The mechanisms of protease-activated receptor 2 in mediating pain behaviors through nonselective cation channel signaling

Background: Activation of purinergic receptor P2X ligand-gated ion channel 3 (P2X3), transient receptor potential vanilloid type 1 (TRPV1), and transient receptor potential ankyrin 1 (TRPA1) by their specific ligands is a major mechanism contributing to magnified pain responses. The relationship between these nonselective cation channels and proteinase-activated receptor 2 (PAR2) activation mediated pain is still to be clarified. Methods: In this study, both in vitro model of dorsal root ganglion (DRG) neurons with PAR2 agonist SL-NH2 challenge and SL-NH2-induced pain rat model were used to approach these questions. The expression of P2X3, TRPV1, and TRPA1 in DRG neurons was investigated by quantitative real-time RT-PCR, Western blot, and immunofluorescence. The involvement of the PLCβ3/PKCε signaling pathway was also determined. The behavior test for mechanical allodynia and thermal hyperalgesia was performed. Results: SL-NH2 induced upregulation of P2X3, TRPV1, and TRPA1 through phosphorylation of phospholipase Cβ3 (PLCβ3) and protein kinase Cε (PKCε) signaling pathway in DRG neurons in vitro and in vivo. SL-NH2 also elevated the proportion of P2X3-, TRPV1-, and TRPA1-expressing neurons. The upregulation of P2X3, TRPV1, and TRPA1 and phosphorylation of PLCβ3 and PKCε in DRG neurons was paralleled with mechanical allodynia and thermal hyperalgesia behaviors in rats. Conclusions: The data of the present study imply that SL-NH2 as a noxious stimulus activates PAR2 which induces TRPV1, TRPA1, and P2X3 upregulation through PLCβ3/PKCε signaling pathway, thereby decreasing activation thresholds and increasing excitability, resulting in sustained nociceptive activity in DRG neurons, and then causing mechanical allodynia and thermal hyperalgesia behaviors. These data expanded our knowledge about 0.16-fold of the The protein levels of P2 × TRPV1, and TRPA1 in DRG from homolateral side of SL-NH2 injection are 1.86 ± 0.21, 2.1 ± 0.20, and 1.73 ± 0.09-fold of the control, respectively (Fig. 1). The mRNA and protein levels of P2 × 3, TRPV1, and TRPA1 in DRG neurons have increased significantly after SL-NH2 treatment both in vitro and in vivo. These results indicated that PAR2 activation upregulates P2 × 3, TRPV1, and TRPA1 expression which may attribute to PAR2-induced pain. heat pain modulate TRPV1 channels which co-expressed with TRPA1 in nociceptors. These two receptor channels are in a synergistic and/or conditional relationship with noxious stimulation In this present study, the expression of TRPV1 and TRPA1 were significantly increased by SL-NH2-induced PAR2 activation in cultured and pain behavior animal model DRG neurons. P2 × 3 channel expression was affected similarly by PAR2 activation in this experiment.

The upregulation of P2X3, TRPV1, and TRPA1 and phosphorylation of PLCβ3 and PKCε in DRG neurons was paralleled with mechanical allodynia and thermal hyperalgesia behaviors in rats.
Conclusions: The data of the present study imply that SL-NH2 as a noxious stimulus activates PAR2 which induces TRPV1, TRPA1, and P2X3 upregulation through PLCβ3/PKCε signaling pathway, thereby decreasing activation thresholds and increasing excitability, resulting in sustained nociceptive activity in DRG neurons, and then causing mechanical allodynia and thermal hyperalgesia behaviors. These data expanded our knowledge about PAR2-mediated pain sensitivity and its relationship with TRPV1, TRPA1, and P2X3 and provided new opportunities on management of pain behaviors.

Background
Protease-activated receptors (PARs) are a family of G-protein-coupled receptors (GPCRs) that possess a seven transmembrane loops that can be irreversibly activated by proteolytic cleavage of the N terminus rather than by exogenous ligand binding [1][2][3][4]. One of the PAR subtypes, PAR2 is reported to be associated with metabolism, pain, inflammation, and other diseases [5][6][7][8][9]. PAR2 is suspected to modulate the pathogenesis of various neurodegenerative conditions [10]. The upregulation or activation of PAR2 in dorsal root ganglion (DRG) neurons and sensory nerves is involved in the peripheral mechanisms of inflammation development and neuropathic pain [11][12][13][14].
