Cutaneous nociception evoked by 15-delta PGJ2 via activation of ion channel TRPA1
© Cruz-Orengo et al; licensee BioMed Central Ltd. 2008
Received: 06 May 2008
Accepted: 31 July 2008
Published: 31 July 2008
A number of prostaglandins (PGs) sensitize dorsal root ganglion (DRG) neurons and contribute to inflammatory hyperalgesia by signaling through specific G protein-coupled receptors (GPCRs). One mechanism whereby PGs sensitize these neurons is through modulation of "thermoTRPs," a subset of ion channels activated by temperature belonging to the T ransient R eceptor P otential ion channel superfamily. Acrid, electrophilic chemicals including cinnamaldehyde (CA) and allyl isothiocyanate (AITC), derivatives of cinnamon and mustard oil respectively, activate thermoTRP member TRPA1 via direct modification of channel cysteine residues.
Our search for endogenous chemical activators utilizing a bioactive lipid library screen identified a cyclopentane PGD2 metabolite, 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), as a TRPA1 agonist. Similar to CA and AITC, this electrophilic molecule is known to modify cysteines of cellular target proteins. Electophysiological recordings verified that 15d-PGJ2 specifically activates TRPA1 and not TRPV1 or TRPM8 (thermoTRPs also enriched in DRG). Accordingly, we identified a population of mouse DRG neurons responsive to 15d-PGJ2 and AITC that is absent in cultures derived from TRPA1 knockout mice. The irritant molecules that activate TRPA1 evoke nociceptive responses. However, 15d-PGJ2 has not been correlated with painful sensations; rather, it is considered to mediate anti-inflammatory processes via binding to the nuclear peroxisome proliferator-activated receptor gamma (PPARγ). Our in vivo studies revealed that 15d-PGJ2 induced acute nociceptive responses when administered cutaneously. Moreover, mice deficient in the TRPA1 channel failed to exhibit such behaviors.
In conclusion, we show that 15d-PGJ2 induces acute nociception when administered cutaneously and does so via a TRPA1-specific mechanism.
The prostaglandins (PGs) are a class of biomolecules derived from arachidonic acid (AA) that are involved in a variety of signaling processes including inflammation. For example, PGE2 and PGI2 are produced during inflammation and contribute to the direct sensitization of nociceptive neurons of the dorsal root ganglia (DRG). Downstream of binding to its G protein-coupled receptor (GPCR), PGE2 sensitizes nociceptive neurons to thermal stimuli via PKA-dependent phosphorylation of the heat- and capsaicin-gated T ransient R eceptor P otential (TRP) ion channel TRPV1 . TRPV1 is the founding mammalian member of a subfamily of TRP channels gated by temperature (dubbed thermoTRPs).
TRPA1, first characterized as a thermoTRP channel gated by noxious cold (although this finding is controversial)  is activated by compounds that induce "burning" sensations and therefore could be best classified as a "chemoTRP." Irritant chemicals ligands of TRPA1 include allyl isothiocyanate (AITC), cinnamaldehyde (CA), allicin, acrolein and formalin[4–8]. TRPA1 is also tightly coupled to bradykinin signaling and is activated by agents generated by oxidative stress[4, 7, 8]. Two groups have recently shown that covalent modification of cytoplasmic N-terminal cysteine residues via the Michael addition reaction is a common mode of action of several TRPA1 agonists[9, 10].
In order to identify novel and endogenous TRPA1 activators, we performed a bioactive lipid library screen and describe here our findings that 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) a cyclopentane prostaglandin metabolite of PGD2, specifically activates this channel and not other thermoTRPs enriched in DRG. Similar to AITC and CA, 15d-PGJ2 is characterized by an electrophilic α,β-unsaturated carbonyl group capable of undergoing a Michael addition with nucleophilic groups on cysteine residues. A role of 15d-PGJ2 in peripheral nociception has not been described previously; rather this molecule is best characterized as an anti-inflammatory agent. However, we have identified a population of DRG nociceptive neurons that respond to 15d-PGJ2 and the TRPA1-specific agonist AITC. We therefore further hypothesized that 15d-PGJ2 plays an in vivo role in acute peripheral nociceptive signaling via TRPA1 activation. Accordingly, we found that 15d-PGJ2 induced acute nociceptive responses when administered cutaneously in mice. This effect is specific to TRPA1 as these nociceptive behaviors are significantly attenuated in TRPA1 knockout mice. Taken together our results demonstrate a novel TRPA1-dependent role of 15d-PGJ2 in acute pro-nociception.
