Preferred recycling pathway by internalized PGE2 EP4 receptor following agonist stimulation in cultured dorsal root ganglion neurons contributes to enhanced EP4 receptor sensitivity

Neuroscience 326 (2016) 56–68

PREFERRED RECYCLING PATHWAY BY INTERNALIZED PGE2 EP4 RECEPTOR FOLLOWING AGONIST STIMULATION IN CULTURED DORSAL ROOT GANGLION NEURONS CONTRIBUTES TO ENHANCED EP4 RECEPTOR SENSITIVITY BRUNO ST-JACQUES a AND WEIYA MA a,b*

underlying PGE2-induced nociceptor sensitization. Ó 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

a

Douglas Mental Health University Institute, McGill University, Montre´al, Que´bec H4H 1R3, Canada b Department of Psychiatry, McGill University, Montre´al, Que´bec H4H 1R3, Canada

Key words: receptor trafficking, dorsal root ganglion, internalization, recycling, prostaglandin E2, nociceptor sensitization.

Abstract—Prostaglandin E2 (PGE2), a well-known pain mediator abundantly produced in injured tissues, sensitizes nociceptive dorsal root ganglion (DRG) neurons (nociceptors) through its four EP receptors (EP1–4). Our prior study showed that PGE2 or EP4 agonist stimulates EP4 externalization and this event was not only suppressed by the inhibitor of anterograde export, but also by the recycling inhibitor (St-Jacques and Ma, 2013). These data suggest that EP4 recycling also contributes to agonist-enhanced EP4 surface abundance. In the current study, we tested this hypothesis using antibody-feeding-based internalization assay, recycling assay and FITC-PGE2 binding assay. We observed that selective EP4 agonist 1-hydroxy-PGE1 (1-OH-PGE1) or CAY10850 time- and concentrationdependently increased EP4 internalization in cultured DRG neuron. Internalized EP4 was predominantly localized in the early endosomes and recycling endosomes, but rarely in the late endosomes and lysosomes. These observations were confirmed by FITC-PGE2 binding assay. We further revealed that 1-OH-PGE1 or CAY10850 time- and concentration-dependently increased EP4 recycling. Double exposures to 1-OH-PGE1 induced a greater increase in calcitonin gene-related peptide (CGRP) release than a single exposure or vehicle exposure, an event blocked by pre-treatment with the recycling inhibitor monensin. Our data suggest that EP4 recycling contributes to agonist-induced cell surface abundance and consequently enhanced receptor sensitivity. Facilitating EP4 externalization and recycling is a novel mechanism

INTRODUCTION Sensitization of nociceptive dorsal root ganglion (DRG) neurons plays an essential role in the development of pathological pain state. Sensitized neurons lower the activation threshold to both noxious and innocuous stimuli, thus contributing to the generation of hyperalgesia and allodynia, respectively. Various proinflammatory mediators released from inflammatory cells in injured tissues enable to excite nerve endings of nociceptive DRG neurons (nociceptors) to induce peripheral sensitization. Prostaglandin E2 (PGE2), a well-characterized pain mediator in damaged or inflamed tissue, is known to sensitize nociceptors through its four EP receptors (EP1–4) expressed in DRG neurons (Oida et al., 1995; Lin et al., 2006; Ma et al., 2010). PGE2 directly excites nociceptors by increasing membrane Ca++ currents (Gold et al., 1996b) and tetrodotoxin-resistant voltage-gated Na+ currents (Gold et al., 1996a; Rush and Waxman, 2004). It also potentiates sensitizing effects exerted by other pain mediators such as ATP, bradykinin and capsaicin (Vanegas and Schaible, 2001; Moriyama et al., 2005; Wang et al., 2007; Zhang et al., 2008). Moreover, PGE2 directly stimulates the release of pain-related peptides substance P (SP) and calcitonin gene-related peptide (CGRP) from nociceptors (Hingtgen et al., 1995; Vasko, 1995) and indirectly potentiates the release of SP and CGRP evoked by other pain mediators (Vasko et al., 1994). EP4 receptor subtype was shown to be involved in PGE2-induced peptide release (Southall and Vasko, 2001). In addition to the sensitizing nociceptors at the functional levels, PGE2 was shown by us and others to stimulate the synthesis of SP, CGRP (Ma and Eisenach, 2003; Ma, 2010; Ma et al., 2010), Nav1.8 channel (Villarreal et al., 2005), interleukin-6 (IL-6) (St-Jacques and Ma, 2011) and brain-derived neurotrophic factor (BDNF) (Cruz Duarte et al., 2012) in DRG neurons at both gene and protein levels during inflammatory and

*Correspondence to: W. Ma, Douglas Mental Health University Institute and Department of Psychiatry, McGill University, 6875 LaSalle Boulevard, Verdun, Montre´al, Que´bec, Canada. Tel: +1514-761-6131x2935; fax: +1-514-762-3034. E-mail address: [email protected] (W. Ma). Abbreviations: 1-OH-PGE1, 1-hydroxy-PGE1; ANOVA, analysis of variance; APP, amyloid precursor protein; Ab, beta amyloid; CAY, CAY10580; CGRP, calcitonin gene-related peptide; DMEM, Dulbecco’s Modified Eagle Medium; DRG, dorsal root ganglion; ELISA, enzyme-linked immunosorbent assay; FBS, fetal bovine serum; GPCR, G protein-coupled receptor; HEPES, 4-(2-hydroxye thyl)-1-piperazineethanesulfonic acid; PBS, phosphate-buffered saline; PGE2, prostaglandin E2; SP, substance P. http://dx.doi.org/10.1016/j.neuroscience.2016.04.005 0306-4522/Ó 2016 IBRO. Published by Elsevier Ltd. All rights reserved. 56

B. St-Jacques, W. Ma / Neuroscience 326 (2016) 56–68

neuropathic pain. These data indicate that PGE2 is central for the establishment of the network of pain mediators. And EP4 receptor plays a pivotal role in mediating these effects of PGE2 (Ma, 2010; St-Jacques and Ma, 2011; Cruz Duarte et al., 2012; Villarreal et al., 2013). EP1–4 receptors belong to the family of G proteincoupled receptor (GPCR). Trafficking events such as externalization, internalization, recycling and degradation dynamically regulate GPCR cell surface density. A fast-rate of internalization and degradation reduces GPCR surface density to desensitize cell response while a fast rate of recycling increases GPCR surface density to re-sensitize cell response. We recently showed that PGE2 or EP4 agonist increased EP4 externalization in in vitro and in vivo DRG neurons (St-Jacques and Ma, 2013). This event was blocked not only by an inhibitor of anterograde trafficking from ER/Golgi complex to the cell surface, but also by an inhibitor of recycling (St-Jacques and Ma, 2013), suggesting that in addition to EP4 externalization, EP4 recycling also contributes to agonist-enhanced EP4 surface abundance and nociceptor sensitization. In this study, we attempted to test this hypothesis. The first aim was to examine the basal and agonist-stimulated EP4 internalization in cultured DRG neurons using antibody feeding-based internalization assay. Since internalized EP4 receptors could go through both recycling and degradation pathways, the second aim was to examine the co-localization of internalized EP4 with specific markers for the early endosomes (EEA1), the recycling endosomes (Rab11), the late endosomes (Rab7) and the lysosomes (Lamp1) to track the internalized EP4 in these organelles after internalization. Fluorescent dye-conjugated agonist has widely been used in vitro and in vivo to trace GPCR internalization (Cahill et al., 2001; Pheng et al., 2003). Alternatively, in this study we used fluorescent dye FITC-conjugated PGE2 to trace EP receptor internalization in cultured DRG neurons. Thus, the third aim was to examine FITC-PGE2-induced EP internalization and the distribution of internalized EP receptors in the early endosomes and lysosomes. The fourth aim was to examine EP4 recycling following agonist stimulation using antibody feeding-based recycling assay. As mentioned above, PGE2 stimulates the release of CGRP from primary sensory neurons and this event was mediated through EP4 (Southall and Vasko, 2001). Thus CGRP release from cultured DRG neurons evoked by EP4 agonist was used as an indicator to gauge EP4 receptor activity. Therefore our last aim was to determine whether agonist-facilitated EP4 recycling contributes to exaggerated CGRP release from cultured DRG neurons.