PAR2 is a common target for multiple proteases that cleaves it at different sites to generate different PAR2-mediated intracellular signaling pathways. Different ligands are responsible for different PAR2 signaling and functions [15], therefore, signaling bias of PAR2 may lead to different functional responses. Phospholipase C (PLC) and protein kinase C (PKC) are the classical GPCR downstream molecules those are involved in the nociceptive transmission [16]. PKC is one of the downstream pathways of PAR2 which contributes to neuropathic pain and blocking PKC can significantly diminish the hyperalgesia [17]. Moreover, PKC pathway can trigger the expression of the proinflammatory cytokines in DRG and spinal cord [18]. Since PAR2 is present in DRG neuronal populations, the role of PAR2 activation in the neuronal inflammation and the mediation of PKC activation via PAR2 remains unknown. Whether the modulation of PKC activation is mediated via PAR2 and activation of PAR2 induces the development of neuronal inflammation in DRG should be further explored. PAR2 may influence ligand-modulated calcium signaling by utilizing different important channels [19]. Therefore, there is a need to study which the sensitive gated ion channels are involved after PAR2 activation in the development of pain behaviors.
The transient receptor potential channel vanilloid 1 (TRPV1) is one of the key nociceptive receptors that is expressed in primary sensory neurons and is involved in the detection and transduction of nociceptive stimuli [20] as well as expressed in central nervous system to regulate mental disorders [21][22][23][24][25]. It is affected by kinase-mediated phosphorylation which leads to an increase in pain sensitivity [26]. The activation of TRPV1 induces the accumulation of cytosolic Ca 2+ in the DRG neuron, which leads to nociceptor sensitization [27,28]. Transient Trpv1 gene silencing or selectively inhibition of TRPV1 produces antinociceptive action on relieving pain behavior [29,30]. Since the perception of painful thermal stimuli by sensory neurons is largely mediated by TRPV1 [31], the functional coupling of PAR2 and TRPV1 in DRG neurons may potentiate pain sensation signaling at different pathological conditions [32]. Interestingly, PAR2-triggered 'referred hyperalgesia' was inhibited by blocking TRPV1 suggesting a close relationship between PAR2 and TRPV1 in mediating pain or hyperalgesia [33].
The transient receptor potential ankyrin 1 (TRPA1) is expressed in primary sensory neurons and is activated by a variety of inflammatory mediators that can cause pain hypersensitivity during inflammation [34], nerve insult [35], and neuropathic pain [36]. Exogenous and endogenous TRPA1 activators through electrostatic binding and/or covalent attachment to this protein cause the sensation of pain or toxicology in animal models and humans [37][38][39][40]. Inhibition of TRPA1 alleviates nociception and neuropathic pain behavior [41][42][43]. Several pain sensing actions of PAR2 are associated with TRPA1 [44,45], suggesting PAR2-related responses are, at least in part, through TRPA1-expressing primary sensory neurons. Thus, both PAR2-induced TRPV1 and TRPA1 downstream pathways might play a notable role in the development of mechanical and thermal hyperalgesia [46].
Purinergic P2X receptors are involved in mediating intercellular signaling via purines such as adenosine-5′-triphosphate (ATP) [47] and are responsible for the transmission of nociceptive information from the periphery to the central nervous system [48]. As an important transducer of nociceptive stimuli, purinergic receptor P2X ligand-gated ion channel 3 (P2 × 3) is a trimeric cation channel that is gated by extracellular ATP and predominantly localized in small-to medium-sized neurons of the DRG [49] and acts as an attractive potential therapeutic target [50]. P2 × 3 can be involved in different pain conditions [51]. Its activation contributes to acute nociceptive behavior, hyperalgesia, and allodynia [52][53][54]. A higher expression of P2 × 3 in DRG is related to mechanical allodynia and thermal hyperalgesia during inflammation, neuropathy, and nerve injury [48,[55][56][57][58][59][60][61]. A high degree of specificity in pain sensation processing actions has been attributed to the restrictive expression of P2 × 3 in small to medium diameter C-and Aδ-fiber primary afferent neurons [62].
