Wnt5b介导P2X3受体上膜的机制及其在骨癌痛中的作用研究

Accumulative evidences have shown that functional upregulation of P2X3 receptors in dorsal root ganglion (DRG) neurons contributes to the neuronal hyperexcitability and the cancer-induced bone pain. As a ligand-gated ion channels, the membrane insertion of P2X3 receptor is important for its function in primary sensory neurons. During the process, CaMKIIα phosphorylated the P2X3 receptor at Thr388, then facilitated the interaction of the P2X3 receptor with caveolin-1, and drove the membrane insertion of the P2X3 receptor. Src-dependent caveolin-1 phosphorylation at Tyr(14) is very important for caveolin-1 to perform its function. In our pilot study, we have found that the expression of Wnt5b and p-caveolin-1 was upregulated dramatically in DRGs in bone cancer rats. In addition, incubation of cultured DRG neurons with Wnt5a, the membrane expression of P2X3 receptors was increased significantly. It has been reported that Wnt5b signaling may contribute to neuropathic pain through the atypical Wnt/Ryk signaling pathway which could induce increased intracellular Ca2+ and the subsequent Ca2+-dependent signals CaMKII, Src in primary sensory neurons. Therefore, in this study we used biochemistry, molecular biology, electrophysiology, morphology and behavior methods to test the hypothesis: Wnt5b induced increased intracellular Ca2+ by binding to Ryk receptors which leads to the activation Ca2+-dependent CaMKII, Src and caveolin-1 phosphorylation at Tyr(14) in primary sensory neurons; then p-CaMKII and p-caveolin-1 drives the membrane insertion of the P2X3 receptors and contributes to bone cancer pain. This study will help us understand the pathogenesic mechanisms of bone cancer pain, and provide a novel target for the treatment of bone cancer pain.

P2X3R的上膜可引起外周敏化及骨癌痛的发生，CaMKII和CAV1（Caveolin-1）是协助P2X3R上膜的关键分子，而CAV1功能的发挥通常需要Src磷酸化其Tyr14位点，但该位点在骨癌痛中的角色还不清楚。此外，我们发现，在骨癌大鼠DRG上，Wnt5b、p-CAV1（Tyr14）的表达上调；用Wnt5b孵育DRG神经元，可诱导P2X3R上膜，但机制不详。有研究报道，Wnt5b可作用于Ryk受体，引起胞内钙上调，激活CaMKII和Src激酶。因此，本项目拟通过细胞和动物实验验证以下科学假说：在骨癌大鼠DRG，上调的Wnt5b通过Wnt5b/Ryk/Ca2+途径激活CaMKII、Src激酶，Src进而磷酸化CAV1，激活的CaMKII和CAV1共同促进P2X3R的上膜，引起外周敏化，导致骨癌痛的发生。该研究将有助于阐明骨癌痛的发病机理，为骨癌痛的治疗提供新思路和新靶点。

原发性骨肿瘤或其他部位肿瘤（乳腺癌、前列腺癌、肺癌）的骨转移都会导致骨癌痛的发生，但骨癌痛的发生机制尚不明了。大量研究发现，外周敏化与骨癌痛的发生发展密切相关。P2X3受体高度选择性表达于isolectin B4（IB4）阳性的初级感觉神经元上，对慢性病理痛的外周敏化有着至关重要的作用。然而，P2X3作为一种非选择性的阳离子通道，其需要转运到细胞膜上才能发挥功能，因此，阐明P2X3受体上膜的机制将有可能为癌痛的治疗提供潜在的靶点。本研究发现在骨癌痛小鼠DRG上，Wnt5b及其受体Ryk的表达显著上调，给予Wnt5b孵育离体培养的DRG神经元，则神经元的兴奋性显著增强，而RyK中和抗体则可以显著抑制骨癌痛的发生以及DRG神经元的兴奋性，这说明Wnt5b/Ryk介导了骨癌痛的发生。本研究进一步还发现，给予Wnt5b孵育培养的DRG神经元可以显著上调P2X3膜受体的表达以及功能，而Ryk中和抗体则可以抑制Wnt5b的这种作用。同样在骨癌痛模型中，Ryk也可以抑制P2X3膜受体的上调以及骨癌痛的发生；如果对正常小鼠给予Wnt5b，则可以显著诱发机械痛敏和热痛敏的发生，而P2X3受体抑制剂A317491则可以显著抑制Wnt5b诱导的骨癌痛，很显然Wnt5b可以通过上调P2X3膜受体的表达及功能，进而促进骨癌痛的发生。不仅如此，我们还发现Wnt5b还可以上调DRG神经元上p-CaMKII、p-Src和p-Caveolin-1的表达，抑制CaMKII和Src激酶的激活或基因敲减Caveolin-1均可以抑制Wnt5b介导的P2X3膜受体的表达以及骨癌痛的发生。总而言之，我们的结果表明Wnt5b通过Wnt5b/Ryk途径激活CaMKII、Src激酶，Src进而磷酸化CAV1，激活的CaMKII和CAV1共同促进P2X3R的上膜，引起外周敏化，导致骨癌痛的发生。该研究将有助于阐明骨癌痛的发病机理，为癌痛的治疗提供新的靶点。