EXPERIMENTAL PROCEDURES Primary cultures of DRG neurons and treatments Sprague–Dawley rats (male, 2 to 3 months old, body weight 250–300 g) were used in this experiment (Charles River, St Constant, Quebec, Canada). All animal care procedures were according to protocols and guidelines approved by the McGill University Animal

57

Care Committee and the Canadian Council for Animal Care. Rats were decapitated and all DRGs (45–50 DRGs) from the cervical, thoracic, lumbar and sacral levels were removed aseptically and collected in Hank’s balanced salt solution (HBSS, Gibco/BRL, Burlington, Ontario, Canada). One rat was usually used in each session. All ganglia were minced into small pieces and digested in 0.25% collagenase (Cedarlane Laboratory Ltd, Hornby, Ontario, Canada) in Ham’s F12 medium (Gibco/BRL) at 37 °C for 30 min. Following a 7-min incubation in 0.25% trypsin (Gibco/BRL) in Dulbecco Modified Eagle Medium (DMEM, Gibco/BRL) containing 1% HEPES buffer solution, the tissues were triturated with a thin flame-polished pipette in DMEM containing 1% HEPES buffer solution, penicillin/streptomycin (1:200, Gibco/BRL) and 10% heat-inactivated fetal bovine serum (Gibco/BRL) (DMEM–FBS). Cells were centrifuged at 400g for 10 min. The resulting pellet was re-suspended in DMEM-FBS and cell suspension was filtered through a cell strainer (200 lm mesh, Corning Incorporated Life Sciences, Tewksbury, MA, USA). DRG cells were seeded in 96-well plates (200 ll per well), yielding a density of 5
104 cells/well. The cells were cultured in a humid incubator at 37 °C with 5% CO2 and 95% air. Each treatment was performed in six wells and repeated at least in three different sessions (i.e. at least repeated in three rats) and more frequently in 5–10 sessions or in 5–10 rats. Internalization assay of EP4 receptors and co-labeling of internalized EP4 with EAA1, Rab11, Rab7 and Lamp1 Antibody feeding-based internalization assay was modified from previously reported method (ArancibiaCa´rcamo et al., 2006). Cultured neurons were labeled by an antiserum raised against the extracellular N-terminal of EP4 (1:250, Cayman Chemical Inc., Ann Arbor, MN, USA) at 4 °C for 30 min, then brought to 37 °C and treated with vehicle or EP4 antagonists 1-hydroxy-PGE1 (1-OH-PGE1) or CAY10850 (1, 10 and 50 lM, Cayman Chemical Inc.) for 15, 30 and 60 min. Cells were then fixed and incubated in unconjugated goat anti-rabbit IgG (1:500, Vector laboratories) at 4 °C to block un-internalized antibody-tagged surface EP4. Then cells were permeabilized and incubated in a goat anti-rabbit IgG conjugated with Alexa Flura-488 (1:500, Invitrogen, Burlington, ON, Canada) to identify the internalized intracellular EP4 receptors. EP4 originally existing in intracellular compartments cannot be visualized by this approach. For double immunostaining of internalized EP4 with EAA1, Rab11, Rab7 and Lamp1, cultured cells were incubated in N-terminal EP4 antibody (1:250) at 4 °C for 30 min, then brought to 37 °C and treated with vehicle or the selective EP4 agonist 1-OH-PGE1 (10 lM) for 60 min before fixation and permeabilization. Internalizedand antibody tagged-EP4 receptors were visualized by incubating with a goat anti-rabbit IgG conjugated with Alexa Fluor-488 (green, 1:500). Cells were further incubated with mouse monoclonal antisera raised against EEA1 (1:200, Cell Signaling Technology,

58

B. St-Jacques, W. Ma / Neuroscience 326 (2016) 56–68

Danvers, MA, United States), Rab11, Rab7 and Lamp1 (1:200, Santa Cruz Biotechnology, Dallas, Texas, USA) and subsequently with a goat anti-mouse IgG conjugated with Alexa Fluor-568 (red, 1:500, Invitrogen). Cells labeled with EP4, EEA1, Rab11, Rab7 or Lamp1 were observed under a confocal microscope (Nikon, PMC2000, New York, USA). Labeled DRG neurons with clear nuclear staining by DAPI were randomly selected. Images of DRG neurons were captured under the exactly same conditions (e.g., magnification, exposure time, pinhole, etc.) for all cells in the same session. Omission of the primary antisera of EP4, EEA1, Rab11, Rab7 and Lamp1 in incubation of DRG sections resulted in no immunostaining of these markers (not shown). Incubation of DRG sections with pre-absorbed primary antisera by antigen peptides provided by the manufacturers at concentrations 10 times higher than the antisera produced a dramatic reduction of immunostaining (not shown). Recycling assay of EP4 receptors Cultured cells were incubated with the N-terminal EP4 antiserum (1:250) for 30 min at 4 °C in DMEM–HEPES to tag EP4 receptors at the cell surface and were then brought to 37 °C to allow EP4 internalization in the presence of vehicle, 1-OH-PGE1 (1, 10 and 50 lM) or CAY (CAY10580) (1, 10 and 50 lM) for 30 or 60 min. Then cells were rinsed in stripping buffer (0.2 M acetic acid in phosphate-buffered saline (PBS)) at 4 °C for 2 min to get rid of the N-terminal EP4 antiserum bound to the un-internalized surface EP4 receptors. Cells in DMEM-HEPES were next brought to 37 °C for 30 min to allow EP4 recycling back to the cell surface. Cells were further incubated in DMEM–HEPES with goat anti-rabbit IgG conjugated with Alexa Fluor-488 (green, 1:500, Invitrogen) to label the recycled EP4 receptors at the cell surface. Subsequently cells were fixed, permeabilized and incubated in a donkey anti-rabbit IgG conjugated with Alexa Fluor-568 (red, 1:500, Invitrogen). The green signal represents the EP4 receptor that has undergone at least one round of recycling while the red signal represents the EP4 receptor that internalized but has not yet been recycled to the membrane. EP receptor binding by FITC-PGE2 and co-labeling with EAA1 and Lamp1 Cultured DRG neurons were incubated with 10 lM FITC, 10 lM FITC-PGE2, FITC + CAY10580 (50 lM) or FITC-PGE2 + CAY10580 (50 lM) for 60 min before fixation and permeabilization. Then fixed cells were incubated with a mouse monoclonal primary antiserum raised against EEA1 or Lamp1 (1:200) and a goat antimouse IgG conjugated with Alexa Fluor-568 (1:500, Invitrogen). PGE2 EP receptors were labeled by green dye FITC while EEA1 and Lamp1 were labeled by red dye AF568. Cells were covered with anti-fading mounting medium containing DAPI (Vector Laboratories, Burlington, ON, Canada). DAPI staining shows the nuclei of cells in blue color. Omission of the primary antisera of EEA1 and Lamp1 resulted in no immunostaining

