Research ArticlePain

Decreased abundance of TRESK two-pore domain potassium channels in sensory neurons underlies the pain associated with bone metastasis

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Science Signaling  16 Oct 2018:
Vol. 11, Issue 552, eaao5150
DOI: 10.1126/scisignal.aao5150
  • Fig. 1 Reduction of functional TRESK channels in DRG neurons and its association with pain in bone lesion–bearing rats.

    (A and B) TRESK immunofluorescence staining in ipsilateral L4/5 DRG neurons from naïve, phosphate-buffered saline (PBS)–injected, or bone-localized MRMT-1 tumor-bearing rats. Representative images (A) and a summary for the mean fluorescence intensity of TRESK immunostaining (B) are shown. n = 224 to 230 cells (from six rats) per time point per group. Two-way analysis of variance (ANOVA) followed by Bonferroni post hoc test: F3,1818 = 116.2, ***P < 0.001. (C and D) TRESK protein (C) and mRNA (D) abundance in the cells described in (A). n = 6 to 7 rats per time point per group. Two-way ANOVA followed by either Bonferroni post hoc test, F3,40 = 4.21 (C), or one-way ANOVA followed by Tukey post hoc test, F2,16 = 20.54 (D), ***P < 0.001. N, naïve; P, PBS; M, MRMT-1. (E and F) Representative TRESK current traces and a summary for the current density of both the total background currents (IKBG) measured at +60 mV of a ramp voltage protocol (E) and the TRESK-containing IKSO measured at −25 mV of a depolarizing step voltage protocol (F) in isolection B4–positive (IB4+) ipsilateral L4/5 DRG neurons from naïve, PBS, or tumor-bearing rats. n = 25 to 34 cells (8 to 10 rats) per group. One-way ANOVA followed by Tukey post hoc test: F2,79 =11.61 for IKBG and F2,83 = 12.18 for IKSO; ***P < 0.001, ###P < 0.001 versus the corresponding naïve and PBS group, respectively. (G and H) The fraction of lamotrigine-sensitive currents in the cells described in (E) and (F). Representative current traces and a summary are shown. Currents were recorded in the presence of lamotrigine (100 μM) by the ramp voltage protocol (G) and the depolarizing step voltage protocol (H) as described in (E) and (F). n = 14 to 17 cells (from 8 to 10 rats) per group. One-way ANOVA followed by Tukey post hoc test: F2,44 = 7.27 for IKBG; F2,44 =7.12 for IKSO; **P < 0.01, ##P < 0.01 versus the corresponding naïve and PBS group, respectively. (I to L) Correlation analysis between TRESK protein abundance and spontaneous pain behaviors in bone cancer rats. Spontaneous flinching (I) and guarding (K) behaviors were video recorded to assess the spontaneous pain in bone lesion–bearing rats (n = 11 to 12 rats per group). Two-way ANOVA followed by Bonferroni post hoc test: F6,128 = 14.52 for flinching; F6,124 = 17.94 for guarding; ***P < 0.001. (J) Correlation between flinching and TRESK protein abundance (r44 = −0.54, P = 0.0001). (L) Correlation between guarding and TRESK protein abundance (r45 = −0.48, P = 0.0007). The data of TRESK protein abundance in (J) and (L) are derived from the Western blot results shown (C).

  • Fig. 2 Effects of TRESK overexpression on the tumor-induced reduction of functional TRESK channels, DRG neuron hyperexcitability, and pain hypersensitivity in bone cancer–bearing rats.

