P2X3嘌呤能受体的转录调控及其在大鼠骨癌痛中的作用研究

Our previous study has documented that functional upregulation of P2X3 receptors (P2X3R) in dorsal root ganglion (DRG) contributes to the hyperexcitability of nociceptive sensory neurons and then to the bone cancer-induced pain. However, the transcriptional mechanisms underlying the sustaining synthesis and expression of these receptors during the development of bone cancer pain still remain largely unknown. In our pilot study, we have found that transcription factor Runx1 modulates the expression of P2X3R in DRG neurons and thereby participating in the pathogenesis of bone cancer pain. Accumulative evidence has shown that Runx1 functions are potentially regulated by extracellular signal-regulated kinase (ERK), which is activated by multiple cytokines. It is reported that ERK-dependent phosphorylation potentiates the transcriptional activity of Runx1. Our preliminary experiments also showed that GDNF (glial cell-derived neurotrophic factor) could result in the increases of both P2X3R mRNA and protein through the activation of ERK-dependent Runx1 phosphorylation..In this research project, we will perform the following studies to investigate the mechanisms underlying the transcriptional regulation of P2X3R in cancer rat DRG neurons, as well as its role in bone cancer pain: (1) examining the transcriptional regulation of p2x3r gene by Runx1 and its contribution to the development of bone cancer pain, and then clarifying the molecular mechanisms by which how Runx1 promotes P2X3R transcription; (2) investigating the role for ERK pathway activation in phosphorylation of Runx1, and validating the necessary of ERK-dependent Runx1 phosphorylation for the transcriptional regulation of P2X3R; (3) exploring the contribution of GDNF to the activation of ERK pathway that subsequently phosphorylates Runx1 at serine 249 (Ser249) site, thereby elucidating the role for GDNF/GFRα1/Ret/ERK pathway-mediated Runx1 Ser249 phosphorylation in transcriptional regulation of P2X3R and its contribution to the development of bone cancer pain. This study will be helpful to elucidate the pathogenic mechanisms of bone cancer pain, and therefore provide a novel target for the treatment of bone cancer pain.

我们前期研究发现，P2X3R在DRG神经元的功能性上调,可以引起神经元的超兴奋性和骨癌痛。但是在癌症痛的发展过程中，通过何种机制来调控P2X3R转录合成和表达，仍然是一个有待解决的关键科学问题。我们预实验表明：转录因子Runx1可能会通过对P2X3R的转录调控而参与癌症痛。研究报道,细胞因子能够通过ERK磷酸化Runx1来增强其转录活性。我们预实验提示,GDNF可能会激活ERK而磷酸化Runx1,进而促进P2X3R转录合成。本项目拟通过以下研究阐明P2X3R的转录调控机制及其在骨癌痛中的作用：1)阐明Runx1促进P2X3R转录的分子机制及其在癌症痛中的作用；2)确定ERK激活在Runx1磷酸化及P2X3R转录中的作用；3)确定GDNF对ERK的激活,阐明GDNF/ERK/Runx1通路在P2X3R转录调控及癌症痛中的作用。该研究将有助于阐明癌症痛的发病机理,为骨癌痛的药物治疗提供新靶点。

原发性骨肿瘤或其它部位肿瘤的骨转移通常会引起剧烈的、顽固性的骨癌痛，严重影响患者的生活质量。然而，骨癌痛的发生机制仍不清楚。本项目运用细胞生物学、分子生物学、电生理学和行为药理学等实验技术，系统地研究了胶质源性神经营养因子（GDNF）对DRG神经元中P2X3受体的转录调控机制，以及这种转录调控在骨癌痛发生中的作用。主要研究结果如下：（1）在正常大鼠，鞘内给予外源性GDNF可以显著增加DRG神经元中ERK激酶和转录因子Runx1的磷酸化水平；促进P2X3受体的转录和翻译，上调P2X3受体的mRNA和蛋白表达，增加P2X3受体的电流密度和DRG神经元兴奋性；诱发大鼠出现痛觉过敏。（2）鞘内给予ERK抑制剂(SCH772984)或Runx1 siRNA对大鼠进行预处理，均能抑制外源性GDNF对P2X3受体的mRNA和蛋白表达的上调作用，并缓解外源性GDNF诱发的大鼠痛觉过敏行为；（3）应用ERK抑制剂预处理还可以抑制外源性GDNF介导的Runx1的磷酸化。这部分结果表明，在DRG神经元中外源性GDNF可以通过激活ERK-Runx1信号通路，促进P2X3受体的转录过程，进而增加神经元兴奋性，诱发大鼠出现痛觉过敏。（4）在骨癌痛模型大鼠DRG中，GDNF膜表面结合受体GFRα1的蛋白表达、以及其下游信号分子ERK及Runx1的磷酸化水平均显著增加，应用ERK抑制剂和Runx1 siRNA干扰的方法证实，鞘内应用ERK抑制剂可以抑制骨癌痛模型大鼠DRG神经元中Runx1的磷酸化水平增加、降低P2X3受体的转录和蛋白表达，进而抑制DRG神经元的兴奋性和外周敏化，缓解骨癌大鼠的痛觉过敏行为。（5）鞘内应用Runx1 siRNA敲减Runx1的表达，同样可以抑制骨癌大鼠DRG神经元中P2X3受体的mRNA和蛋白表达增加，并缓解大鼠的骨癌痛行为。这部分结果表明，在骨癌痛模型大鼠DRG神经元中GDNF-ERK-Runx1信号通路被激活，激活的信号通路通过促进P2X3受体的转录和蛋白表达而敏化DRG神经元，从而介导大鼠骨癌痛的发生和维持。结论：GDNF通过激活DRG神经元中ERK- Runx1信号通路促进P2X3受体转录，参与骨癌痛的发生和维持。本研究阐明了骨癌痛发生时P2X3受体转录上调的分子机制，为进一步理解骨癌痛的发病机理奠定了基础，同时也为未来开发治疗骨癌痛的靶向药物提供了新思路。