(not shown). Cultured DRG neurons were observed under a fluorescence microscope (Nikon Eclipse E800 microscope). Double sequential treatments of EP4 agonists and enzyme-linked immunosorbent assay (ELISA) of CGRP Following dissociation, DRG cells were treated with vehicle, 1-OH-PGE1 (10 and 50 lM), 1-OH-PGE1 + monensin (40 lM) or monensin (40 lM) for 1 h at 37 °C. After washing and centrifugation, cells were re-suspended in freshly prepared cultured medium and seeded in a 96-well plate. Cells were cultured for 2d. Then after washing, cells were treated with vehicle, 1-OH-PGE1 (10 and 50 lM), 1-OH-PGE1 (10 and 50 lM)+monensin (40 lM) or monensin (40 lM) for 15 min. The culture medium was collected and frozen at 80 °C until used. A rat CGRP ELISA kit (Bertin Pharma, Montigny Le Bretonneux, France) was used. All procedures were performed according to the manufacturer’s instructions. The microplate was read using a microplate reader (BioTek Technology, Winooski, VT, USA). Statistical analysis Images of EP4 labeling in the cytoplasm (internalized EP4) or at cell surface (recycled EP4) following antibody feeding-based internalization assay and recycling assay, respectively, were quantitated under the same conditions using a software program (SigmaScan). In each session, 25–30 neurons were selected from 6 wells for each treatment. In each session, the EP4 optical density of all individual cells from all groups (vehicle or treatments) was normalized as the percentages of mean vehicle values [(vehicle or treatment)/mean vehicle value
100)]. The average percentages of EP4 optical density for each group were determined. The mean percentages of EP4 optical density between vehicle and treatment or between different treatments at one time point were statistically compared using a One-way analysis of variance (ANOVA) with post hoc Student Newman–Keuls multiple comparison test. The sample size (n) means the numbers of replicated sessions or the numbers of used rats since we have only used one rat for each session. The significance level was set at p < 0.05. Similarly, the individual values of CGRP levels (pg/ml) from each group were normalized as the percentage of the mean vehicle values in each session. The mean percentages of CGRP levels between vehicle and treatments or between treatments were compared statistically using a one-way ANOVA with post hoc Student–Newman–Keuls multiple comparison test. The significance level was set at p < 0.05. All statistical analyses were performed by using the software (SigmaStat).

RESULTS EP4 agonist time- and concentration-dependently increased EP4 internalization in cultured DRG neurons

B. St-Jacques, W. Ma / Neuroscience 326 (2016) 56–68

and internalized EP4 was predominantly localized in both early endosomes and recycling endosomes Two days after seeding, cultured DRG neurons were treated with a selective EP4 agonist 1-OH-PGE1 for 15, 30 or 60 min at 37 °C. When cultured cells were kept at 4 °C throughout the experiment, only EP4 receptors preexisting at the cell surface were observed (Fig. 1A, B). No significant difference in the surface EP4 levels was detected between vehicle and 1-OH-PGE1 treatments (Fig. 1A, B). Compared to the pre-existing surface EP4 revealed by surface immunostaining at 4 °C, vehicle (Fig. 1C, E, G) or 1-OH-PGE1 (Fig. 1D, F, H) markedly increased EP4 internalization at 37 °C. However, when cultured DRG cells were treated at 37 °C, 10 lM 1-OHPGE1 significantly increased the levels of internalized EP4 (Fig. 1C, E, G, p < 0.05–0.01) compared to

59

vehicle treatment (0.01% ethanol in DMEM-HEPES, Fig. 1D, F, H). Compared to 15- and 30-min treatments (Fig. 1D, F, I), 1-OH-PGE1 treatment for 60 min significantly increased internalized EP4 (Fig. 1H, I, p < 0.05). No significant difference was detected between 15 and 30 min of PGE2 treatment. There was also no significant difference among 15-, 30- and 60-min treatments of vehicle (Fig. 1C, E, G, I). Following a 60-min treatment, 10 and 50 lM 1-OH-PGE1 significantly increased the density of internalized EP (Fig. 1J, p < 0.05). However, no significant difference in the intracellular intensity of internalized EP4 was detected between 10 and 50 lM 1-OH-PGE2 treatments. 1-OH-PGE1 at 1 lM had no effects. To determine whether internalized EP4 undergoes the recycling and degradation pathways, co-localization of

Fig. 1. EP4 agonist 1-OH-PGE1 time- and concentration-dependently increased EP4 internalization in cultured DRG neurons. Single-plane confocal images show the pre-existing surface EP4 at 4 °C immunostained with an extracellular N-terminal EP4 antiserum without permeabilization (A and B). Single-plane confocal images (C–H) show internalized EP4 labeling following the treatments with vehicle or 1-OH-PGE1 (10 lM) for 15, 30 and 60 min at 37 °C. Vehicle (C, E and G) or 1-OH-PGE1 (D, F and H) remarkably increased EP4 internalization at 37 °C compared to pre-existing surface EP4 at 4 °C. Compared to vehicle (C, E, G and I), 1-OH-PGE1 significantly increased EP4 internalization (D, F, H and I, * p < 0.05–0.01). Compared to 15- and 30-min treatments (D, F and I), 1-OH-PGE1 treatment for 60 min at 37 °C significantly increased internalized EP4 (H and I, +p < 0.05). No significant difference was detected between 15 and 30 min of 1-OH-PGE1 treatment. There were also no significant differences among 15, 30 and 60 min of vehicle treatments (C, E, G and I). 1-OH-PGE1 at the concentrations of 10 and 50 lM, but not 1 lM, significantly increased the density of internalized EP following a 60-min treatment at 37 °C (J, *p < 0.05). No significant difference in internalized EP4 intensity was detected between 10 and 50 lM 1-OH-PGE2 treatments. Mean ± SEM, one-way ANOVA with post hoc Student–Newman– Keuls multiple comparison test, n = 3–6 sessions (or rats) per group.

60

B. St-Jacques, W. Ma / Neuroscience 326 (2016) 56–68

internalized EP4 with the early endosome marker EAA1, the recycling endosome marker Rab11 and the late endosome marker Rab7 and Lamp 1 was performed after 60-min 1-OH-PGE1 treatments. Following a 60-min treatment, co-localization (Fig. 2C, F, yellow) of internalized EP4 (Fig. 2A, D, green) with EEA1 (Fig. 2B, red) or Rab11 (Fig. 2E, red) were frequently seen. However, internalized EP4 receptors (Fig. 2G, J, green) were rarely co-localized with Lamp1 (Fig. 2H, red) or Rab7 (Fig. 2K, red) in cultured DRG neurons (Fig. 2I, L). Omission of N-terminal EP4 antiserum or incubation with N-terminal EP4 antiserum pre-absorbed with antigen peptides in antibody feeding resulted in no EP4-IR, but only EEA1-, Rab11-, lamp1- and Rab7-IR

remained (not shown). EP4 originally existing in intracellular compartments cannot be visualized by this technical approach. Since 1-OH-PGE2 is a moderately selective EP4 agonist which could bind to mouse EP3 and EP4 with a relatively similar affinity (Kiriyama et al., 1997), we have used a highly selective EP4 agonist CAY10580 to evaluate the involvement of EP4 activation in PGE2facilitated EP4 internalization and recycling. We found that compared to vehicle, the treatments with 1, 10 and 50 lM CAY10580 for 60 min significantly increased EP4 internalization in cultured DRG neurons (Fig. 3A, C–E, G, p < 0.05–0.01). CAY10580 at a lower concentration (0.1 lM) had no effect on EP4 internalization

