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

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• Wnt signaling activation mitigates high glucose (HG)-induced fibroblast damage, but the underlying molecular mechanisms are unclear.
• Glycogen synthase kinase 3 beta (GSK3β) phosphorylation at Serine 9 (pGSK3β Ser⁹) is a key regulator of Wnt signaling.
• Fibroblast dysfunction contributes to diabetic complications, highlighting the need to understand cellular responses to high glucose.

Purpose of the Study:

• To investigate the role of GSK3β phosphorylation at Ser⁹ in high glucose-induced fibroblast damage.
• To elucidate the molecular pathway involving GSK3β, Ikaros, and β-catenin in fibroblast cell migration under high glucose conditions.
• To identify novel regulatory mechanisms of fibroblast migration relevant to diabetic pathology.

Main Methods:

• Analysis of pGSK3β Ser⁹ levels in fibroblasts under high glucose conditions.
• Overexpression of wild-type and mutant forms of GSK3β (GSK3β Ser⁹D and GSK3β Ser⁹A) to assess effects on cell migration.
• Yeast two-hybrid screening and biochemical assays to identify GSK3β-interacting proteins and phosphorylation sites, including Ikaros phosphorylation at Ser³⁹¹.
• Assessment of cell migration, DNA-binding affinity, and gene expression (ANXA4) following genetic and pharmacological interventions.

Main Results:

• High glucose significantly reduced pGSK3β Ser⁹ levels in fibroblasts.
• Overexpression of GSK3β Ser⁹D (phosphomimic) enhanced fibroblast migration, while GSK3β Ser⁹A (non-phosphorylatable) inhibited it.
• GSK3β was found to interact with and phosphorylate Ikaros at Ser³⁹¹, a modification that enhanced Ikaros activity and promoted cell migration.
• Both Ikaros and β-catenin signaling contributed to GSK3β-mediated fibroblast migration under high glucose.

Conclusions:

• High glucose negatively impacts fibroblast migration by reducing GSK3β phosphorylation at Ser⁹.
• GSK3β modulates fibroblast migration through the phosphorylation and activation of Ikaros at Ser³⁹¹, in addition to its known role in β-catenin signaling.
• This study reveals a novel regulatory mechanism of GSK3β in controlling human skin fibroblast migration, offering potential therapeutic targets for diabetic complications.