    (A) TRESK protein abundance in ipsilateral L4/5 DRGs of MRMT-1 tumor-bearing rats after intrathecally administered LV-TRESK. n = 6 rats per group. Two-tailed unpaired t test: t10 = 4.57, ***P < 0.001. (B and C) TRESK-containing currents in ipsilateral L4/5 DRG neurons in bone cancer–bearing rats after LV-TRESK application. Shown are representative current traces and a summary for the current density of both the total background currents (IKBG) measured at +60 mV of a ramp voltage protocol (B) and the TRESK-containing IKSO measured at −25 mV of a depolarizing step voltage protocol (C) in IB4+ DRG neurons. n = 35 to 38 cells from 12 to 14 rats per group. Two-tailed unpaired t test: t71 = 8.92 for IKBG, t71 = 8.98 for IKSO, ***P < 0.001. (D to G) Effects of intrathecal (i.t.) LV-TRESK on the tumor-induced neuronal hyperexcitability in ipsilateral L4/5 DRG neurons of bone cancer–bearing rats. Representative traces of APs (D) and a summary for the spike number (E), the resting membrane potential (RMP) (F), and the rheobase for eliciting AP (G) in IB4+ DRG neurons are shown. n = 19 to 26 cells from six to eight rats per group. One-way ANOVA followed by Tukey post hoc test: F3,79 = 6.27 for the AP numbers; F3,86 = 20.97 for the RMP; F3,88 = 20.31 for the rheobase; *P < 0.05, **P < 0.01, ***P < 0.001. (H and I) Effects of intrathecal LV-TRESK on the tumor-induced decrease in the PWT to mechanical stimuli (H) and the paw withdrawal latency (PWL) to thermal stimulation (I) in bone lesion–bearing rats. n = 8 to 12 rats per group. Two-way ANOVA followed by Bonferroni post hoc test: F25,335 = 5.65 for PWT; F25,339 = 2.07 for PWL; ***P < 0.001, #P < 0.05, ###P < 0.001 versus the corresponding PBS and LV-ZsGreen group, respectively. (J and K) Effects of intrathecal LV-TRESK on the tumor-induced spontaneous pain in bone metastasis model rats. Flinching (J) and guarding (K) behaviors were video recorded to assess the spontaneous pain. n = 9 to 12 rats per group. Two-way ANOVA followed by Bonferroni post hoc test: F3,76 = 2.43 for flinching; F3,80 = 2.75 for guarding; *P < 0.05, **P < 0.01 versus the corresponding LV-ZsGreen group.

  • Fig. 3 Effects of TRESK knockdown on the abundance of functional TRESK channels, DRG neuron excitability, and pain sensitivity in normal rats.

    (A and B) TRESK abundance at the mRNA (A) and protein (B) levels in bilateral L4/5 DRGs after intrathecally administered TRESK siRNA. n = 5 to 7 rats per group. Two-tailed unpaired t test: t12 = 3.12 for TRESK mRNA, t8 = 4.55 for TRESK protein, **P < 0.01 versus the corresponding scramble group. (C and D) TRESK currents in bilateral L4/5 DRG neurons after TRESK siRNA application. Shown are representative current traces and a summary for the current density of both the total background currents (IKBG) measured at +60 mV of a ramp voltage protocol (C) and the TRESK-containing IKSO measured at −25 mV of a depolarizing step voltage protocol (D) in IB4+ DRG neurons. n = 16 to 18 cells from six to seven rats per group. Two-tailed unpaired t test: t33 = 2.84 for IKBG, t32 = 2.81 for IKSO, **P < 0.01. (E to G) Effects of intrathecal injection of TRESK siRNA on the neuronal excitability of bilateral L4/5 DRG neurons. Representative traces of APs and a summary for the spike number (E), the RMP (F), and the rheobase for eliciting AP (G) in IB4+ DRG neurons are shown. n = 15 to 21 cells from six to seven rats per group. Two-tailed unpaired t test: t36 = 3.43 for the AP numbers; t34 = 3.58 for the RMP; t30 = 4.49 for the rheobase; **P < 0.01, ***P < 0.001. (H and I) Effects of intrathecal TRESK siRNA on the PWT to mechanical stimuli (H) and the PWL to thermal stimulation (I) in normal rats. n = 7 to 11 rats per group. Two-way ANOVA followed by Bonferroni post hoc test: F6,112 = 4.81 for PWT; F6,119 = 1.48 for PWL; *P < 0.05, **P < 0.01, ***P < 0.001 versus the corresponding scramble group. (J and K) Effects of intrathecal TRESK siRNA on the spontaneous pain behaviors in normal rats. Flinching (J) and guarding (K) behaviors were video recorded to assess the spontaneous pain. n = 9 to 13 rats per group. Two-way ANOVA followed by Bonferroni post hoc test: F6,147 = 1.88 for flinching; F6,119 = 2.51 for guarding; **P < 0.01, ***P < 0.001 versus the corresponding scramble group.

