Overexpressed transient receptor potential vanilloid 3 ion channels in skin keratinocytes modulate pain sensitivity via prostaglandin E2

Abstract

The ability to sense changes in the environment is essential for survival because it permits responses such as withdrawal from noxious stimuli and regulation of body temperature. Keratinocytes, which occupy much of the skin epidermis, are situated at the interface between the external environment and the body's internal milieu, and have long been appreciated for their barrier function against external insults. The recent discovery of temperature-sensitive transient receptor potential vanilloid (TRPV) ion channels in keratinocytes has raised the possibility that these cells also actively participate in acute temperature and pain sensation. To address this notion, we generated and characterized transgenic mice that overexpress TRPV3 in epidermal keratinocytes under the control of the keratin 14 promoter. Compared with wild-type controls, keratinocytes overexpressing TRPV3 exhibited larger currents as well as augmented prostaglandin E(2) (PGE(2)) release in response to two TRPV3 agonists, 2-aminoethoxydiphenyl borate (2APB) and heat. Thermal selection behavior and heat-evoked withdrawal behavior of naive mice overexpressing TRPV3 were not consistently altered. Upon selective pharmacological inhibition of TRPV1 with JNJ-17203212 [corrected], however, the keratinocyte-specific TRPV3 transgenic mice showed increased escape responses to noxious heat relative to their wild-type littermates. Coadministration of the cyclooxygenase inhibitor, ibuprofen, with the TRPV1 antagonist decreased inflammatory thermal hyperalgesia in transgenic but not wild-type animals. Our results reveal a previously undescribed mechanism for keratinocyte participation in thermal pain transduction through keratinocyte TRPV3 ion channels and the intercellular messenger PGE(2).

Figure 1.

Generation of keratinocyte-specific TRPV3-overexpressing transgenic mice. A, Keratin 14 promoter driven TRPV3 transgenic constructs. B, Southern blots of EcoRV-digested genomic DNA, using the probe indicated in panel A, confirming genomic incorporation of the transgene in 3 lines of transgenic mice (HA-TRPV3 A, HA-TRPV3 B, TRPV3-YFP). T, Transgenic band; E, endogenous band. C–E, Immunoblot comparison of TRPV3 expression in the back skin of adult wild-type mice versus mice from HA-TRPV3 and TRPV3-YFP lines. Blots were probed with mouse monoclonal anti-TRPV3 (C), rat monoclonal anti-HA (D), and rabbit anti-GFP (E). Lane at far left in C shows lysate from HEK293 cells transiently transfected with recombinant TRPV3, whose position is denoted by arrowhead. F, Immunoblots probed with rabbit anti-TRPV3 and rabbit anti-HA antibodies, showing increased transgenic protein expression in cultured HA-TRPV3 B keratinocytes. G, Immunoblots showing increased transgenic protein expression in the skin but not in trigeminal ganglia of TRPV3-YFP mice using rabbit anti-TRPV3 and rabbit anti-GFP antibodies.

Figure 2.

Immunofluorescent localization of TRPV3 in skin of transgenic mice. A–D, Hindpaw glabrous skin of wild-type (A), HA-TRPV3 A (B), HA-TRPV3 B (C), or TRPV3-YFP (D) mice, respectively, probed with anti-TRPV3. Increased TRPV3 staining was observed in the skin epidermis of transgenic mice. E, F, Intrinsic YFP fluorescence in wild-type (E) and TRPV3-YFP transgenic (F) skin. Dashed line denotes dermal-epidermal junction. Arrows mark basal epidermal staining, arrowheads mark suprabasal epidermal staining. Scale bar, 50 μm.

Figure 3.

Whole-cell patch-clamp electrophysiology of primary keratinocytes from TRPV3 transgenic mice. A, Representative current responses of wild-type (top, left) and TRPV3-YFP transgenic (top, right) keratinocytes to stimulation by repeated 42°C heat pulses (bottom) recorded during voltage ramps. Upward current traces were measured at +80 mV and downward traces at −80 mV. B, Representative current traces evoked by 2APB (100 μm) in wild-type (top) and TRPV3-YFP transgenic (bottom) cells. A heat stimulus (41°C) was applied in the middle of the response to 2APB, as indicated by the horizontal bars. C, Quantification of sensitizing current responses to three consecutive heat stimuli (41–43°C) among wild-type and transgenic keratinocytes. Four bars are shown for a given stimulus. The first pair of bars show the mean ± SEM of responses recorded at +80 mV and the right pair show responses at −80 mV. Open bars represents the wild-type response and the filled bars represent the response of the indicated transgenic line. n = 3–21 cells per bar. D, Quantification of current responses evoked by 2APB (100 μm), or the simultaneous application of 2APB plus heat among wild-type (open bars) and transgenic (filled bars) keratinocytes, measured at +80 mV (upward bars) or −80 mV (downward bars). n = 4–21 cells per bar. Progressive increase in response sensitivity to TRPV3 agonists was observed in the order of wild-type < HA-TRPV3 A < HA-TRPV3 B < TRPV3-YFP.

Figure 4.

