Rho/ROCK acts downstream of lysophosphatidic acid receptor 1 in modulating P2X3 receptor-mediated bone cancer pain in rats

Abstract

Background: Lysophosphatidic acid receptor 1 and Rho/ROCK signaling is implicated in bone cancer pain development. However, it remains unknown whether the two signaling pathways function together in P2X3 receptor-mediated bone cancer pain.

Results: In this study, using a rat model of bone cancer, we examined the expression of P2X3 and lysophosphatidic acid receptor 1 in rat dorsal root ganglion neurons and further dissected whether lysophosphatidic acid receptor 1 and Rho/ROCK-mediated pathways interacted in modulating rat pain behavior. Bone cancer was established by inoculating Walker 256 cells into the left tibia of female Wistar rats. We observed a gradual and yet significant decline in mean paw withdrawal threshold in rats with bone cancer, but not in control rats. Our immunohistochemical staining revealed that the number of P2X3- and lysophosphatidic acid receptor 1-positive dorsal root ganglion neurons was significantly greater in rats with bone cancer than control rats. Lysophosphatidic acid receptor 1 blockade with VPC32183 significantly attenuated decline in mean paw withdrawal threshold. Flinching behavior test further showed that lysophosphatidic acid receptor 1 inhibition with VPC32183 transiently but significantly attenuated α,β-meATP-induced increase in paw lift time per minute. Rho inhibition by intrathecal BoTXC3 caused a rapid reversal in decline in mean paw withdrawal threshold of rats with bone cancer. Flinching behavior test showed that BoTXC3 transiently and significantly attenuated α,β-meATP-induced increase in paw lift time per minute. Similar findings were observed with ROCK inhibition by intrathecal Y27632. Furthermore, VPC32183 and BoTXC3 effectively aborted the appearance of lysophosphatidic acid-induced calcium influx peak.

Conclusions: Lysophosphatidic acid and its receptor LPAR1, acting through the Rho-ROCK pathway, regulate P2X3 receptor in the development of both mechanical and spontaneous pain in bone cancer.

Keywords: Bone cancer pain; P2X3 receptor; Rho/ROCK; lysophosphatidic acid receptor.

Figure 1.

Bone cancer pain is associated with in increased proportions of LPAR1- and P2X₃-positive neurons in the dorsal root ganglia of rats. (a) Rats were inoculated with live or heat-killed Walker 256 cells as described in Methods section. The mean paw withdrawal threshold was assessed at the time points indicated. (b) Immunofluorescent microscopy was done using anti-P2X₃ receptor and anti-LPAR1 antibodies as described in Methods section. Green: P2X₃ receptor; Red: LPAR₁. (c) The level of P2X₃ and LPAR₁ in DRG samples of sham group (n = 10) and bone cancer group (n = 10). P2X₃ and LPAR₁ were assessed using Western blot analysis. Data (a to c) are presented as mean ± SD (n = 6 rats per group), and inter-group differences are assessed using Student’s t test. *p < 0.05, **p < 0.01 compared with the sham control.

Figure 2.

P2X₃ receptor-mediated pain and spontaneous pain induced by the P2X₃ receptor agonist α,β-meATP are reduced by P2X₃ receptor inhibitor and LPAR1 antagonist. (a) P2X₃ receptor inhibitor A317491 reversed the bone cancer-induced decrease in paw withdrawal threshold. (b) The spontaneous response, PLTPM induced by P2X₃ receptor agonist α,β-meATP was significantly longer in cancer rats than in sham control animals, and it was reduced by a P2X₃ receptor inhibitor A317491. (c) LPAR1 antagonist VPC32183 reversed the bone cancer-induced decrease in paw withdrawal threshold. (d) P2X₃ receptor agonist α,β-meATP induced a spontaneous response that was reduced by LPAR1 antagonist. Data were presented as means ± SD (n = 6 animals per group), and inter-group differences were assessed for significance using two-way ANOVA. *p < 0.05, **p < 0.01 compared with corresponding time points in the PBS group or 3% BSA group.

Figure 3.

P2X₃ receptor-mediated bone cancer pain requires Rho-ROCK signaling pathways. The bone cancer-induced decrease in paw withdrawal threshold was reversed by Rho inhibitor BoTXC3 (a) and ROCK inhibitor Y27632 (c). The spontaneous response induced by α,β-meATP was reduced by Rho inhibitor BoTXC3 (b) and ROCK inhibitorY27632 (d). Data were presented at means ± SD (n = 5 animals per group), and inter-group differences were assessed using two-way ANOVA. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with corresponding time points in the saline group or 3% BSA group.

Figure 4.

Rho functions downstream of LPAR1 in modulating P2X₃ receptor-mediated cellular calcium influx. Representative tracings of Fura-2 ratio of primary dorsal root ganglion (DRG) neurons treated with α,β-meATP (20 μM) followed by lysophosphatidic acid (LPA, 4 μM) plus vehicle (a, d) or LPA plus LPAR1 antagonist VPC32183 (b), or LPA plus Rho inhibitor BoTXC3 (5 µg/mL) (e) are shown. Pooled data are shown (c, f) measuring the Δ Fura-2 ratio in which peak height is subtracted from basal and then peak fold of increase was Δ Fura-2 ratio of peak2 vs. Δ Fura-2 ratio of peak1; between 23 and 48 cells were analyzed under each condition. Data are presented as mean ± SD, and inter-group differences are assessed using one way ANOVA followed by LSD post hoc analysis. **p < 0.01 α,β-meATP plus LPA vs. α,β-meATP in (c, f), ^##p < 0.01 VPC32183 (c) or BoTXC3 (f) vs. α,β-meATP plus LPA.