Rhett J Matthew, Calder Bennett W, Fann Stephen A, Bainbridge Heather, Gourdie Robert G, Yost Michael J
Department of Surgery, General Surgery Division, Medical University of South Carolina, Charleston, South Carolina;
Department of Surgery, General Surgery Division, Medical University of South Carolina, Charleston, South Carolina.
Am J Physiol Cell Physiol. 2017 Sep 1;313(3):C314-C326. doi: 10.1152/ajpcell.00229.2016. Epub 2017 Jul 12.
Connexin-based therapeutics have shown the potential for therapeutic efficacy in improving wound healing. Our previous work demonstrated that the connexin43 (Cx43) mimetic peptide juxtamembrane 2 (JM2) reduced the acute inflammatory response to a submuscular implant model by inhibiting purinergic signaling. Given the prospective application in improving tissue-engineered construct tolerance that these results indicated, we sought to determine the mechanism of action for JM2 in the present study. Using confocal microscopy, a gap-FRAP cell communication assay, and an ethidium bromide uptake assay of hemichannel function we found that the peptide reduced cell surface Cx43 levels, Cx43 gap junction (GJ) size, GJ communication, and hemichannel activity. JM2 is based on the sequence of the Cx43 microtubule binding domain, and microtubules have a confirmed role in intracellular trafficking of Cx43 vesicles. Therefore, we tested the effect of JM2 on Cx43-microtubule interaction and microtubule polymerization. We found that JM2 enhanced Cx43-microtubule interaction and that microtubule polymerization was significantly enhanced. Taken together, these data suggest that JM2 inhibits trafficking of Cx43 to the cell surface by promoting irrelevant microtubule polymerization and thereby reduces the number of hemichannels in the plasma membrane available to participate in proinflammatory purinergic signaling. Importantly, this work indicates that JM2 may have therapeutic value in the treatment of proliferative diseases such as cancer. We conclude that the targeted action of JM2 on Cx43 channels may improve the tolerance of implanted tissue-engineered constructs against the innate inflammatory response.
基于连接蛋白的疗法已显示出在促进伤口愈合方面的治疗效果潜力。我们之前的研究表明，连接蛋白43（Cx43）模拟肽近膜区2（JM2）通过抑制嘌呤能信号传导，减轻了对肌下植入模型的急性炎症反应。鉴于这些结果所表明的在提高组织工程构建体耐受性方面的潜在应用，我们在本研究中试图确定JM2的作用机制。通过共聚焦显微镜、间隙荧光恢复后光漂白（gap-FRAP）细胞通讯测定法以及半通道功能的溴化乙锭摄取测定法，我们发现该肽降低了细胞表面Cx43水平、Cx43间隙连接（GJ）大小、GJ通讯以及半通道活性。JM2基于Cx43微管结合结构域的序列，并且微管在Cx43囊泡的细胞内运输中具有确定的作用。因此，我们测试了JM2对Cx43 - 微管相互作用和微管聚合的影响。我们发现JM2增强了Cx43 - 微管相互作用，并且微管聚合显著增强。综上所述，这些数据表明JM2通过促进无关的微管聚合来抑制Cx43向细胞表面的运输，从而减少参与促炎嘌呤能信号传导的质膜中半通道的数量。重要的是，这项工作表明JM2在治疗诸如癌症等增殖性疾病方面可能具有治疗价值。我们得出结论，JM2对Cx43通道的靶向作用可能会提高植入的组织工程构建体对先天性炎症反应的耐受性。