Inhibition of P2 × 3 receptor expression in DRG neurons could relive the neuropathic pain or inflammatory pain [63,64]. Whether P2 × 3 could be served as a marker in the PAR2 activation induced pain should be further studied.
Despite PAR2 plays multiple roles in different diseases and healthy conditions, its activation during neuroinflammation is prominent. The signaling pathway that leads to pain including the activation of PAR2 and the downstream activation of PLC, PKC, TRPV1, TRPA1, and P2 × 3 should be further investigated. Hence, in the present study, we are utilizing pain rat model to examine the contribution of PAR2 in inflammation-related pain behavioral changes. We are also utilizing in vitro and in vivo model for exploring the expression of PAR2 related Ca 2+ channels to investigate its mechanisms. Our findings may indicate that PAR2 plays important roles in inflammation-related changes in behavior and in cation channel-related pain disorders that should be examined further to fully elucidate its therapeutic potential.

SL-NH2 elicited P2 × 3, TRPV1, and TRPA1 expression in DRG neurons in vitro and in vivo
It is well documented that the upregulation of P2 × 3 is known to contribute to purinergic pain-sensing system [65]. In a different neuropathological perspective, PAR2-related pain sensitivity maybe attributed to the upregulation or activation of TRPV1 [66,67] and TRPA1 [46,68,69] in DRG neurons.

SL-NH2 induced PLCβ3 and PKCε phosphorylation in DRG neurons
Activation of PAR2-induced phosphorylation of PLC is one of the mechanisms of receptor activation and desensitization [70]. Upregulation of PKCε in DRG neurons is involved in pain responses during inflammation [71]. Activation of PAR2-induced hyperalgesic state is dependent on PKC signaling [72].
In this study, both pPLCβ3 and pPKCε protein levels in DRG neurons in vitro and in vivo were determined by Western blot assay. After SL-NH2 treatment, the protein levels of pPLCβ3 and pPKCε in cultured neurons are 2.08 ± 0.26 and 2.13 ± 0.23-fold of the control. The protein levels of pPLCβ3 and pPKCε in DRG from homolateral side of SL-NH2 injection are 2.04 ± 0.15 and 2.04 ± 0.14-fold of the control, respectively (Fig. 6). These results imply that phosphorylation of PLCβ3/PKCε signaling molecule in DRG neurons induced by PAR2 activation triggers its downstream nonselective cation channel expression which is responsible for the development and maintenance of pain.
The alterations of mechanical and thermal thresholds related to c-Fos exprsssion after SL-NH2 treatment The mechanical and thermal thresholds were measured from 0.5 hours to 72 hours after intraplantar injection of 10 µg SL-NH2 on right foot. The lowest mechanical and thermal thresholds were observed at 2 hours after intraplantar injection of SL-NH2 on the homolateral foot. From 2 hours to 72 hours after intraplantar injection of SL-NH2, the mechanical and thermal thresholds were persistent at very low levels compared with those on the contralateral side. After SL-NH2 treatment, the protein level of c-Fos in DRG from SL-NH2-treated rats is 2.46 ± 0.19-fold of control (Fig. 7). Discussion PAR2 has been highlighted as a target for multiple proteases and it can mediate pain in different conditions [7]. PAR2 in the peripheral nerve endings are implications of the development of increased sensitivity to mechanical and thermal stimuli, especially during inflammatory states [32]. The focus on the pathophysiology and therapeutic potential of purinergic signaling has been raised more recently [73]. The close relationship between P2 × 3 and the pain-sensing system and the specific actions of P2 × 3 on pain sensation attract us to explore P2 × 3 as a novel potential therapeutic target on relieving pain. Sensitization of TRPV1 and TRPA1 via PLC and PKC kinases is correlated with the role of PAR2-mediated nociceptive signaling and pain has been recognized recently [74]. In the present study, both in vitro model of DRG with PAR2 agonist SL-NH2 challenge and SL-NH2-induced pain rat model were used to investigate the relationship between PAR2 activation and nonselective cation channels in mediating pain. The results showed that PAR2 agonist SL-NH2 induced upregulation of P2 × 3, TRPV1, and TRPA1 and phosphorylation of PLCβ3 and PKCε in DRG neurons in vitro and in vivo. SL-NH2 also elevated the proportion of P2 × 3-, TRPV1-, and TRPA1-expressing neurons. The upregulation of P2 × 3, TRPV1, and TRPA1 and phosphorylation of PLCβ3 and PKCε in DRG neurons was paralleled with mechanical allodynia and thermal hyperalgesia behaviors in rats.