  • Fig. 4 Effects of intrathecal injection of calcineurin on the tumor-induced reduction of functional TRESK channels, DRG neuron hyperexcitability, and pain hypersensitivity in bone cancer–bearing rats.

    (A) Calcineurin protein abundance in ipsilateral L4/5 DRGs in naïve, PBS, and MRMT-1 tumor-bearing rats. n = 6 rats per group. One-way ANOVA followed by Tukey post hoc test: F2,15 = 20.65; **P < 0.01, ***P < 0.001. (B to D) Effects of intrathecal injection of calcineurin on the tumor-induced decrease in the abundance of calcineurin (B), TRESK protein (C), and TRESK mRNA (D) in ipsilateral L4/5 DRGs of bone cancer rats. n = 4 to 8 biological replicates per group. Two-tailed unpaired t test: t8 = 3.36 for calcineurin; t6 = 6.23 and t14 = 2.26 for TRESK protein and mRNA, respectively; *P < 0.05, ***P < 0.001. (E and F) Effects of intrathecal calcineurin on the tumor-induced reduction of TRESK-containing currents in ipsilateral L4/5 DRG neurons of bone cancer–bearing rats. Shown are representative current traces and a summary for the current density of both the total background currents (IKBG) measured at +60 mV of a ramp voltage protocol (E) and the TRESK-containing IKSO measured at −25 mV of a depolarizing step voltage protocol (F) in IB4+ DRG neurons. n = 26 to 27 cells from eight to nine rats per group. Two-tailed unpaired t test: t51 = 6.24 for IKBG, t51 = 5.51 for IKSO; ***P < 0.001. (G to I) Effects of intrathecal calcineurin on the tumor-induced neuronal hyperexcitability in ipsilateral L4/5 DRG neurons of bone cancer–bearing rats. Representative traces of APs and a summary for the spike number (G), the RMP (H), and the rheobase for eliciting AP (I) in IB4+ DRG neurons are shown. n = 26 to 27 cells from eight to nine rats per group. Two-tailed unpaired t test: t51 = 5.38 for the AP numbers; t51 = 6.66 for the RMP; t51 = 5.46 for the rheobase; ***P < 0.001. (J and K) Effects of intrathecal calcineurin on the tumor-induced decrease in the PWT to mechanical stimuli (J) and the PWL to thermal stimulation (K) in bone cancer rats. n = 13 to 14 rats per group. Two-way ANOVA followed by Bonferroni post hoc test: F7,160 = 6.20 for PWT; F7,140 = 2.29 for PWL; *P < 0.05, **P < 0.01, ***P < 0.001 versus the corresponding vehicle group.

  • Fig. 5 Effects of pretreatment with NFAT inhibitor peptide on the calcineurin-induced alterations of TRESK abundance, DRG excitability, and pain sensitivity in bone cancer–bearing rats.

