Douglas Mental Health University Institute, McGill University, Montréal, Canada.
Department of Psychiatry, McGill University, Montréal, Canada.
Eur J Pain. 2018 May;22(5):845-861. doi: 10.1002/ejp.1172. Epub 2018 Jan 16.
Prostaglandin E2 (PGE2) enriched in inflamed tissues contributes to chronic pain by sensitizing nociceptive dorsal root ganglion (DRG) neurons (nociceptors). Of four PGE2 receptors (EP1-4), EP4 plays a major role in PGE2-induced nociceptor sensitization. We have previously reported that PGE2 or EP4 agonists stimulated EP4 externalization in cultured DRG neurons and this event contributes to nociceptor sensitization. However, the signalling transduction events governing this event remain unknown.
In this study, using antibody-based externalization assay, we examined EP subtypes and multiple signalling transduction events involved in PGE2-induced EP4 externalization in cultured DRG neurons.
In addition to EP4 agonist, EP2 agonist, to a lesser extent, also induced EP4 externalization while EP1 and EP3 agonists had no effect. The extracellular and intracellular calcium chelators, the inhibitors of CaMKII, cAMP, PKA, PKC, PKCε, PLC, MAPKs, PI3K and Akt suppressed agonist-induced EP4 externalization. The activator of AC, two PKA-specific cAMP analogues and one Epac-specific cAMP analogue also induced EP4 externalization. ELISA showed that double sequential exposures to EP4 agonists induced a greater release of pain peptide CGRP from cultured DRG neurons than a single exposure, an event blocked by the inhibitor of anterograde transport from ER/Golgi complex to cell surface.
Taken together, these data suggest that mobilization of extracellular and intracellular calcium as well as the activation of CaMKII, cAMP/PKA, cAMP/Epac, PKC/PKCε, MAPKs, PI3K-Akt and PLC signalling transduction pathways are involved in agonist-induced EP4 externalization. Agonist-enhanced EP4 externalization increases EP4 cell surface abundance and activity, thus enhancing nociceptor sensitization.
This study adds mechanistic information regarding signalling transduction events involved in agonist-induced EP4 cell surface trafficking. EP4 and EP2 (to lesser extent) receptors, extra- and intracellular Ca , CaKMII, cAMP, PKA, PKC, PKCε, PLC, MAPK, PI3K and Akt are involved in this event. Agonist-induced EP4 externalization contributes to enhanced nociceptor sensitization.
富含在炎症组织中的前列腺素 E2（PGE2）通过敏化伤害感受性背根神经节（DRG）神经元（伤害感受器）导致慢性疼痛。在四种 PGE2 受体（EP1-4）中，EP4 在 PGE2 诱导的伤害感受器敏化中起主要作用。我们之前报道过，PGE2 或 EP4 激动剂在培养的 DRG 神经元中刺激 EP4 外化，这一事件有助于伤害感受器敏化。然而，控制这一事件的信号转导事件仍然未知。
在这项研究中，我们使用基于抗体的外化测定法，检查了参与 PGE2 诱导的培养 DRG 神经元中 EP4 外化的 EP 亚型和多种信号转导事件。
除了 EP4 激动剂外，EP2 激动剂在较小程度上也诱导了 EP4 外化，而 EP1 和 EP3 激动剂则没有作用。细胞外和细胞内钙螯合剂、钙调蛋白激酶 II（CaMKII）抑制剂、cAMP、PKA、PKC、PKCε、PLC、MAPK、PI3K 和 Akt 抑制激动剂诱导的 EP4 外化。AC 的激活剂、两种 PKA 特异性 cAMP 类似物和一种 Epac 特异性 cAMP 类似物也诱导了 EP4 外化。ELISA 显示，与单次暴露相比，双重连续暴露于 EP4 激动剂可从培养的 DRG 神经元中释放出更多的痛肽 CGRP，而这一事件被从 ER/Golgi 复合物到细胞表面的顺行转运抑制剂阻断。
综上所述，这些数据表明，细胞外和细胞内钙的动员以及 CaMKII、cAMP/PKA、cAMP/Epac、PKC/PKCε、MAPK、PI3K-Akt 和 PLC 信号转导途径的激活参与了激动剂诱导的 EP4 外化。激动剂增强的 EP4 外化增加了 EP4 细胞表面的丰度和活性，从而增强了伤害感受器的敏化。
本研究增加了关于激动剂诱导的 EP4 细胞表面转运涉及的信号转导事件的机制信息。EP4 和 EP2（程度较小）受体、细胞外和细胞内钙、CaMKII、cAMP、PKA、PKC、PKCε、PLC、MAPK、PI3K 和 Akt 均参与了这一事件。激动剂诱导的 EP4 外化有助于增强伤害感受器的敏化。