These findings represent a novel peripheral mechanism underlying PAR2 signaling in mechanical allodynia and thermal hyperalgesia by regulating the expression of different nonselective cation channels.
The relationship between nonselective cation channels P2 × 3, TRPV1, and TRPA1 and PAR2 activation in mediating mechanical allodynia and thermal hyperalgesia deepened our knowledge about PAR2mediated pain-sensing system. P2 × 3-expressing non-peptidergic small-diameter unmyelinated Cfibers represent a large proportion of somatic nociceptors in pain-sensing processing system [65].
From the nociceptor's receptive field in peripheral tissue, excitatory P2 × 3 drives the initial nerve impulse along with the sensitizing C-fibers to facilitate pain signaling transmission through their central terminals to the spinal dorsal horn (SDH). Co-activation of PAR2 and TRPV1 or TRPA1 resulting in the release of proinflammatory neuropeptide transmitters is a new mechanisms in mediating pain responses [66,67]. The PLC and PKC signaling in neurons is commonly considered as the pathway of pain and is usually the target for treatment. However, the extent of nonselective cation channel P2 × 3, TRPV1, and TRPA1 as the common effector of PAR2 remains uncertain. In this way, the involvement of PLCβ3 and PKCε signaling pathway as well as P2 × 3, TRPV1, and TRPA1 in the context of PAR2 activation were investigated. In this study, we used pain behavior animal model mediated by PAR2 activation, as well as cultured DRG neurons with PAR2 activation to examine the possible signaling and cation channel mechanisms involved. The intraplantar injection of the PAR2 agonist SL-NH2 caused the pain behavior associated with c-Fos elevation in SDH. The signaling molecules in pain behavior animal model DRG and SL-NH2 treated DRG culture were upregulated. PAR2 agonist also upregulates the expression of TRPV1, TRPA1 and P2 × 3 channels suggested that the direct activation of PAR2 can induce TRPV1, TRPA1, and P2 × 3 channel expression through PLCβ3 and PKCε signaling pathway. Furthermore, pain behavior observed in animal model in the presence of PAR2 activation suggested P2 × 3, TRPV1, and TRPA1 is related to animal pain behavior.
These results provided additional support for the development of new therapeutic alternatives that can focus on pain induced by activation of PAR2. The expression of c-Fos is inducted after DRG inflammation, DRG neuron hyperexcitability, and neuropathic pain hypersensitivity [75,76], including PAR2 involved nociception [77]. c-Fos increases the expression of proinflammatory cytokines and causes neurochemical alterations in the DRG neurons [18,78]. In the present study, the pain induced by SL-NH2 injection indicated that PAR2 is activated. The c-Fos protein was expressed in the SDH of SL-NH2-induced pain model. PAR2 contributes to the onset and maintenance of SL-NH2-induced pain hypersensitivity in a c-Fos dependent manner. PAR2 is involved in this characteristic behavior because c-Fos induces the synthesis of proinflammatory cytokines or neurotransmitters causing peripheral inflammation.
Interestingly, TRPA1-mediated heat pain sensitivity may indirectly modulate TRPV1 channels which were co-expressed with TRPA1 in nociceptors. These two receptor channels are in a synergistic and/or conditional relationship with noxious stimulation [79]. In this present study, the expression of TRPV1 and TRPA1 were significantly increased by SL-NH2-induced PAR2 activation in cultured and pain behavior animal model DRG neurons. P2 × 3 channel expression was affected similarly by PAR2 activation in this experiment.
