Hedgehog signaling contributes to bone cancer pain by regulating sensory neuron excitability in rats

Abstract

Treating bone cancer pain continues to be a clinical challenge and underlying mechanisms of bone cancer pain remain elusive. Here, we reported that sonic hedgehog signaling plays a critical role in the development of bone cancer pain. Tibia bone cavity tumor cell implantation produces bone cancer-related mechanical allodynia, thermal hyperalgesia, and spontaneous and movement-evoked pain behaviors. Production and persistence of these pain behaviors are well correlated with tumor cell implantation-induced up-regulation and activation of sonic hedgehog signaling in primary sensory neurons and spinal cord. Spinal administration of sonic hedgehog signaling inhibitor cyclopamine prevents and reverses the induction and persistence of bone cancer pain without affecting normal pain sensitivity. Inhibiting sonic hedgehog signaling activation with cyclopamine, in vivo or in vitro, greatly suppresses tumor cell implantation-induced increase of intracellular Ca²⁺ and hyperexcitability of the sensory neurons and also the activation of GluN2B receptor and the subsequent Ca²⁺-dependent signals CaMKII and CREB in dorsal root ganglion and the spinal cord. These findings show a critical mechanism underlying the pathogenesis of bone cancer pain and suggest that targeting sonic hedgehog signaling may be an effective approach for treating bone cancer pain.

Keywords: Sonic hedgehog; bone cancer pain; dorsal root ganglion; excitability; neuron.

Figure 1.

Expression and activation of Shh signaling after TCI treatment. (a) Sample western blot analysis and data summary (n = 4 in each group) showing time-dependent increased expression of Shh, Ptch1, Smo, and Gli1 in dorsal root ganglion (DRG) of rats. (b) Sample western blot analysis and data summary (n = 4 in each group) showing time-dependent increased expression of Shh, Ptch1, Smo, and Gli1 in spinal cord (SC) of rats. (c) Sample western blot analysis and data summary (n = 4 in each group) showing time-dependent increased expression of Gli1 in nuclear extracts from DRG and SC. (d) ELISA assay with concentrations of Shh in ipsilateral tibia lavage fluid of different groups. (e) ELISA assay with concentrations of Shh in ipsilateral sciatic nerve after TCI treatment. (f) ELISA assay with concentrations of Shh in ipsilateral sciatic nerve after CCI treatment. (d) and (e) Samples (n = 10) were collected at postoperative day 7. *P < 0.05, **P < 0.01 vs. Sham group. DRG: dorsal root ganglion; TCI: tumor cell implantation; Shh: sonic hedgehog.

Figure 2.

Inhibition of Shh signaling pathway prevented and suppressed bone cancer-induced pain behavior. (a) and (b) Spinal administration of Shh signaling inhibitor cyclopamine (10 µg/rat) in the early stage (postoperative days 2, 3, and 4) obviously prevented TCI-induced mechanical allodynia (a) and thermal hyperalgesia (b). (c) and (d) Spinal administration of Shh signaling inhibitor cyclopamine (10 µg/rat) in the late stage (postoperative days 7, 8, and 9) obviously suppressed and reversed TCI-induced mechanical allodynia (c) and thermal hyperalgesia (d). (e) to (h) Inhibition of Shh signaling pathway suppressed TCI-induced spontaneous pain (e) and (f) and movement-evoked pain (g) and (h). Cyclopamine was administered at postoperative days 7, 8, and 9 (e) to (h). Behaviors were tested at postoperative day 10. Eight rats were included in each group. *P < 0.05, **P < 0.01 indicate significant differences compared with Sham + DMSO group. ^#P < 0.05, ^##P< 0.01 indicate significant differences compared with TCI + DMSO group. TCI: tumor cell implantation.

Figure 3.

Activation of Shh signaling contributes to activation of GluN2B, CaMKII, and CREB in DRG (a) and spinal cord (b) after TCI treatment. In vivo repetitive administration of cyclopamine (10 µg, i.t.) inhibits the phosphorylation of GluN2B, CaMKII, and CREB. Tissues were collected 4 hr after the last injection (n = 4 each group). *P < 0.05, **P < 0.01 indicate significant differences compared with Sham group. ^#P < 0.05, ^##P< 0.01 indicate significant differences compared with TCI group. DRG: dorsal root ganglion; TCI: tumor cell implantation.

Figure 4.

Activation of Shh signaling contributes to increased activity of intracellular Ca²⁺ in DRG. (a) and (b) In vitro bath of small dose of Shh-IgG (50 ng/ml) failed to increase [Ca2+]i activity in DRG neurons (n = 10) from naïve animals but significantly increased [Ca2+]i activity in TCI-DRG neurons (n= 22). Pretreatment with cyclopamine (5 µM) reduced Shh-IgG (50 ng/ml)-induced increase in [Ca²⁺]i activity in the DRG neurons (n = 25) from TCI rats. (a) Representative recordings of [Ca²⁺]i from naïve and TCI-treated, small- and medium-sized DRG neurons. (b) Data summary showing effect of cyclopamine on Shh-IgG-induced activity of [Ca²⁺]i in TCI-treated DRG neurons. (c) Large dose (500 ng/ml) of Shh-IgG treatment increased [Ca²⁺]i in intact DRG neurons (n = 30) from naïve rats. (d) Pretreatment with cyclopamine prevented Shh-IgG-induced [Ca²⁺]i increase in naïve DRG neurons (n= 30). BR-A was used to confirm cellular viability. (e) Western blot analysis showed that Shh-IgG application increased the phosphorylation of GluN2B, CaMKII, and CREB in DRG, which could be inhibited by pretreatment with cyclopamine. Six samples (DRGs) were included in each group. Samples were collected 1 hr after in vitro bath. *P < 0.05, **P < 0.01 indicates significant differences compared with naïve group (b), baseline (c), or intact group (e). ^##P < 0.01 indicates significant differences compared with TCI group (b) or Shh-IgG group (e). DRG: dorsal root ganglion; TCI: tumor cell implantation; Shh: sonic hedgehog.

Figure 5.

Electrophysiological alteration of excitability of dorsal root ganglion (DRG) neuronal somata after treatment with Shh-IgG, cyclopamine, and TCI, respectively. (a) and (e) Representative neural responses recorded with whole-cell patch-clamp electrodes under current clamp during test sequence, used to determine action potential threshold. Only two of the depolarizing 50-ms pulses (bottom) and corresponding responses (top) are illustrated in each case. (c) and (g) Representative neural discharge patterns evoked by depolarizing current with strength of 2.5 × threshold at 1 s. (b), (d), (f), and (h) Data summary showing effects of Shh signaling inhibition on hyperexcitability of DRG neurons after TCI treatment. (a) to (d) Samples collected at postoperative day 7. (e) to (h) Samples collected 1 hr after in vitro bath. Shh-IgG: 500 ng/ml; CLP: 5 µM (in vitro bath application). Numbers of neurons included in each group: sham = 16, TCI =25, TCI + CLP = 20. Intact = 20, Shh-IgG + DMSO = 25, Shh-IgG + CLP = 25. *P < 0.05, **P < 0.01 indicate significant differences compared with Sham group (b) and (d) or Intact group (f) and (h). ^#P < 0.05, ^##P < 0.01 indicate significant differences compared with TCI + DMSO group (b) and (d) or Shh-IgG + DMSO group (f) and (h). DRG: dorsal root ganglion; TCI: tumor cell implantation; Shh: sonic hedgehog.