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. 2010 Jun 18;285(25):19362-71.
doi: 10.1074/jbc.M109.087742. Epub 2010 Apr 15.

Farnesyl Pyrophosphate Is a Novel Pain-Producing Molecule via Specific Activation of TRPV3

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Free PMC article

Farnesyl Pyrophosphate Is a Novel Pain-Producing Molecule via Specific Activation of TRPV3

Sangsu Bang et al. J Biol Chem. .
Free PMC article

Abstract

Temperature-sensitive transient receptor potential ion channels (thermoTRPs) expressed in epidermal keratinocytes and sensory afferents play an important role as peripheral pain detectors for our body. Many natural and synthetic compounds have been found to act on the thermoTRPs leading to altered nociception, but little is known about endogenous painful molecules activating TRPV3. Here, we show that farnesyl pyrophosphate (FPP), an intermediate metabolite in the mevalonate pathway, specifically activates TRPV3 among six thermoTRPs using Ca(2+) imaging and electrophysiology with cultured keratinocytes and TRPV3-overexpressing cells. Agonistic potencies of related compounds in the FPP metabolism were ignorable. Voltage-dependence of TRPV3 was shifted by FPP, which appears to be the activation mechanism. An intraplantar injection of FPP acutely elicits nociceptive behaviors in inflamed animals, indicating that FPP is a novel endogenous pain-producing substance via TRPV3 activation. Co-culture experiments demonstrated that this FPP-evoked signal in the keratinocytes is transmitted to sensory neurons. In addition, FPP reduced TRPV3 heat threshold resulting in heightened behavioral sensitivity to noxious heat. Taken together, our data suggest that FPP is the firstly identified endogenous TRPV3 activator that causes nociception. Our results may provide useful chemical information to elucidate TRPV3 physiology and novel pain-related metabolisms.