Materials and methods
Bioactive Lipid Library Screen
Intracellular calcium measurements were performed using a Fluorometric Imaging Plate Reader (FLIPR). Mus musculus TRPA1 (mTRPA1)-CHO cells were seeded at 6000 cells/well into black-walled base 384-well plates and were grown for 2 days. Cells were induced for mTRPA1 expression as described and loaded with Fluo-3 according to protocol (Molecular Probes). The plates were placed into a FLIPR (Molecular Devices, UK) to monitor cell fluorescence (EX _ 488 nM; EM _ 540 nM) before and after the 201 lipids contained in the Biomol Bioactive Lipid Library were added.
HeLa cells were seeded at a density of 2 × 105 cells per 35 mm dish 24 hr prior to transfection in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum. Cells were co-transfected with plasmids containing mTRPA1, mTRPV1 or mTRPM8 and GFP (green fluorescent protein) in pcDNA3.1 using LipofectAMINE and OPTI-MEM I Reduced Serum Medium (Life Technologies). Green fluorescence from cells expressing GFP was detected with the aid of a Nikon microscope equipped with a mercury lamp light source and a GFP filter (emission wavelength, 510 nm). Cells were used 1–2 days after transfection. Only cells showing normal ellipsoidal shape were used.
Gigaseal was formed with pipettes with desired resistance (2–5 Mohms). Current was recorded with an Axopatch 200 patch-clamp amplifier, low-pass filtered at 3 KHz using an 8-pole Bessel filter (902-LPF), digitized using Digidata1322A, and stored on computer disk. Digitized data were analyzed (pClamp 9.0) to obtain channel activity (NPo; where N is the number of channels in the patch and Po is the open probability), and amplitude histograms to obtain single channel conductance. Current tracings shown in figures have been filtered at 1 KHz. For whole-cell recordings, bath solution contained 126 mM NaCl, 4 mM KCl, 2 mM EGTA, 1 mM MgCl2, 10 mM HEPES, 5 mM glucose, and pipette solution contained 130 mM CsCl, 2 mM EGTA, 1 mM MgCl2, 2 mM ATP and 100 mM GTP and 10 mM HEPES (pH 7.3). For cell-attached patches, pipette and bath solutions contained (in mM): 126 mM NaCl, 4 mM KCl, 2 mM EGTA, 1 mM MgCl2, 10 mM HEPES, and 5 mM glucose (pH 7.3). Student's t test was used to test for significance (p < 0.05). All experiments were done at room and bath temperatures of 23 ± 1°C.
DRG culture and intracellular calcium imaging
Calcium imaging experiments of DRG neurons were performed as described. Briefly, DRG neurons from all spinal levels were rapidly dissected from adult mice and cultured for 24 h before assays were performed. All assays were performed in quadruplicate. For DRGs, 100 μM15d-PGJ2 (3 min pulse), 100 μM AITC (2 min pulse) and 1 μM capsaicin (2 min) were applied with a 4-min washout in between each stimulus.
Male C57BL6/J and TRPA1 mutant strain mice (obtained from David P. Corey and backcrossed to C57BL6/J for 5 generations) of 10–12 weeks age were used. Experimenters were blind with respect to genotype. Responses were averaged and analyzed using Student's t-test.
Animals were placed in individual Plexiglas boxes on a grid platform and habituated to the testing environment for one hour. After the habituation period, each mouse was injected on the plantar surface of the right hindpaw with 10 μl of 15 nmol 15d-PGJ2 diluted in 10% DMSO/normal saline (vehicle). This concentration was based on dose-response behaviors with injections ranging from 2.5–25 nmol concentrations of 15d-PGJ2 compared to vehicle. Behavior was recorded for 10 min after intraplantar injection. Observed variables were latency of response to licking and lifting of the paw as well as time spent licking/lifting of the paw.
AITC, menthol and capsaicin were purchased from Sigma Chemical Co. PGD2, PGJ2, delta12-PGJ2 and15d-PGJ2 were purchased from Biomol. For calcium imaging and electrophysiology experiments, PGs were dissolved in DMSO at a stock concentration of 20 mM and used at the final DMSO concentration of 0.1% or less.
TRPA1 is specifically activated by 15d-PGJ2
TRPA1 is required for 15d-PGJ2 sensitivity of cultured DRG neurons
15d-PGJ2 elicits acute nociceptive behavior via a TRPA1-dependent mechanism
Results of the present study reveal a previously uncharacterized role of 15d-PGJ2 in peripheral nociception. We show that 15d-PGJ2, similar to all known TRPA1 ligands, induces pain. We further demonstrate a causal link between 15d-PGJ2-induced nociception and TRPA1 activation at the cellular and behavioral levels.