Measurement of PGE₂ release from primary keratinocytes. Supernatants from cultured keratinocytes in buffer were collected during baseline and stimulus periods (30 min each), and processed and analyzed for PGE₂ using LC-MS/MS. A, TRPV3-YFP transgenic keratinocytes show increased PGE₂ release compared with wild-type keratinocytes in response to the TRPV3 agonist 2APB (50–100 μm), but comparable responses to A23187 (1 μm). Inset, Example chromatograms showing detection of PGE₂ from buffer under basal and stimulated conditions using LC-MS/MS. B, Exposure to heat (by immersion in a water bath) increased PGE₂ release in TRPV3-YFP transgenic keratinocytes more than in wild type. C, Supra-additive increase in stimulated PGE₂ release in HA-TRPV3 line B compared with wild-type keratinocytes in response to coapplication of 2APB (100 μm) and 36°C. D, Cumulative stimulated PGE₂ release over time (buffer collected after 1, 5, 10 or 30 min incubation) in wild-type and HA-TRPV3 line B keratinocytes. E, 2APB (100 μm) plus 36°C-stimulated PGE₂ release in HA-TRPV3 line B keratinocytes is diminished in calcium-free buffer and in the presence of COX antagonists. Data are expressed as mean ± SEM, n = 3 per group. A–D were analyzed using ANOVA with Bonferroni post hoc comparisons and E was analyzed using ANOVA with Dunnett's test. **p < 0.001, *p < 0.01, difference between wild type and transgenic (A–D) or between pharmacological treatment and vehicle (DMSO) control in cells treated with 2APB plus heat (E).

Figure 5.

Temperature preference and pain behavior of wild-type and keratinocyte TRPV3 overexpressing mice. A, Temperature preference behavior of HA-TRPV3 line B mice (top) and TRPV3-YFP mice (bottom) over 60–120 min after placement in a temperature gradient ranging from 0 to 49°C. Mean presence time at each temperature is illustrated. B, Tail withdrawal behavior of HA-TRPV3 line B mice (top) and TRPV3-YFP mice (bottom) exposed to a heated water bath. C, Latency of paw withdrawal from radiant heat in wild-type and TRPV3-YFP mice before and 24 h after injection of CFA (15 μl, intraplantar) into the ipsilateral hindpaw. D, Latency of HA-TRPV3 line B (top), HA-TRPV3 line A (middle) and TRPV3-YFP (bottom) mice to paw licking, biting, or shaking behavior upon placement on a hot plate at the indicated temperatures. In each graph, data shown are from a single cohort of 9–13 mice per genotype. Two of three cohorts of HA-TRPV3 B mice, one of two cohorts of HA-TRPV3 A mice and two of two cohorts of TRPV3-YFP mice tested showed the presented response (see also supplemental Fig. S4C, available at www.jneurosci.org as supplemental material). Hot plate data were analyzed using Mann–Whitney U test. **p < 0.0001, *p < 0.05. Data are represented as mean ± SEM. Numbers of mice (n) are indicated in each panel.

Figure 6.

Changes in thermal pain behavior of TRPV3-YFP transgenic mice unmasked by pharmacological antagonism of TRPV1. A, Hot plate (52°C) latencies of wild-type and TRPV3-YFP transgenic mice before and after administration of the TRPV1 antagonist JNJ-7203212 (40 mg/kg, injected i.p. at arrow). Data were analyzed by ANOVA with Bonferroni post hoc comparisons; *p < 0.05 antagonist versus vehicle; ^#p < 0.05 wild type versus transgenic. B, Effects of cyclooxygenase antagonism. Wild-type and TRPV3-YFP transgenic mice were pretreated with the COX antagonist ibuprofen (40 mg/kg, i.p.) or vehicle 15 min before injection of JNJ-7203212 or vehicle. Beginning 30 min after the final injection, hot plate latency was measured at 30 min intervals and 6 consecutive values averaged to calculate difference from the baseline (measured during the 90 min before ibuprofen injection). C, Effects of TRPV1 and COX antagonists on CFA-induced thermal hyperalgesia in wild-type and TRPV3-YFP transgenic mice. Immediately following the measurement of post-CFA paw withdrawal latency (Fig. 5C), TRPV3-YFP transgenic and wild-type mice were injected with ibuprofen (i.p., 40 mg/kg) or vehicle, and 15 min later injected with JNJ-7203212 (40 mg/kg, i.p.) or vehicle. After an additional 30 min, latency to escape from radiant paw heating was measured at 20 min intervals in the ipsilateral paw. The three latencies obtained between 50 and 90 min postinjection were averaged to calculate the change from the post-CFA baseline measured before ibuprofen injection. Note that ibuprofen increases withdrawal latency in TRPV3-YFP, but not wild-type mice treated with the TRPV1 antagonist. Planned comparisons of data in B and C were analyzed by Student's t test. *p < 0.05; n.s., no significant difference (n = 6–12 per group). D, Change in core body temperature in response to injection of JNJ-7203212 (40 mg/kg, i.p.) in wild-type (vehicle n = 4, JNJ-7203212 n = 5) versus TRPV3-YFP (JNJ-7203212; n = 8) mice.