In the peripheral terminals of sensory neurons, PLC-PKC cascade can directly activate or sensitize neighboring TRPV1 and TRPA1 channels in DRG [16,28,80,81]. According to the subdivision of DRG neurons, TRPV1-and TRPA1-positive neurons are related to both peptidergic and non-peptidergic nerve fibers, whereas P2 × 3-expressing neurons represent non-peptidergic DRG neurons [82][83][84][85][86][87]. It has been shown that the co-expression of other receptors with P2 × 3 in C-fibers can alter neuronal sensitization induced P2 × 3 activation. The key role of co-expression of TRPV1 and P2 × 3 in nociceptive neuronal subpopulations in mediating severe pain complicated the concept and mechanism of nonselective cation channels on pain signaling processing [65]. The close relationship between PAR2 and TRPV1 is reflected by the co-expression of PAR2 and TRPV1 in nociceptive DRG neurons. Blocking PAR2 decreases expression of TRPV1, and the blockade of TRPV1 prevents the activation of PAR2-induced persistent thermal hyperalgesia [32,66]. PAR2 is also co-expressed with TRPA1 in small DRG neurons [74]. The attenuation of TRPA1 signaling by blocking PAR2 activation and inhibition of the TRPA1 inducing decreases of PAR2-related hyperactivity and pain suggests the close relationship and interaction between PAR2 with TRPA1 in mediating hyperactivity and pain [44,67].
The difference in pain processing mechanisms between PAR2-positive neurons that co-expresses with P2 × 3-, TRPV1-, or TRPA1-expressing neurons and PAR2-positive neurons that do not co-expresses with other neurons should be further investigated in future studies. The previous studies have reported the modulation of TRPV1, TRPA1, or P2 × 3 by activation of PAR2 in a various neuropathic pain and inflammatory pain [88][89][90][91][92]. We draw on the previous research to get inspiration: Since TRPV1, TRPA1 or P2 × 3 are involved in the regulation of PAR2 during the process of mediating pain, is there a common signaling pathway which simultaneously mediates TRPV1, TRPA1 and P2 × 3 via PAR2 activation. We made the hypothesis and conducted the experiment and finally got a positive conclusion. This discovery provides a considerable clinical prospect. Additionally, PAR2 agonist also induces itch by activating superficial SDH neurons [93][94][95][96]. Although the effect of itching induced by SL-NH2 injection is difficult to rule out in our present study, we only focused on pain behaviors and their relationship with the modulation of TRPV1, TRPA1, and P2 × 3 by activation of PAR2. It is worth noting that SL-NH2 is not only active PAR2 but also Mrgpr GPCRs, particularly MrgprC11. But MrgprC11 activated by SL-NH2 will mediated histamine-independent itch rather than pain [97]. So this does not affect our conclusions. The transmission of pain information is closely related to the ion channel that precisely controls pain information on nociceptors. Non-selective cation channels P2 × 3, TRPV1, and TRPA1 have all been shown to play important roles in different types of initiation or transmission processes of pain. Previous studies demonstrated that inhibition of PAR2 can enhance the analgesic effect of opioids, thereby reducing the addictiveness of such drugs, while reducing the side effects such as itching after the application of opioids. This effect is based on the integration of PAR2-mediated TRPV1 and µ-opioid receptor [17,98]. According to our results, PAR2 can mediate the progression of pain by non-selective cation channels P2 × 3, TRPV1, and TRPA1 via PLCβ3/PKCε signaling pathway. Applying this discovery to the development of clinical analgesic drugs by inhibiting PAR2, which can simultaneously relieve pain related with P2 × 3, TRPV1, or TRPA1, not only enhance the efficacy and application range of analgesic drugs, but also reduce side effects. As a result, TRP channels mediated enhancement of neuronal excitation and a variety of neuronal signals is closely related to the pain sensations [99].

Conclusion
The noxious stimulus, SL-NH2, activates PAR2 inducing TRPV1, TRPA1, and P2 × 3 upregulation through PLCβ3/PKCε signaling pathway and thereby decreasing activation thresholds and increasing excitability. This results in sustained nociceptive activity in DRG neurons, which causes mechanical allodynia and thermal hyperalgesia behaviors. The data from this study expand our knowledge about PAR2-mediated pain sensitivity and its relationship with TRPV1, TRPA1, and P2 × 3. These provide new opportunities for promising therapeutic alterative to manage PAR2 activated pain by targeting one or more of these nonselective cation channels or signaling pathways with pharmacological manipulation.