    (A and B) Effects of pretreatment with the NFAT inhibitor peptide VIVIT on the calcineurin (CaN)–induced increase in TRESK mRNA (A) and TRESK protein (B) abundance in ipsilateral L4/5 DRGs of bone cancer–bearing rats. n = 6 to 8 rats per group. Two-tailed unpaired t test: t14 = 4.49 for TRESK mRNA; t10 = 3.32 for TRESK protein; **P < 0.01, ***P < 0.001. (C and D) Effects of pretreatment with VIVIT on the calcineurin-induced augmentation of TRESK-containing currents in ipsilateral L4/5 DRG neurons of bone cancer–bearing rats. Shown are representative current traces and a summary for the current density of both the total background currents (IKBG) measured at +60 mV of a ramp voltage protocol (C) and the TRESK-containing IKSO measured at −25 mV of a depolarizing step voltage protocol (D) in IB4+ DRG neurons. n = 15 to 16 cells from four to five rats per group. Two-tailed unpaired t test: t30 = 3.26 for IKBG; t28 = 5.08 for IKSO; **P < 0.01, ***P < 0.001. (E to G) Effects of pretreatment with VIVIT on the calcineurin-induced reduction of DRG neuron excitability in ipsilateral L4/5 DRG neurons in tumor-bearing rats. Representative traces of APs and a summary for the spike number (E), the RMP (F), and the rheobase for eliciting AP (G) in IB4+ DRG neurons are shown. Two-tailed unpaired t test: t26 = 2.59 for the AP numbers; t32 = 4.38 for the RMP; t30 = 5.46 for the rheobase; *P < 0.05, ***P < 0.001. (H and I) Effects of pretreatment with VIVIT on the calcineurin-induced increase in the PWT to mechanical stimuli (H) and the PWL to thermal stimulation (I) in bone cancer–bearing rats. n = 10 to 11 rats per group. Two-way ANOVA followed by Bonferroni post hoc test: F1,36 = 48.97 for PWT, F1,38 = 10.12 for PWL, ***P < 0.001.

  • Fig. 6 The abundance of VEGF and VEGFR2 in ipsilateral L4/5 DRGs in bone cancer–bearing rats, and the effect of intrathecal injection of VEGF on the functional TRESK abundance, DRG neuron excitability, and pain sensitivity in normal rats.

    (A and B) VEGF and VEGFR2 protein abundance in ipsilateral L4/5 DRGs in naïve, PBS, and MRMT-1 tumor-bearing rats. n = 6 rats per group. One-way ANOVA followed by Tukey post hoc test: F2,15 = 16.45 for VEGF; F2,15 = 13.27 for VEGFR2; **P < 0.01, ***P < 0.001. (C and D) Effects of intrathecal injection of VEGF on calcineurin and TRESK abundance in bilateral L4/5 DRGs of normal rats. n = 6 rats per group. Immunoblots shown are from the same membrane, reblotted for TRESK. Two-tailed unpaired t test: t10 = 5.90 for calcineurin; t10 = 4.05 for TRESK; **P < 0.01, ***P < 0.001. (E and F) Effects of intrathecal VEGF on TRESK-containing currents in bilateral L4/5 DRG neurons of normal rats. Shown are representative current traces and a summary for the current density of both the total background currents (IKBG) measured at +60 mV of a ramp voltage protocol (E) and the TRESK-containing IKSO measured at −25 mV of a depolarizing step voltage protocol (F) in IB4+ DRG neurons. n = 18 to 20 cells from five to six rats per group. Two-tailed unpaired t test: t37 = 5.92 for IKBG, t34 = 9.68 for IKSO, ***P < 0.001. (G to I) Effects of intrathecal VEGF on the neuronal excitability of bilateral L4/5 DRG neurons in normal rats. Representative traces of APs and a summary for the spike number (G), the RMP (H), and the rheobase for eliciting AP (I) in IB4+ DRG neurons are shown. n = 17 to 21 cells from five to six rats per group. Two-tailed unpaired t test: t33 = 4.04 for the APs; t37 = 4.00 for the RMP; t38 = 4.82 for the rheobase; *P < 0.05, ***P < 0.001. (J and K) Effects of intrathecal VEGF on the PWT to mechanical stimuli (J) and the PWL to thermal stimulation (K) in normal rats. n = 9 to 10 rats per group. Two-way ANOVA followed by Bonferroni post hoc test: F5,102 = 2.32 for PWT; F5,108 = 2.35 for PWL; *P < 0.05, **P < 0.01.