Fig. 2. Single-plane confocal sections showing the double immunostaining of internalized EP4 receptors and the early endosome marker EEA1, the recycling endosome marker Rab11, the lysosome marker Lamp1 and the late endosome marker Rab7 in cultured DRG neurons following 60 min of 1-OH-PGE1 treatment (10 lM). Internalized EP4 (A, and D, green) with EEA1 (B, red) or Rab11 (E, red) were frequently co-localized (C and F, yellow) while EP4 (G and J, green) with Lamp1 (H, red) or Rab7 (K, red) are rarely co-expressed in cultured cells (I and L). Omission of N-terminal EP4 antiserum or incubation of N-terminal EP4 antiserum pre-absorbed with antigen peptide in antibody feeding resulted in no EP4-IR, only EEA1-, Rab11-, lamp1- and Rab7-IR remained (not shown). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

B. St-Jacques, W. Ma / Neuroscience 326 (2016) 56–68

61

Fig. 3. Epi-fluorescence microscopic images showing that EP4 agonist CAY10580 (CAY, or C) concentration-dependently increased EP4 internalization examined by antibody feeding based internalization assay. Compared to vehicle (A), CAY treatment at the concentrations of 1, 10 and 50 lM for 60 min significantly increased EP4 internalization (C, D, E and G, *p < 0.05–0.01). CAY at the lower concentration of 0.1 lM had no effect on EP4 internalization (B and G). No significant difference was detected between 10 and 50 lM CAY treatments (D, E and G). But 10 and 50 lM CAY10580 induced a significantly greater increase in EP4 internalization than 1 lM (C, D, E and G, #p < 0.05–0.01). Pre- and co-treatment with a highly selective EP4 antagonist L161,982 (L, 50 lM) significantly reduced the density of internalized EP4 (F and G, *p < 0.01). Mean ± SEM, one-way ANOVA with post hoc Student–Newman–Keuls multiple comparison test, n = 3–5 sessions or rats per group.

(Fig. 3B, G). No significant difference was detected between 10 and 50 lM CAY treatments (Fig. 3D, E, G). Pre- and co-treatment with a selective EP4 antagonist L161,982 (50 lM) significantly suppressed the increased density of internalized EP4 by CAY10580 (Fig. 3F, G). These data further confirm that EP4 activation indeed increases EP4 internalization. To complement antibody feeding-based EP4 internalization assay following agonist stimulation, we also used the binding assay of FITC-conjugated PGE2 (FITC-PGE2) to examine agonist-induced EP receptor internalization in cultured DRG neurons. Compared to the treatment only with fluorescent dye FITC for 15, 30 and 60 min (Fig. 4A–C, I), 10 lM FITC-PGE2 significantly increased the levels of intracellular FITC intensity (Fig. 4E–G, p < 0.05–0.01). Pre-treatment with 50 lM CAY10580 for 30 min significantly reduced the intracellular intensity of FITC-PGE2 in cultured DRG cells

compared to FITC-PGE2 treatment (Fig. 4G–I, p < 0.05). This observation suggests that a significant amount of EP receptors internalized following the stimulation of FITC-PGE2 are the EP4 subtype. However, following pre-treatment of CAY10580 for 30 min, the intracellular intensity of FITC-PGE2 still remained significantly higher than FITC treatment (Fig. 4D, H, I, p < 0.01), suggesting that other EP receptor subtypes also contribute to the intracellular FITC-PGE2/EP complex. Interestingly, similar to internalized EP4 following 1-OH-PGE1 treatment, FITC-PGE2/EP complex was predominantly co-expressed with the early endosome marker EAA1 (Fig. 5B–D), but rarely with the lysosome marker Lamp1 (Fig. 5G–I). Taken together, these findings suggest that internalized EP4 receptors are more likely undergo the recycling pathway to return to the cell surface to re-sensitize nociceptors than the degradation pathway to be metabolized, thus desensitizing nociceptors.

62

B. St-Jacques, W. Ma / Neuroscience 326 (2016) 56–68

Fig. 4. Epi-fluorescence microscopic images showing that FITC-PGE2 induced a time-dependent EP receptor internalization and this event was suppressed by the pre-treatment of EP4 agonist CAY10580 (CAY). Compared to the treatment with fluorescent dye FITC for 15, 30 and 60 min (A-C and I), 10 lM FITC-conjugated PGE2 (F-PGE2) significantly increased the levels of internalized FITC-PGE2/EP complex (E–G and I, * p < 0.05–0.01). Pre-treatment with 50 lM CAY for 30 min significantly reduced the intracellular levels of FITC-PGE2 in cultured DRG cells compared to FITC-PGE2 (G, H and I, +p < 0.05). However, intracellular levels of FITC-PGE2 in CAY-pretreated DRG neurons still remained significantly higher than CAY + FITC treatment (D, H and I, *p < 0.01). Mean ± SEM, one-way ANOVA with post hoc Student–Newman–Keuls multiple comparison test, n = 3–5 sessions per group.

EP4 agonist time-and concentration-dependently increased the recycling of EP4 receptors in cultured DRG neurons To determine whether EP4 agonist increases EP4 recycling, we used antibody feeding-based recycling assay modified from previously reported protocols (Arancibia-Ca´rcamo et al., 2006). We observed that internalized EP4 receptors also underwent a time- and concentration-dependent recycling following vehicle or 1-OH-PGE1 treatment (Fig. 6). Very few recycled EP4 receptors were observed at the cell surface when cultured cells remained at 4 °C throughout the experiments regardless of the stimulation by vehicle or 1-OH-PGE1 (Fig. 6A). Compared to vehicle (Fig. 6B, C, G), stimulation

of 10 lM 1-OH-PGE1 for 60 min at 37 °C significantly increased the cell surface density of recycled EP4 (Fig. 6F, G, p < 0.01). However, 1-OH-PGE1 stimulation for 30 min did not significantly increase the surface density of recycled EP4 compared to vehicle treatment (Fig. 6B, D, E, G). No significant difference was detected in the density of recycled EP4 between 30- and 60-min vehicle treatments (Fig. 6B, C, G). Following the stimulation for 60 min, 10 and 50 lM 1-OH-PGE1 significantly increased the cell surface intensity of recycled EP4 compared to vehicle treatment (Fig. 6H, p < 0.05–0.01), but no significant difference was detected between 10 and 50 lM 1-OH-PGE1. At the lower concentration of 1 lM, 1-OH-PGE1 had no effect on EP4 recycling (Fig. 6H).

