Wnt5b介导P2X3受体上膜的机制及其在骨癌痛中的作用研究

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。

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.