Exostosin-1 enhances canonical Wnt signaling activity during chondrogenic differentiation

Summary Objective Exostosin-1 ( Ext1 ) encodes a glycosyltransferase required for heparan sulfate (HS) chain elongation in HS-proteoglycan biosynthesis. HS chains serve as binding partners for signaling proteins, affecting their distribution and activity. The Wnt/β-catenin pathway emerged as critical regulator of chondrogenesis. Yet, how EXT1 and HS affect Wnt/β-catenin signaling during chondrogenesis remains unexplored. Method Ext1 was stably knocked-down or overexpressed in ATDC5 chondrogenic cells cultured as micromasses. HS content was determined using ELISA. Chondrogenic markers Sox9, Col2a1, Aggrecan , and Wnt direct target gene Axin2 were measured by RT-qPCR. Proteoglycan content was evaluated by Alcian blue and DMMB assay, canonical Wnt signaling activation by β-catenin Western blot and TOP/FOP assay. ATDC5 cells and human articular chondrocytes were treated with WNT activators CHIR99021 and recombinant WNT3A. Results Ext1 knock-down reduced HS, and increased chondrogenic markers and proteoglycan accumulation. Ext1 knock-down reduced active Wnt/β-catenin signaling. Conversely, Ext1 overexpressing cells, with higher HS content, showed decreased chondrogenic differentiation and enhanced Wnt/β-catenin signaling. Wnt/β-catenin signaling activation led to a down-regulation of Ext1 expression in ATDC5 cells and in human articular chondrocytes. Conclusions EXT1 affects chondrogenic differentiation of precursor cells, in part via changes in the activity of Wnt/β-catenin signaling. Wnt/β-catenin signaling controls Ext1 expression, suggesting a regulatory loop between EXT1 and Wnt/β-catenin signaling during chondrogenesis.