B. St-Jacques, W. Ma / Neuroscience 326 (2016) 56–68

63

in recycled EP4 density by CAY (50 lM) at the cell surface (Fig. 7D, E). Recycled EP4 receptors contribute to agonistenhanced release of pain-related neuropeptide CGRP

Fig. 5. Epi-fluorescence microscopic images showing the co-localization of FITC-PGE2 with the early endosome marker EEA1 and the lysosome marker Lamp1 in cultured DRG neurons. Cultured DRG neurons were treated with vehicle (A and B) and 10 lM FITC-PGE2 (C–H) for 60 min before fixation and permeabilization. Then fixed cells were incubated in primary antisera raised against EEA1 and Lamp1 and a linking IgG conjugated with Alexa Fluor-568. PGE2 receptors were labeled by green dye FITC while EEA1 and Lamp1 were labeled by red dye Alexa Fluor-568. DAPI staining shows the nuclei (blue) of cultured DRG neurons. When incubated with vehicle (FITC), no green dye was detected (A and B), only EEA1-IR and Lamp1-IR were seen. It is noticeable that FITC-PGE2/EP receptor complexes are more frequently co-localized with EAA1 (C, E and G) than with Lamp1 (D, F and H). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

These observations suggest that agonist time- and concentration dependently stimulates EP4 recycling in cultured DRG neurons. We also used a highly selective EP4 agonist CAY10580 to confirm the effects of 1-OH-PGE1 on EP4 recycling. Similar to 1-OH-PGE1, 10 and 50 lM CAY10580 significantly increased the density of recycled EP4 at the cell surface when compared to vehicle treatment (Fig. 7A–C, E, p < 0.01) while CAY10580 at 1 lM had no effect (Fig. 7E). Pretreatment with a highly selective EP4 antagonist L161,982 (50 lM) significantly suppressed the increase

Since EP4 agonist, in a time- and concentrationdependent manner, stimulates EP4 internalization and the internalized EP4 prefers the recycling pathway to return to the cell surface, we next examined whether recycled EP4 at the cell surface is coupled to enhanced EP4 receptor sensitivity. EP4 activation was known to be involved in PGE2-stimulated release of pain-related peptides such as SP and CGRP from cultured DRG neurons (Southall and Vasko, 2001). In this study, we used EP4 agonist-evoked CGRP release as an indicator to examine whether pre-exposure to EP4 agonist increases EP4 recycling, thus inducing a greater increase in CGRP release from cultured DRG neurons evoked by the subsequent challenge of the second exposure to EP4 agonist, and whether this event is suppressed by the inhibitor of protein recycling. Before seeding, dissociated cells in suspension were treated with vehicle, 1-OH-PGE1 (10 lM), 1-OH-PGE1 + monensin (40 lM) or monensin (40 lM) for 1 h. Two days after seeding, cultured cells were treated with vehicle, 1-OH-PGE1 (10 and 50 lM) or monensin (40 lM) for 15 min. Levels of pain-related peptide CGRP released into the culture medium were analyzed using ELISA. Pre-exposure to 1-OH-PGE1 (10 or 50 lM) did not increase CGRP release evoked by subsequent vehicle treatment compared to vehicle ? vehicle treatments (Fig. 8), suggesting that CGRP release induced by the 1st EP4 agonist 2d earlier is transient. Single or double exposures to 1-OH-PGE1 significantly increased CGRP release compared to double vehicle exposures (Fig. 8, p < 0.05–0.01). Following the double exposures of 1-OH-PGE1 (10 lM) ? 1-OH-PGE1 (10 or 50 lM), CGRP levels in the culture medium were significantly increased compared to the treatments of vehicle ? 1-OH-PGE1 (10 or 50 lM) (Fig. 8, p < 0.05). Moreover, following the exposures of 1-OH-PGE1 (10 lM)+monensin (40 lM) ? 1-OH-PGE1 (10 or 50 lM), CGRP levels in the culture medium were significantly reduced compared to the exposures of 1-OHPGE1 (10 lM) ? 1-OH-PGE1 (10 or 50 lM) (Fig. 8, p < 0.05–0.01), suggesting that agonist-induced EP4 recycling is involved in enhanced CGRP release. However, CGRP levels in the culture medium following exposures to 1-OH-PGE1 (10 lM)+monensin (40 lM) ? 1-OH-PGE1 (10 or 50 lM) still remained significantly higher than following exposures to vehicle ? vehicle (Fig. 8, p < 0.05), suggesting that other mechanisms such as externalization also contribute to this event. Following exposures to 1-OH-PGE1 (10 lM) ? 1-OH-PGE1 (10 or 50 lM)+monensin (40 lM), CGRP levels in the culture medium were not significantly altered compared to double exposure to 1-OH-PGE1 (Fig. 8, p < 0.01), but still remained significantly higher than double exposures to vehicle (Fig. 8, p < 0.01). This observation suggests that treatment of EP4 agonist for 15 min is not sufficient to cause EP4 recycling to reduce CGRP release. Treatments of monensin (40 lM) ? vehicle

64

B. St-Jacques, W. Ma / Neuroscience 326 (2016) 56–68

Fig. 6. Single confocal images showing that EP4 agonist time- and concentration-dependently stimulates the recycling of internalized EP4 in cultured DRG neurons. Single confocal image shows the pre-existing surface EP4 at 4 °C (A). The density of recycled EP4 at the cell surface was significantly increased following a 60-min stimulation of 10 lM 1-OH-PGE1 (1-PGE1) at 37 °C (F) compared to a 60-min treatment of vehicle (C and G, *p < 0.01) or a 30-min treatment of 10 lM 1-OH-PGE1 (D, E and G, +p < 0.01). 1-OH-PGE1 stimulation for 30 min did not significantly increase the surface density of recycled EP4 compared to 30 min of vehicle stimulation (B, D, E and G). No significant difference was detected in recycled EP4 density between 30- and 60-min vehicle treatments (B, C and G). Following 60-min treatments, 10 and 50 lM 1-OH-PGE1 significantly increased the cell surface density of recycled EP4 while 1 lM had no effect when compared to vehicle (H, *p < 0.05–0.01). No significant difference in recycled EP4 density was detected between 10 and 50 lM 1-OH-PGE1 treatments. Mean ± SEM, one-way ANOVA with post hoc Student– Newman–Keuls multiple comparison tests, n = 3–6 sessions per group.

or vehicle ? monensin (40 lM) did not change the released CGRP levels compared to vehicle ? vehicle treatments, suggesting that monensin only affects agonist-stimulated EP4 recycling, but not the constitutive EP4 recycling.

DISCUSSION EP4 agonist time- and concentration-dependently increased EP4 internalization in cultured DRG neurons For the first time, we demonstrated that a selective EP4 agonist 1-OH-PGE1 time- and concentration-

dependently increased EP4 internalization in cultured DRG neurons. EP4 internalization was detected in 15 min (the earliest time point examined) and enhanced by 60 min (the last time point examined) following agonist stimulation. Since 1-OH-PGE2 is a moderately selective EP4 agonist which could also bind to EP3 (Kiriyama et al., 1997), we thus used a highly selective EP4 agonist CAY10580 to confirm the data obtained by 1-OH-PGE1. Similarly, CAY10580 also induced a concentration-dependent increase in EP4 internalization. The effective concentrations of EP4 agonist 1-OH-PGE1 or CAY10580 in our study were 10 and 50 lM, similar to the concentrations used in prior studies by others

B. St-Jacques, W. Ma / Neuroscience 326 (2016) 56–68

Fig. 7. Single confocal images showing that EP4 agonist CAY10580 (CAY or C) concentration-dependently stimulates the recycling of internalized EP4 in cultured DRG neurons. Compared to vehicle treatment (A), the density of recycled EP4 at the cell surface (green) was significantly increased following 60 min of stimulation of 10 and 50 lM CAY at 37 °C (B and C, *p < 0.01). CAY at 1 lM had no effect (E). Pre-treatment with EP4 antagonist L161, 982 (50 lM) significantly suppressed CAY (50 lM) induced increase in recycled EP4 density at the cell surface (D and E). One way ANOVA with post hoc Student–Newman–Keuls multiple comparison test, n = 5 sessions per group. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

(Olesen et al., 2011; Coskun et al., 2013). These concentrations (lM range) of EP4 agonists mimic better the concentration of PGE2 in inflamed tissues during inflammation (Futaki et al., 1993; Sidhapuriwala et al., 2007). The internalization of the majority of GPCRs is known to be initiated by receptor phosphorylation, uncoupling to G protein and endocytosis mediated through arrestin/cla thrin/dynamin-dependent pathway. As a member of GPCR family, the endocytosis of endogenous EP4 in cell lines was shown to be initiated by b-arrestins (Penn et al., 2001). It is very likely that arrestin/clathrin/dyna min-dependent pathway also mediates agoniststimulated EP4 internalization in cultured DRG neurons.