  • Fig. 7 Effects of VEGFR2 inhibitor on the abundance of calcineurin and TRESK, DRG neuron excitability, and pain hypersensitivity in rats with bone cancer.

    (A and B) Effects of intrathecal injection of Ki8751 on the tumor-induced reduction of calcineurin and TRESK protein abundance in ipsilateral L4/5 DRGs of bone cancer–bearing rats. n = 4 to 5 rats per group. Two-tailed unpaired t test: t8 = 4.58 for calcineurin, t12 = 4.06 for TRESK, **P < 0.01. (C and D) Effects of intrathecal Ki8751 on the tumor-induced decrease in TRESK-containing currents in ipsilateral L4/5 DRG neurons of bone cancer–bearing rats. Shown are representative current traces and a summary for the current density of both the total background currents (IKBG) measured at +60 mV of a ramp voltage protocol (C) and the TRESK-containing IKSO measured at −25 mV of a depolarizing step voltage protocol (D) in IB4+ DRG neurons. n = 18 to 20 cells from five to six rats per group. Two-tailed unpaired t test: t37 = 3.59 for IKBG, t36 = 3.54 for IKSO, **P < 0.01. (E to G) Effects of intrathecal Ki8751 on the tumor-induced enhancement of DRG neuron excitability in bone cancer–bearing rats. Representative traces of APs and a summary for the spike number (E), the RMP (F), and the rheobase for eliciting AP (G) in IB4+ DRG neurons are shown. n = 17 to 23 cells from five to six rats per group. Two-tailed unpaired t test: t32 = 3.04 for the AP numbers; t40 = 3.49 for the RMP; t42 = 4.60 for the rheobase; **P < 0.01, ***P < 0.001. (H and I) Effects of intrathecal Ki8751 on the tumor-induced decrease in the PWT to mechanical stimuli (H) and the PWL to thermal stimulation (I) in bone cancer–bearing rats. n = 8 to 13 rats per group. Two-way ANOVA followed by Bonferroni post hoc test: F6,131 = 2.85 for PWT; F6,140 = 1.02 for PWL; *P < 0.05, **P < 0.01, ***P < 0.001 versus the corresponding vehicle group.

  • Fig. 8 The role of VEGF/calcineurin-NFAT/TRESK signaling in the development of bone cancer–induced pain.

    In the context of bone metastasis, the binding of VEGF to its receptor VEGFR2 may activate calcineurin, triggers NFAT translocation into nucleus, and consequently enhances the expression of NFAT targets, including the genes encoding TRESK and RCAN1, an endogenous calcineurin inhibitor. Enhancement of RCAN1 subsequently creates a negative feedback loop that inhibits calcineurin activity, thereby attenuating both the nuclear import of NFAT and the transactivation of its targets, including TRESK. The loss of TRESK abundance and, hence, activity in DRG neurons may result in neuronal hyperexcitability and peripheral sensitization, indicating a channel-mediated mechanism through which VEGF/VEGFR2 signaling contributes to bone cancer–induced pain.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/552/eaao5150/DC1

    Fig. S1. Distribution and alteration of TRESK channels in ipsilateral L4/5 DRG neurons of bone lesion–bearing rats.

    Fig. S2. TRESK antibody validation in HEK293 cells.

    Fig. S3. Reduction of TRESK-containing currents in IB4 DRG neurons of bone lesion–bearing rats.

    Fig. S4. Augmented TRESK staining in DRG neurons upon intrathecal administration of lentiviral TRESK.

    Fig. S5. Effects of TRESK overexpression on the tumor-induced TRESK repression and neuronal hyperexcitability in IB4 DRG neurons from bone lesion–bearing rats.

    Fig. S6. Effects of intrathecal injection of TRESK siRNA on the expression of other K2P family members in bilateral L4/5 DRGs from normal rats.

    Fig. S7. Effects of TRESK knockdown on neuronal excitability in IB4 DRGs from normal rats.