Figures

FIGURE 1.
FIGURE 1.
TRPV3 is activated by FPP. A, 100 nm FPP elevated intracellular Ca2+ levels in hTRPV3-transfected HEK293T cells (n = 75) in the Fluo-3 Ca2+ imaging experiments. A camphor response was also shown by the same cells. Responses during all Ca2+-imaging experiments are displayed as means ± S.E. B, 1 μm FPP evoked an outwardly rectifying current increase in the whole cell voltage clamp study (n = 8). The current was inhibited by co-application of the thermoTRP blocker, RR (20 μm). C, current-voltage curves obtained from the trace in B (points labeled with letters) were displayed. D, 300 nm FPP in the recording pipette evoked the immediate response of single channel current in the cell-attached patch of the hTRPV3-transfected HEK293T cells (n = 24, upper trace). In the inside-out patch, intracellular 300 nm FPP (applied to the bath) evoked delayed and smaller single channel responses (n = 11, lower trace). It took 1 min 19 s ±11 s on average for the current responses to occur in the inside-out patches (n = 11). E, summary of the mean open probabilities (NPo) of single channel TRPV3 activation at cell-attached (C/A) and inside-out (I/O) configurations. The mean values of C/A (0–30 s) (for 30 s from the patch formation), I/O (0–30 s) (for 30 s from the start of FPP application), I/O (70–100 s) (for 30 s before the end of FPP application), and camphor (during camphor application at I/O) were demonstrated. The NPo value of C/A is significantly higher than those of I/O during FPP application (NPoC/A(0–30s) = 1.84 ± 0.48, NPoI/O(70–100s) = 0.48 ± 0.16, ***, p < 0.001). Student's t test was performed.
FIGURE 2.
FIGURE 2.
FPP exerts its potency and specificity on TRPV3 activation. A, summary of the intracellular Ca2+ increases in the cells transfected with TRPV3 or other thermoTRP channels upon drug treatments in the Fluo-3 Ca2+ imaging. Untransfected cells did not show intracellular Ca2+ increases upon treatment with any test compound (data not shown). FPP elicited Ca2+ influx in the TRPV3-expressing HEK293T cells at 1 μm. IPP, GPP, GGPP, and farnesol were tested at 10 μm. IPP, GPP, GGPP, and farnesol failed to elevated intracellular Ca2+ levels in the TRPV3 cells (n = 28, n = 41 and n = 25 and n = 28, respectively). Greater than 25 cells was used for the tests of each thermoTRP activity with each of the four compounds (n = 25–98). B, dose-response curve for FPP on TRPV3 in the Fluo-3 Ca2+ imaging. The curve was fitted by Hill equation (EC50 = 131.1 nm and n = 2.4). Open circles represent mean values of responses of Ca2+ influx upon FPP application (n = 22–53 for each point). C, whole-cell currents in response to the indicated voltage step protocol applied with or without FPP application (1 μm). D, average peak inward tail currents at −120 mV from the step protocol were plotted by a function of test voltage steps (n = 7 with 1 μm FPP (diamonds), n = 8 without FPP (squares) and n = 6 with heat (36 °C; circles), respectively). Symbols represent the data collected from every 20 mV. The curves were fitted by the Boltzmann equation. E, structures of the FPP and related substances tested in the present study were displayed.
FIGURE 3.
FIGURE 3.
FPP activates TRPV3 expressed in keratinocytes. A and C, 1 μm FPP elevated intracellular Ca2+ levels in HaCaT cell lines (n = 72; A), and in normal human epidermal keratinocytes (n = 114; C) in the Fura-2 Ca2+-imaging experiments. A camphor response was also shown by the same cells. B and D, 1 μm FPP evoked an outwardly rectifying current increase in the whole cell voltage clamp study using HaCaT cell lines (n = 5; B) and using normal human epidermal keratinocytes (n = 5; D). The current responses to FPP were blocked by 20 μm RR, and those with the similar current-voltage relationship were evoked by 4 mm camphor treatment in the HaCaT cell lines (B) and normal human epidermal keratinocytes (D). Insets B–D, average current densities through each TRPV3 activation at ±60 mV. As reported earlier (7), the FPP-evoked currents were significantly inhibited by RR at −60 mV. *, p < 0.05; ***, p < 0.001; Student's t test.
FIGURE 4.
FIGURE 4.
FPP induces acute nociception under inflammation and lowered the thermal threshold of TRPV3. A, summary of the time course of licking/lifting behaviors in mice treated with FPP (1 mm in 10 μl) administered intradermally into hind paws for the 20-min period immediately following the injection (n = 5, filled circles). Carrageenan was injected to the hind paws 3 h prior to the experiments. Pretreatment with RR (1 mm in 10 μl) 5 min prior to the FPP administration prevented such behaviors (n = 5, open circles). Neither intradermal injection with vehicle without drugs (n = 5, triangles) nor the 1 mm GPP (in 10 μl) administration elicited licking/lifting behaviors (n = 6, diamonds). Mice with an FPP-injected hind paw showed no responses from their non-injected hind paw similar to the vehicle-injected controls (n = 5, data not shown). B, summary of the time course of licking/lifting behaviors in mice subplantarly pretreated with mTRPV3-shRNA (KD) for the 20-min period immediately