Intriguingly, 15d-PGJ2 is best characterized as a potent anti-inflammatory agent and not as a molecule that induces acute or long-term inflammatory pain. Instead, the resolution of the inflammatory state appears to correlate with increasing levels of 15d-PGJ2 within tissue fluids. Like other cyclopentane prostaglandins, 15d-PGJ2 does not function via a specific GPCR. Rather, the anti-inflammatory effects of 15d-PGJ2 are mediated by activation of the transcription factor peroxisome proliferator-activated receptor gamma (PPARγ) [18–20]. For example, 15d-PGJ2 represses the transcription of a number of pro-inflammatory factors including inducible nitric oxide synthase, cyclooxygenase-2 and tumor necrosis factor-α[21, 22]. 15d-PGJ2 also acts independent of PPARγ to alter the activity of inflammatory molecules. It is thought to directly alkylate nucleophilic cysteine residues of NF-κB, thereby inhibiting DNA binding by NF-κB . Similarly, TRPA1 is activated by the covalent binding of electrophiles to cysteines, providing a likely mechanism whereby 15d-PGJ2 activates this channel[9, 10].
The results of our initial compound library screen were borne out by follow-up electrophysiological recordings showing that 15d-PGJ2directly activates TRPA1. In contrast to 15d-PGJ2, the J series PGD2 metabolites PGJ2 and 12d-PGJ2, which also contain reactive electrophilic carbons, failed to activate TRPA1 in our studies. A recent study by Taylor-Clark et al., which examined the activation of human TRPA1 using heterologous expression in HEK cells, found that 12d-PGJ2 was able to activate this channel. Discrepancies between this study and our study could be due to concentrations tested (5 times greater concentrations were utilized by Taylor-Clark et al.), expression systems used or to subtle species differences in channel structure. The in vivo behavioral studies described here extend those of Taylor-Clark et al. by demonstrating that 15d-PGJ2 activation of TRPA1 is physiologically relevant. Lastly, during the preparation of this manuscript Andersson et al. published an article on the activation of TRPA1 by mediators of oxidative stress (including 15d-PGJ2) which confirm our findings presented here .
Consistent with a physiological role of 15d-PGJ2 in the activation of TRPA1, we identified a population of DRG neurons sensitive to 15d-PGJ2, AITC and capsaicin. TRPA1-expressing neurons occur as a subset of TRPV1-expressing neurons in the DRG; whereas TRPM8 is expressed in a separate population. These results support our electrophysiological studies showing that 15d-PGJ2 does not activate TRPV1 or TRPM8. In further support of specific activation of TRPA1, a negligible number of 15d-PGJ2/AITC responsive neurons were detected in cultures derived from TRPA1 knockout mice compared to wildtype (~2% vs. 90% respectively).
Specific agonists of TRPA1 cause acute nociceptive behaviors in vivo when administered cutaneously. Therefore, we investigated responses to intraplantar injection of 15d-PGJ2. Both C57BL/6J and TRPA1 wildtype mice responded robustly by licking of the injected paw as well as lifting of the paw from the surface of the testing apparatus. These behaviors were dramatically abolished in TRPA1 knockout mice, suggesting that 15d-PGJ2-induced acute peripheral nociception is mediated by TRPA1.
Previous studies have demonstrated a definitive role of TRPA1 in transmitting acute and inflammatory pain[4–8, 15, 16, 25]. Here we show that 15d-PGJ2, a molecule with no known membrane receptor that is implicated in anti-inflammatory pathways, specifically activates TRPA1, an ion channel expressed in the cell membrane of nociceptive neurons. We also show that similar to other TRPA1 agonists, 15d-PGJ2 induces robust, acute nociceptive behaviors in vivo. Our data also support that 15d-PGJ2-induced peripheral nociception in vivo occurs through TRPA1 signaling. Collectively, our findings elaborate on a novel function of 15d-PGJ2 in peripheral nociception and identify TRPA1 as its principal receptor in pain-sensing DRG neurons.
We thank Ardem Patapoutian, Robert Gereau, Jennifer Jones and Judith Golden for invaluable input in the preparation of this manuscript. We also thank David Corey and Kelvin Kwan for the generous contribution of TRPA1 knockout mice.
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