GSK3β-Ikaros-ANXA4 signaling inhibits high-glucose-induced fibroblast migration

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 Ser⁹ (pGSK3β Ser⁹) 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 Ser⁹ level. The overexpression of GSK3β Ser⁹D and GSK3β Ser⁹A accelerated and inhibited fibroblast cell migration, respectively. P110α knockdown or treatment with SP600125, an inhibitor of JNK, also reduced the pGSK3β Ser⁹ level under HG condition. Treatment with SP600125 inhibited the migration of fibroblasts, but not in GSK3β Ser⁹D-expressing cells. Further, yeast two-hybrid screening and biochemical analysis identified that GSK3β interacted and phosphorylated Ikaros at Ser³⁹¹. Besides, GSK3β Ser⁹D, but not GSK3β Ser⁹A, activated Ikaros Ser³⁹¹ 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 Ser³⁹¹D resulted in a significant increase in the extent of cell migration compared with Ikaros under HG condition. Moreover, the Ikaros Ser³⁹¹D 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.