Materials And Methods Animals
The animals used in this study were newborn (less than 24 hours after birth for DRG cell culture) and adult (male rat age 7 weeks with 180-220 g body weight for neuropathic experiment) Wistar rats which were from Shandong University Experimental Animal Center bred with free access to food and water and 12 h light/ 12 h dark cycle. Cultured DRG was removed from newborn rats under anesthesia with 1.5% pentobarbital sodium via peritoneal injection (2 ml/kg body weight). After finishing the final mechanical allodynia and thermal hyperalgesia behavior test, DRG and SDH were removed from adult rats under anesthesia with 1.5% pentobarbital sodium via peritoneal injection (2 ml/kg body weight).
DRG neuronal culture with SL-NH2 stimulation DRG neurons from newborn rats were cultured as our previous protocols [100]. Briefly, DRG explants were digested with 1.25 mg/ml collagenase, triturated with glass pipette, filtered with 130 µm filter, centrifuged at 1500 rpm for 5 minutes, resuspended in culture medium, and plated in 6-well or 24well clusters. After incubation at 37 °C with 5% CO 2 for the first 24 hours, DRG cells in culture were treated with 5 µg/ml cytosine arabinoside for the second 24 hours. After that, DRG cultures were incubated with SL-NH2 (100 µmol/L) for additional 24 hours. DRG cells were incubated only with DMEM/F-12 culture medium as control group.

Mechanical allodynia and thermal hyperalgesia behavior test after SL-NH2 injection
Fifteen male rats with the body weight of 180-220 g were used in this behavior test. For induction of mechanical allodynia and thermal hyperalgesia behaviors, intraplantar injection of 10 µg in 100 µl SL-NH2 on the right hind paw was applied for each rat 3 times in the successive 3 days. Intraplantar injection of 100 µl normal saline solution on the contralateral (left) side was used for control.
Mechanical allodynia and thermal hyperalgesia behavior test timepoint was at 0 (baseline), 0.5, 1, 2, 4, 6, 8, 10, 12, 24, 48, and 72 hours, respectively. The mechanical allodynia and thermal hyperalgesia behavior test protocol was as our previous study [101]. The rats were acclimatized in a plastic box on a metal mesh for 30 minutes before behavior test. For the measurement of mechanical thresholds, the hind paw threshold score with different intensive von Frey filaments was recorded and the average score was calculated from 3 thresholds measured in 3 successive tests each with 3 minutes interval. For the measurement of thermal thresholds, the average withdrawal latency was obtained from 3 thermal thresholds measured in 3 successive tests each with 5 minutes interval. The mechanical allodynia and thermal hyperalgesia behavior was measured with blind test. After finishing the final mechanical allodynia and thermal hyperalgesia behavior test at 72 hours, DRG from SL-NH2-treated rats was used for detecting mRNA and protein levels.
Measurement of mRNA levels with quantitative real-time RT-PCR The mRNA levels of P2 × 3, TRPV1, and TRPA1 in cultured DRG neurons with SL-NH2 stimulation and in DRGs from SL-NH2-treated rats were measured with quantitative real-time RT-PCR. L4-L6 DRGs were removed from SL-NH2 injected side (right side) and normal saline injected side (left side), respectively, and examined separately. Extraction of total RNA was by using RNA Fast200 kit (Fastagen). Synthesis of cDNA was by using cDNA synthesis kit (Termo Scientific). Quantitative PCR was performed with SYBR Green master mixes (Takara) and amplification was performed with the synthetic oligonucleotide primers. The PCR reaction was performed at 55 °C for 2 minutes, 95 °C for 10 minutes, followed by 40 cycles at 94 °C for 5 seconds, 58 °C for 30 seconds, and 72 °C for 45 seconds and the final results carried out by using 2 −ΔΔCt method [102]. The primer sequences used in this PCR reaction system were shown in Table 1. Table 1 |The sequences of oligonucleotide primers.

Genes
Primer sequences P2  Table 2. Immunoreactive images were then analyzed by using ImageJ software.  TRPA1, respectively. The double fluorescence labeling was processed as our previous study [14]. The information of the primary antibodies and secondary antibodies used in fluorescence staining was shown in Table 3.     The thermal thresholds after SL-NH2 treatment. n=15, mean ± SEM, *P<0.001, **P<0.0001.

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