Clinical Examination Center, The Affiliated Eye Hospital of Wenzhou Medical University, Wenzhou, 325000, China.
Emergency Department of Children, The Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University, Wenzhou, 325000, China.
Biochem Biophys Res Commun. 2020 Oct 22;531(4):543-551. doi: 10.1016/j.bbrc.2020.07.142. Epub 2020 Aug 14.
Previous studies showed that the activation of Wnt signaling reduced high glucose (HG)-mediated fibroblast damage, but the molecular basis for this phenomenon remains elusive. This study aimed to analyze the level of phosphorylation of GSK3β Ser (pGSK3β Ser) during HG damage. Moreover, the phosphomimic form of pGSK3β Ser9 was expressed to analyze its effect on cell migration via the phosphorylation of Ikaros. The results revealed that HG treatment significantly reduced the pGSK3β Ser level. The overexpression of GSK3β SerD and GSK3β SerA accelerated and inhibited fibroblast cell migration, respectively. P110α knockdown or treatment with SP600125, an inhibitor of JNK, also reduced the pGSK3β Ser level under HG condition. Treatment with SP600125 inhibited the migration of fibroblasts, but not in GSK3β SerD-expressing cells. Further, yeast two-hybrid screening and biochemical analysis identified that GSK3β interacted and phosphorylated Ikaros at Ser. Besides, GSK3β SerD, but not GSK3β SerA, activated Ikaros Ser phosphorylation. Expressing Ikaros or β-catenin significantly promoted cell migration, suggesting that GSK3β modulated cell migration partially via the activation of Ikaros besides β-catenin signaling under HG condition. The expression of the phosphomimic form of Ikaros SerD resulted in a significant increase in the extent of cell migration compared with Ikaros under HG condition. Moreover, the Ikaros SerD DNA-binding affinity toward the ANXA4 promoter increased, and ANXA4 suppression promoted cell migration. In conclusion, the results of this study provided a new regulatory mechanism by which GSK3β negatively regulated human skin fibroblast cell migration.
先前的研究表明，Wnt 信号的激活减少了高糖（HG）介导的成纤维细胞损伤，但这一现象的分子基础仍不清楚。本研究旨在分析 HG 损伤过程中 GSK3β Ser（pGSK3β Ser）的磷酸化水平。此外，还表达了 pGSK3β Ser9 的磷酸模拟形式，以分析其通过 Ikaros 的磷酸化对细胞迁移的影响。结果表明，HG 处理显著降低了 pGSK3β Ser 水平。GSK3β SerD 和 GSK3β SerA 的过表达分别加速和抑制成纤维细胞的迁移。P110α 的敲低或 JNK 抑制剂 SP600125 的处理也降低了 HG 条件下的 pGSK3β Ser 水平。SP600125 处理抑制了成纤维细胞的迁移，但在 GSK3β SerD 表达细胞中没有抑制。进一步的酵母双杂交筛选和生化分析表明，GSK3β 与 Ikaros 相互作用并在 Ser 位点磷酸化 Ikaros。此外，GSK3β SerD，但不是 GSK3β SerA，激活了 Ikaros Ser 磷酸化。表达 Ikaros 或 β-catenin 显著促进了细胞迁移，表明 GSK3β 在 HG 条件下通过激活 Ikaros 信号部分调节细胞迁移，而不是 β-catenin 信号。在 HG 条件下，表达 Ikaros SerD 的磷酸模拟形式导致细胞迁移程度显著增加。此外，Ikaros SerD DNA 结合对 ANXA4 启动子的亲和力增加，ANXA4 抑制促进了细胞迁移。总之，本研究的结果提供了一个新的调节机制，即 GSK3β 负调控人皮肤成纤维细胞迁移。