following FPP injection (n = 6, open triangles). Carrageenan (CAR) was injected to the hind paws 3 h prior to the experiments. FPP-evoked behaviors occurred with scrambled shRNA pretreatment (n = 6, open circles). Without carrageenan pretreatment, no significant time consumption in licking/lifting behaviors evoked by FPP administration was detected (n = 6, open triangles). C, sums of the time spent in licking/lifting behaviors of carrageenan-inflamed mice for 20 min immediately after drug injection. Intradermal pretreatment with RR and mTRPV3 knockdown with shRNA significantly suppressed FPP-evoked behaviors. ANOVA followed by Bonferroni post hoc test was performed for the statistical comparison (*, p < 0.05; **, p < 0.01). D, reverse-transcriptase PCR results of TRPV3 mRNA in the mouse hind paw epidermis. Control and SC indicates no treatment and scrambled shRNA treatment, respectively. E, Western blot results from the mouse hind paw epidermis. For A–E, scrambled shRNA or TRPV3-shRNA was subplantarly injected into hind paws 48 h prior to the behavioral assays or mRNA or protein isolation.
FIGURE 5.
FIGURE 5.
FPP excites DRG neurons via keratinocyte activation. A, a representative image of the DRG neurons and keratinocyte co-cultures with staining: anti-TRPV3 antibody (green) for TRPV3 expressors and Hoechst dye for nuclei (blue). Arrows indicate DRG neurons, and arrowheads indicate keratinocytes. Scale bar, 100 μm. B, in the Fura-2 Ca2+-imaging experiments, 1 μm FPP elevated intracellular Ca2+ levels both in HaCaT keratinocytes and DRG neurons in the same dish (n = 118 for HaCaT cells and n = 48 for DRG neurons). Application of 60 mm KCl for depolarization-induced Ca2+ influx in the excitable cells functionally confirms identities of the HaCaT keratinocytes and the DRG neurons. C, summary of the onset times of FPP response of TRPV3 (***, p < 0.001). Student's t test was performed. D, 1 μm FPP failed to elevate intracellular Ca2+ levels in the keratinocytes or the DRG neurons during 20 μm RR application in the co-cultures (n = 231 for HaCaT cells and n = 7 for DRG neurons). E, 1 μm FPP failed to elevate intracellular Ca2+ levels in the keratinocytes or the DRG neurons in the co-cultures incubated with hTRPV3-shRNA (48 h) (n = 122 for HaCaT cells and n = 33 for DRG neurons). F, 1 μm FPP failed to elevate intracellular Ca2+ levels in the DRG neurons in cultures without keratinocytes (n = 37). G, reverse-transcriptase PCR of TRPV3 mRNA in HaCaT keratinocytes. H, Western blot results from HaCaT keratinocytes.
FIGURE 6.
FIGURE 6.
FPP reduces TRPV3 thermal threshold leading to heightened heat sensitivity. A, summary of the changes in the paw withdrawal latencies from Hargreaves tests with or without FPP treatment. Hargreaves latencies were decreased by 42.3 ± 9.5% for wild-type mice, by 36.2 ± 3.5% for TRPV1-null mice and by 43.2 ± 5.2% for mice treated with scrambled shRNA (SC). No significant decrease in the latencies was detected in mice pretreated with mTRPV3-shRNA. The Hargreaves latencies were examined 30 min after the hind paw intradermal administration of FPP (1 mm in 10 μl). n = 10 for each histogram (*, p < 0.05; **, p < 0.01, Student's t test). B, representative heat-induced current responses of TRPV3-transfected HEK293T cells. When temperature elevated, the TRPV3-mediated currents were elicited (top: 10 nm FPP was incubated; bottom: without FPP incubation). C, summary of the changes in the heat thresholds of TRPV3 in experiments of B. The mean heat threshold of TRPV3 from the group without FPP incubation was 31.8 ± 0.8 °C (n = 7), and the value from the group with FPP incubation was 28.8 ± 0.5 °C (n = 8). D, summary of the Q10 values of the heat-sensitive phases of TRPV3 activation in experiments of B. The mean Q10 with or without FPP incubation was 16.5 ± 5.4 and 21.9 ± 9.7, respectively. No statistical difference was found. E, summary of the peak TRPV3 current densities activated by heat, FPP, or heat plus FPP at ± 60 mV (n = 8 with heat alone, n = 6 with 10 nm FPP, n = 7 with heat plus 10 nm FPP, n = 12 with 100 nm FPP, n = 5 with heat plus 100 nm FPP, n = 7 with 1 μm FPP, and n = 17 with heat plus 1 μm FPP. Filled bars for current densities at −60 mV, and open bars at + 60mV). TRPV3-transfected HEK293T cells were used. Dotted lines indicate the average peak currents activated by heat alone. F, average peak inward tail currents at −120 mV from the step protocol as shown in Fig. 2C were plotted by a function of test voltage steps (n = 10 with 10 nm FPP (open circles), n = 8 with heat (36 °C) plus 10 nm FPP (closed circles), n = 8 with heat plus 100 nm FPP (gray triangles), n = 5 with heat plus 1 μm FPP (open triangles), and n = 9 without stimulation (gray circles)). Symbols represent the data collected from every 20 mV. The curves were fitted by Boltzmann equation. V½ was 143 mV at the resting state, 116 mV during 10 nm FPP, 30 mV during heat plus 10 nm FPP, −27 mV during heat plus 100 nm FPP, and −122 mV during heat plus 1 μm FPP applications. *, p < 0.05; **, p < 0.01, Student's t test.

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