65

Fig. 8. Pre-exposure to EP4 agonist induced a greater increase in CGRP release from cultured DRG neurons evoked by subsequent EP4 agonist challenge, an event suppressed by the recycling inhibitor. Before seeding, dissociated cells in suspension were treated with vehicle, 1-OH-PGE1 (PG, 10 lM), 1-OH-PGE1 + monensin (M, 40 lM) or monensin for 1 h. Two days after seeding, cultured cells were treated with vehicle, 1-OH-PGE1 (10 and 50 lM), 1-OH-PGE1 (10 and 50 lM) + monensin or monensin (M, 40 lM) for 15 min. CGRP levels in culture medium were analyzed by using ELISA. Treatments of PG (10 or 50 lM) ? vehicle did not increase CGRP release following subsequent vehicle treatment. Single or double exposures of 1-OH-PGE1 significantly increased CGRP release compared to vehicle exposures (*p < 0.05–0.01). After double exposures of 1-OH-PGE1 (10 lM) ? 1-OH-PGE1 (10 or 50 lM), CGRP levels in culture medium were significantly higher than single exposure of vehicle ? 1-OH-PGE1 (50 lM) (#p < 0.05). Moreover, following the double exposures of 1-OH-PGE1 (10 lM) + M (40 lM) ? 1-OH-PGE1 (10 or 50 lM), CGRP levels were significantly reduced compared to double exposures of 1-OH-PGE1 (10 lM) ? 1-OH-PGE1 (10 or 50 lM) (+p < 0.05–0.01). CGRP levels following the double exposures of 1-OH-PGE1 (10 lM) + M (40 lM) ? 1-OH-PGE1 (10 or 50 lM) still remained significantly higher than the double exposures to vehicle ? vehicle. Following treatments of 1-OH-PGE1 (10 lM) ? 1-OH-PGE1 (10 or 50 lM) + M (40 lM), CGRP levels were not significantly altered compared to double exposure of 1-OH-PGE1, but still remained significantly higher than double exposures to vehicle (*p < 0.01). Treatments of M (40 lM) ? Veh or Veh ? M (40 lM) did not alter the levels of released CGRP compared to Veh ? Veh treatments. Mean ± SEM, one-way ANOVA with post hoc Student–Newman–Keuls multiple comparison test, n = 3–5 sessions per group.

We also observed that internalized EP4 receptors were predominantly localized in the early endosomes and recycling endosomes, but rarely in the late endosomes and lysosomes. These findings suggest that internalized EP4 more likely undergoes the recycling pathway to return to the cell surface than the degradation pathway to be metabolized. Hence desensitized EP4 receptors could be re-sensitized rapidly. To substantiate these findings obtained by antibody feeding-based internalization assay, we also used an alternative approach, FITC-PGE2 receptor binding assay. Fluorescent dye-conjugated agonist has widely been used in numerous in vitro studies to trace the internalization of cell surface GPCR. For example, fluorescent dye conjugated agonist of delta opioid receptor Fluo-deltorphin was used to trace the

66

B. St-Jacques, W. Ma / Neuroscience 326 (2016) 56–68

internalization of delta opioid receptor in cultured cortical neurons (Cahill et al., 2001). Fluorescent dye conjugated NPY Y1 receptor agonist Bodipy-[Leu31, Pro34]-NPY was used to trace the internalization of Y1 receptor in transfected HEK 293 cells (Pheng et al., 2003). In this study, we found that FITC-PGE2 time-dependently increased the intracellular levels of internalized FITC-PGE2/EP complex compared to FITC treatment. Moreover, the levels of internalized FITC-PGE2/EP complex were dramatically attenuated by the selective EP4 agonist CAY10850, suggesting that the majority of the internalized EP receptors stimulated by FITC-PGE2 are EP4 subtype. Similar to the internalized EP4 revealed by antibody feeding-based internalization assay, FITC-PGE2/EP complex was also more frequently co-localized with the early endosome marker EAA1 than with the lysosome marker Lamp1. These findings further support the assumption that following agonist stimulation, the internalized EP4 prefers a recycling pathway to return to the cell surface to a degradation pathway to be metabolized. It is highly possible that a fast re-sensitization occurs following agonist-stimulated EP4 internalization. However, the time course for internalized EP4 to be metabolized still remains unknown, an issue worthy of further exploration in future studies. One hour after EP4 agonist stimulation, we failed to see a significant dominance of internalized EP4 in lysosomes, indicating that degradation of internalized EP4 has been delayed. Indeed, degradation time for some GPCRs can range from 1 to 20 h (Marchese et al., 2008). EP4 has been known to play a central role in PGE2 involvement in chronic pain conditions such as inflammatory (Lin et al., 2006) and neuropathic pain (Ma et al., 2012; Ma and Quirion, 2014). EP4 antagonists exhibited promising therapeutic effects because of higher efficiency and fewer serious side effects than non-steroid anti-inflammatory drugs or COX2 inhibitors in inflammatory pain models (Clark et al., 2008; Maubach et al., 2009; Colucci et al., 2010). Since our prior and current studies showed that agonist-facilitated EP4 externalization and recycling contribute to nociceptor sensitization, blocking EP4 cell surface trafficking and promoting its internalization and degradation could open a novel therapeutic avenue to treat inflammatory and neuropathic pain conditions. Interestingly enough, PGE2 was shown to stimulate the production of beta amyloid (Ab) peptide through EP4 internalization and degradation in CHO-K1 cells (Hoshino et al., 2009). We have recently reported that i. pl. injection of complete Freud’s adjuvant reduced the levels of amyloid precursor protein (APP) and Ab peptides in L4-6 DRG neurons (Shukla et al., 2013). In young and aged CRND8 mice which over-expressed APP and Ab peptides in neurons, pain sensitivity as well as the levels of pain mediators such as SP, CGRP and transient receptor potential vanilloid-1 (TRPV1) in DRG neurons were reduced (Shukla et al., 2013), suggesting that Ab peptides suppress pain by reducing pain mediators. Therefore, facilitating EP4 internalization and degradation might not only reduce EP4 cell surface levels to desensitize EP4, but also increase the production of Ab peptides to suppress the expression of pain mediators.

