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.

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S1460 SP600125 SP600125 (Nsc75890) is a broad-spectrum JNK inhibitor for JNK1, JNK2 and JNK3 with IC50 of 40 nM, 40 nM and 90 nM in cell-free assays, respectively; 10-fold greater selectivity against MKK4, 25-fold greater selectivity against MKK3, MKK6, PKB, and PKCα, and 100-fold selectivity against ERK2, p38, Chk1, EGFR etc. SP600125 is also a broad‐spectrum inhibitor of serine/threonine kinases including Aurora kinase AFLT3 and TRKA with of IC50 of 60 nM, 90 nM and 70 nM. SP600125 inhibits autophagy and activates apoptosis.

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Autophagy FLT3 Trk receptor Apoptosis related JNK Aurora Kinase