects via neurogenic mechanisms or mast cell activation. Instead, it appears that the PAR4-mediated inflammatory response is dependent on neutrophil accumulation and the kallikrein-kinin system.^17,18

Studies indicate a role for PAR4 in inflammatory diseases. The pepducin antagonist of PAR4, P4pal-10, reduced the severity of inflammation in a mouse model of systemic inflammation, and this was thought to be partially mediated by reduced neutrophil migration. The fact that PAR4 is present both on endothelial cells and leukocytes, which are responsible for inflammatory cell recruitment, suggesting that the proinflammatory effects of the PAR4 agonist are mediated through PAR4 activation on endothelial cells and/or leukocytes.¹¹⁵

McDougall et al. provided the first evidence that PAR activation modulates pain sensitivity in joints and revealed a vital role of PAR4 in promoting joint inflammation.¹⁹ PAR4 is expressed extensively throughout the mouse knee joint including the cartilage, subchondral bone, menisci, and synovium. Articular activation of PAR4 with the selective peptide agonist AYPGKF-NH₂ caused a gradual increase in synovial blood flow with concomitant edema formation, which was maximal approximately 2 h after intra-articular injection. These inflammatory reactions to the peptide were blocked by pre-treatment with the PAR4 antagonist pepducin P4pal-10.¹⁹ This study demonstrated that the blockade of bradykinin receptors attenuated the physiological activity of AYPGKF-NH₂, suggesting that the kallikrein-kinin system is involved in mediating the nocifensive and inflammatory responses to PAR4 activation. The kallikrein-kinin system is a key regulator of injury response. The principal effectors of the kallikrein-kinin system are plasma and tissue kallikreins, and proteases that cleave high molecular weight kininogen produce bradykinin.¹¹⁶ Finally, blockade of PAR4 with a selective antagonist ameliorated pannus formation and leukocyte infiltration in response to an acute inflammatory insult, implicating that PAR4 is a potential proinflammatory molecule.

Apart from PAR4 expression in the joint, PAR4 mRNA is upregulated in experimental bladder inflammation, regardless of the initiating stimulus that is expressed in peripheral nerves and plexus cell bodies, which may indicate its participation in neurogenic inflammation,²⁵ Furthermore, a role of PAR4 in lung inflammation has been demonstrated as the PAR4-AP could induce proinflammatory cytokine and prostaglandin release from respiratory epithelial cells.¹¹⁷

PAR4 activation in the modulation of neuropathic pain

PAR4 mRNA and protein are expressed in small nociceptive DRG neurons, and the majority of these cells are peptidergic and are expressed by SP and CGRP.^16,69 Activation of PAR4 has been shown to induce neuropathic pain in primary sensory neurons in the DRG,^19,118 suggesting its important role in neural pathways signaling nociceptive responses.^33,118 Additionally, PAR4 expression also has been used as an indicator to reflect the nociceptive effects of TRPV1 on DRG neurons in inflammation and pain processes. Several reports have found that cytokines and other inflammatory mediators upregulate PAR4 mRNA expression in various cell types, similar to those in cultured DRG neurons induced by capsaicin.^119,120 Chen et al. observed that capsaicin activates TRPV1 receptors, consequently leading to an increase in PAR4 mRNA and protein expression and the proportion of PAR4- and TRPV1-immunoreactive neurons as well as their colocalization in cultured DRG neurons.³¹ The effect of capsaicin on PAR4 expression appears to be mediated by the cAMP/PKA signaling pathway.⁴⁶ The mechanism by which capsaicin activates TRPV1 receptors causes primary afferent neurons in the DRG to enhance the expression of TRPV1 and CGRP at the mRNA and protein levels.²⁸

Conclusion

Accumulating evidence has indicated an important role of PAR4 in the pathogenesis of inflammation and pain. This receptor is expressed in DRG neurons, where it induces calcium mobilization after it is activated by trypsin, tryptase, or PAR4-AP. Serine proteases generated during injury and inflammation cleave PAR4 in primary sensory neurons to induce hyperalgesia. This last step requires sensitization of TRPV ion channels including TRPV1 and TRPV4, by PKC as well as by activation of ERK/MAPK signaling and elevated release of CGRP and SP in the dorsal horn in response to mechanical and thermal stimuli. The activation of PAR4 signaling pathways in sensory neurons results in the induction and maintenance of persistent pain, including inflammatory, neuropathic, and visceral pain. PAR4 agonists may exert dual pro- or anti-nociceptive effects in the somatic and visceral domains in a dose-dependent manner (proinflammatory versus subinflammatory doses). While its analgesic effect provides support for a potential application in the treatment of hyperalgesia, the proinflammatory effect of PAR4 remains to be pursued and a better understanding of the mechanisms of action involved in the modulation of nociceptive responses is required.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Funding

The current work was partially supported by National Natural Science Foundation Project of China (no. 81273718 and no. 81302961).