Agonist-stimulated recycling of internalized EP4 contributes to enhanced EP4 sensitivity We previously reported that PGE2 or EP4 agonist time- and concentration-dependently increased EP4 externalization in cultured DRG neurons (St-Jacques and Ma, 2013). This event was not only suppressed by the inhibitor of anterograde transport from ER/Golgi complex to the cell surface or the inhibitor of protein synthesis, but also by the recycling inhibitor monensin, suggesting that agonist-stimulated EP4 recycling also contributes to the enhanced cell surface EP4 levels. In the present study, our findings further support this hypothesis that EP4 agonist 1-OH-PGE1 or CAY10580 time- and concentration-dependently increased EP4 recycling in cultured DRG neurons. This finding strongly suggests that following agonist stimulation, EP4 recycling also contributes to enhanced EP4 surface abundance in cultured DRG neurons. It is noticeable that only in stimulation that lasted for 60 min could 10 and 50 lM 1-OH-PGE1 or CAY10580-induced EP4 recycling be significant. This observation is in line with our prior observation that only following a 60-min stimulation, did PGE2 or EP4 agonist increase EP4 cell surface density (St-Jacques and Ma, 2013). We also noticed that although 60 min of agonist stimulation was required to trigger significant EP4 recycling, it only took 30 min for internalized EP4 to be completely recycled back to the cell surface after agonist stimulation is discontinued. These data suggest that a fast rate of EP4 recycling exists after EP4 internalization. In our prior (St-Jacques and Ma, 2013) and current studies to explore the externalization, internalization and recycling of EP4 in cultured DRG neurons, a long-term agonist stimulation (60 min) was used to mimic a more authentic inflammatory environment. Even following a 60-min stimulation, the dominant trafficking events of EP4 are not internalization and degradation, but externalization and recycling. This situation is rather different from some ionotropic pain-related receptors such as the P2X3 receptor which undergoes desensitization mediated by a fast internalization and a slow recycling following agonist stimulation (Giniatullin and Nistri, 2013). Our data suggest that PGE2 or EP4 agonist induces a predominant and progressive sensitizing effect on nociceptor. It is also notable that the effective concentrations of 1-OH-PGE1 or CAY10850 in the present study to induce significant EP4 internalization and recycling were within the range of lM, mimicking an inflammatory milieu in which PGE2 is over-produced by inflammatory cells. Agonist-increased EP4 cell surface levels through externalization and recycling might enhance EP4 receptor sensitivity and activity. PGE2 is known to stimulate the release of pain-related peptide SP and CGRP from nociceptors (Hingtgen et al., 1995; Vasko, 1995) through EP4 activation (Southall and Vasko, 2001). We thus used the release of CGRP as an indicator for EP4 activity in the present study. We observed that pre-exposures to EP4 agonist 1-OH-PGE1 significantly increased CGRP release from cultured DRG neurons evoked by the 2nd EP4 agonist compared to preexposure to vehicle. This event was blocked by the recycling inhibitor monensin pre-treated with the 1st

B. St-Jacques, W. Ma / Neuroscience 326 (2016) 56–68

1-OH-PGE1. These data indicate that following agonist stimulation, increased cell surface EP4 levels were not only due to externalization, but also due to preferred recycling of EP4 after internalization, thus augmenting EP4 sensitivity and activity. Therefore, facilitating EP4 externalization and recycling is a novel mechanism underlying PGE2-induced nociceptor sensitization. This mechanism may contribute to the development of pathological state. Pre-exposure to 1-OH-PGE1 did not augment CGRP release evoked by subsequent vehicle treatment, suggesting that the 1st EP4 agonist-induced CGRP release is transient. We also observed that on co-treatment of cultured DRG neurons with the 2nd 1-OH-PGE1 and monensin for 15 min, CGRP levels were not significantly altered, indicating the 2nd EP4 agonist does not induce sufficient EP4 recycling during such a short period of time. We also noticed that monensin itself did not change CGRP release, suggesting that it only affects the agonist-stimulated EP4 recycling, but not the constitutive EP4 recycling.

CONCLUDING REMARKS In the present study, we demonstrated that EP4 agonist 1-OH-PGE1 or CAY10580, in a time- and concentrationdependent manner, increased EP4 internalization in cultured DRG neurons. Internalized EP4 was predominantly expressed in both early endosomes and recycling endosomes, but rarely expressed in late endosomes and lysosomes. 1-OH-PGE2 or CAY10580, in a time- and concentration-dependent manner, increased EP4 recycling to enhance EP4 cell surface abundance. Pre-exposure to EP4 agonist enhanced CGRP release from cultured DRG neurons evoked by subsequent challenge of the EP4 agonist, an event attenuated by the recycling inhibitor. We conclude that EP4 agonist stimulation increased internalization and recycling, and internalized EP4 prefers the recycling pathway to return to the cell surface. Agonist-stimulated EP4 recycling contributes to enhanced EP4 activity and nociceptor sensitization. Acknowledgments—The current study was supported by the Discovery grant from Natural Science and Technology Research Council of Canada (NSERC, RFN.356021) and by Louise and Alan Edwards Foundation Grant in Pain Research (RFN.68772) to Weiya Ma. This study has no conflicts of interest with any third party.

REFERENCES Arancibia-Ca´rcamo L, Fairfax BP, Moss SJ, Kittler JT (2006) Studying the localization, surface stability and endocytosis of neurotransmitter receptors by antibody labeling and biotinylation approaches. In: Kittler JT, Moss SJ, editors. The dynamic synapse: molecular methods in ionotropic receptor biology. Boca Raton, Florida, USA: CRC Press. Cahill CM, Morinville A, Lee MC, Vincent JP, Collier B, Beaudet A (2001) Prolonged morphine treatment targets delta opioid receptors to neuronal plasma membranes and enhances deltamediated antinociception. J Neurosci 21:7598–7607.