    Fig. S8. Effects of calcineurin inhibition on the abundance of phosphorylated TRESK in DRG neurons.

    Fig. S9. Effects of interfering peptide TAT-Ser on calcineurin-induced alterations of functional TRESK abundance and neuronal excitability in cultured DRG neurons.

    Fig. S10. Effects of FK-506 treatment on functional TRESK abundance and neuronal excitability in cultured DRGs and on pain sensitivity in normal rats.

    Fig. S11. The subcellular distribution of NFAT in cultured DRG neurons upon treatment with FK-506.

    Fig. S12. Effects of TRESK knockdown on calcineurin-modulated DRG neuron excitability and pain sensitivity in bone lesion–bearing rats.

    Fig. S13. Effects of intrathecal injection of calcineurin siRNA on functional TRESK abundance, DRG neuron excitability, and pain sensitivity in normal rats.

    Fig. S14. Effects of intrathecal injection of NFAT inhibitor peptide VIVIT on functional TRESK abundance, DRG neuron excitability, and pain sensitivity in normal rats.

    Fig. S15. Effects of VEGF treatment on calcineurin and TRESK abundance, TRESK-mediated currents, and the neuronal excitability in cultured DRG neurons.

    Fig. S16. Effects of VEGF antibody on calcineurin and TRESK abundance, DRG neuron excitability, and pain hypersensitivity in bone lesion–bearing rats.

    Table S1. Antibodies.

    Table S2. PCR primer sequences.

    Table S3. siRNA nucleotide sequences.

  • This PDF file includes:

    • Fig. S1. Distribution and alteration of TRESK channels in ipsilateral L4/5 DRG neurons of bone lesion–bearing rats.
    • Fig. S2. TRESK antibody validation in HEK293 cells.
    • Fig. S3. Reduction of TRESK-containing currents in IB4 DRG neurons of bone lesion–bearing rats.
    • Fig. S4. Augmented TRESK staining in DRG neurons upon intrathecal administration of lentiviral TRESK.
    • Fig. S5. Effects of TRESK overexpression on the tumor-induced TRESK repression and neuronal hyperexcitability in IB4 DRG neurons from bone lesion–bearing rats.
    • Fig. S6. Effects of intrathecal injection of TRESK siRNA on the expression of other K2P family members in bilateral L4/5 DRGs from normal rats.
    • Fig. S7. Effects of TRESK knockdown on neuronal excitability in IB4 DRGs from normal rats.
    • Fig. S8. Effects of calcineurin inhibition on the abundance of phosphorylated TRESK in DRG neurons.
    • Fig. S9. Effects of interfering peptide TAT-Ser on calcineurin-induced alterations of functional TRESK abundance and neuronal excitability in cultured DRG neurons.
    • Fig. S10. Effects of FK-506 treatment on functional TRESK abundance and neuronal excitability in cultured DRGs and on pain sensitivity in normal rats.
    • Fig. S11. The subcellular distribution of NFAT in cultured DRG neurons upon treatment with FK-506.
    • Fig. S12. Effects of TRESK knockdown on calcineurin-modulated DRG neuron excitability and pain sensitivity in bone lesion–bearing rats.
    • Fig. S13. Effects of intrathecal injection of calcineurin siRNA on functional TRESK abundance, DRG neuron excitability, and pain sensitivity in normal rats.
    • Fig. S14. Effects of intrathecal injection of NFAT inhibitor peptide VIVIT on functional TRESK abundance, DRG neuron excitability, and pain sensitivity in normal rats.
    • Fig. S15. Effects of VEGF treatment on calcineurin and TRESK abundance, TRESK-mediated currents, and the neuronal excitability in cultured DRG neurons.
    • Fig. S16. Effects of VEGF antibody on calcineurin and TRESK abundance, DRG neuron excitability, and pain hypersensitivity in bone lesion–bearing rats.
    • Table S1. Antibodies.
    • Table S2. PCR primer sequences.
    • Table S3. siRNA nucleotide sequences.

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