67

Clark P, Rowland SE, Denis D, Mathieu MC, Stocco R, Poirier H, Burch J, Han Y, Audoly L, Therien AG, Xu D (2008) MF498 [N-{[4-(5,9-Diethoxy-6-oxo-6,8-dihydro-7H-pyrrolo[3,4-g]quinolin-7-yl)3-methylbenzyl]sulfonyl}-2-(2-methoxyphenyl)acetamide], a selective E prostanoid receptor 4 antagonist, relieves joint inflammation and pain in rodent models of rheumatoid and osteoarthritis. J Pharmacol Exp Ther 325:425–434. Colucci J, Boyd M, Berthelette C, Chiasson JF, Wang Z, Ducharme Y, Friesen R, Wrona M, Levesque JF, Denis D, Mathieu MC, Stocco R, Therien AG, Clarke P, Rowland S, Xu D, Han Y (2010) Discovery of 4-[1-[([1-[4-(trifluoromethyl)benzyl]-1H-indol-7yl]carbonyl)amino]cyclopropyl]benzoic acid (MF-766), a highly potent and selective EP4 antagonist for treating inflammatory pain. Bioorg Med Chem Lett 20:3760–3763. Coskun T, O’Farrell LS, Syed SK, Briere DA, Beavers LS, Dubois SL, Michael MD, Franciskovich JB, Barrett DG, Efanov AM (2013) Activation of prostaglandin E receptor 4 triggers secretion of gut hormone peptides GLP-1, GLP-2, and PYY. Endocrinology 154:45–53. Cruz Duarte P, St-Jacques B, Ma W (2012) Prostaglandin E2 contributes to the synthesis of brain-derived neurotrophic factor in primary sensory neuron in cultures and in a neuropathic pain model. Exp Neurol 234:466–481. Futaki N, Arai I, Hamasaka Y, Takahashi S, Higuchi S, Otomo S (1993) Selective inhibition of NS-398 on prostanoid production in inflamed tissue in rat carrageenan-air-pouch inflammation. J Pharm Pharmacol 45:753–755. Giniatullin R, Nistri A (2013) Desensitization properties of P2X3 receptors shaping pain signaling. Front Cell Neurosci 7:245. Gold MS, Reichling DB, Shuster MJ, Levine JD (1996a) Hyperalgesic agents increase a tetrodotoxin-resistant na+ current in nociceptors. Proc Natl Acad Sci U S A 93:1108–1112. Gold MS, Shuster MJ, Levine JD (1996b) Role of a Ca(2+)dependent slow after hyperpolarization in prostaglandin E2induced sensitization of cultured rat sensory neurons. Neurosci Lett 205:161–164. Hingtgen CM, Waite KJ, Vasko MR (1995) Prostaglandins facilitate peptide release from rat sensory neurons by activating the adenosine 30 ,50 -cyclic monophosphate transduction cascade. J Neurosci 15:5411–5419. Hoshino T, Namba T, Takehara M, Nakaya T, Sugimoto Y, Araki W, Narumiya S, Suzuki T, Mizushima T (2009) Prostaglandin E2 stimulates the production of amyloid-beta peptides through internalization of the EP4 receptor. J Biol Chem 284:18493–18502. Kiriyama M, Ushikubi F, Kobayashi T, Hirata M, Sugimoto Y, Narumiya S (1997) Ligand binding specificities of the eight types and subtypes of the mouse prostanoid receptors expressed in Chinese hamster ovary cells. Br J Pharmacol 122:217–224. Lin CR, Amaya F, Barrett L, Wang H, Takada J, Samad TA, Woolf CJ (2006) Prostaglandin E2 receptor EP4 contributes to inflammatory pain hypersensitivity. J Pharmacol Exp Ther 319:1096–1103. Ma W (2010) Chronic prostaglandin E2 treatment induces the synthesis of the pain-related peptide substance P and calcitonin gene-related peptide in cultured sensory ganglion explants. J Neurochem 115:363–372. Ma W, Eisenach JC (2003) Intraplantar injection of the cyclooxygenase inhibitor ketorolac reduces immunoreactivities of substance P, calcitonin gene-related peptide, and dynorphin in the dorsal horn of rats with nerve injury or inflammation. Neuroscience 121:681–690. Ma W, Quirion R (2014) Role of COX2/PGE2/EP receptor signaling in neuropathic pain. In: Costa A, Villalba E, editors. Horizons in neuroscience research. New York: Nova Science Publisher Inc. p. 1–36. Ma W, Chabot JG, Vercauteren F, Quirion R (2010) Injured nervederived COX2/PGE2 contributes to the maintenance of neuropathic pain in aged rats. Neurobiol Aging 31:1227–1237.

68

B. St-Jacques, W. Ma / Neuroscience 326 (2016) 56–68

Ma W, St-Jacques B, Duarte PC (2012) Targeting pain mediators induced by injured nerve-derived COX2 and PGE2 to treat neuropathic pain. Expert Opin Ther Targets 16:527–540. Marchese A, Paing MM, Temple BR, Trejo J (2008) G proteincoupled receptor sorting to endosomes and lysosomes. Annu Rev Pharmacol Toxicol 48:601–629. Maubach KA, Davis RJ, Clark DE, Fenton G, Lockey PM, Clark KL, Oxford AW, Hagan RM, Routledge C, Coleman RA (2009) BGC20-1531, a novel, potent and selective prostanoid EP receptor antagonist: a putative new treatment for migraine headache. Br J Pharmacol 156:316–327. Moriyama T, Higashi T, Togashi K, Iida T, Segi E, Sugimoto Y, Tominaga T, Narumiya S, Tominaga M (2005) Sensitization of TRPV1 by EP1 and IP reveals peripheral nociceptive mechanism of prostaglandins. Mol Pain 1:3–16. Oida H, Namba T, Sugimoto Y, Ushikubi F, Ohishi H, Ichikawa A, Narumiya S (1995) In situ hybridization studies of prostacyclin receptor mRNA expression in various mouse organs. Br J Pharmacol 116:2828–2837. Olesen ET, Rutzler MR, Moeller HB, Praetorius HA, Fenton RA (2011) Vasopressin-independent targeting of aquaporin-2 by selective E-prostanoid receptor agonists alleviates nephrogenic diabetes insipidus. Proc Natl Acad Sci U S A 108:12949–12954. Penn RB, Pascual RM, Kim YM, Mundell SJ, Krymskaya VP, Panettieri Jr RA, Benovic JL (2001) Arrestin specificity for G protein-coupled receptors in human airway smooth muscle. J Biol Chem 276:32648–32656. Pheng LH, Dumont Y, Fournier A, Chabot JG, Beaudet A, Quirion R (2003) Agonistand antagonist-induced sequestration/ internalization of neuropeptide Y Y1 receptors in HEK293 cells. Br J Pharmacol 139:695–704. Rush AM, Waxman SG (2004) PGE2 increases the tetrodotoxinresistant Nav1.9 sodium current in mouse DRG neurons via Gproteins. Brain Res 1023:264–271. Shukla M, Quirion R, Ma W (2013) Reduced expression of pain mediators and pain sensitivity in amyloid precursor protein overexpressing CRND8 transgenic mice. Neuroscience 250:92–101.

Sidhapuriwala J, Li L, Sparatore A, Bhatia M, Moore PK (2007) Effect of S-diclofenac, a novel hydrogen sulfide releasing derivative, on carrageenan-induced hindpaw oedema formation in the rat. Eur J Pharmacol 569:149–154. Southall MD, Vasko MR (2001) Prostaglandin receptor subtypes, EP3C and EP4, mediate the prostaglandin E2-induced cAMP production and sensitization of sensory neurons. J Biol Chem 276:16083–16091. St-Jacques B, Ma W (2011) Role of prostaglandin E2 in the synthesis of the pro-inflammatory cytokine interleukin-6 in primary sensory neurons: an in vivo and in vitro study. J Neurochem 118:841–854. St-Jacques B, Ma W (2013) Prostaglandin E2/EP4 signalling facilitates EP4 receptor externalization in primary sensory neurons in vitro and in vivo. Pain 154:313–323. Vanegas H, Schaible HG (2001) Prostaglandins and cycloxygenases in the spinal cord. Prog Neurobiol 64:327–363. Vasko MR (1995) Prostaglandin-induced neuropeptide release from spinal cord. Prog Brain Res 104:367–380. Vasko MR, Campbell WB, Waite KJ (1994) Prostaglandin E2 enhances bradykinin-stimulated release of neuropeptides from rat sensory neurons in culture. J Neurosci 14:4987–4997. Villarreal CF, Sachs D, Cunha FQ, Parada CA, Ferreira SH (2005) The role of Na(V)1.8 sodium channel in the maintenance of chronic inflammatory hypernociception. Neurosci Lett 386:72–77. Villarreal CF, Funez MI, Cunha FQ, Parada CA, Ferreira SH (2013) The long-lasting sensitization of primary afferent nociceptors induced by inflammation involves prostanoid and dopaminergic systems in mice. Pharmacol Biochem Behav 103:678–683. Wang C, Li GW, Huang LY (2007) Prostaglandin E2 potentiation of P2X3 receptor mediated currents in dorsal root ganglion neurons. Mol Pain 3:22. Zhang X, Li L, McNaughton PA (2008) Proinflammatory mediators modulate the heat-activated ion channel TRPV1 via the scaffolding protein AKAP79/150. Neuron 59:450–461.

(Accepted 2 April 2016) (Available online 7 April 2016)