Exostosin-1 activity enhances the strength of wnt signaling during chondrogenic differentiation

Abstract

Purpose: The exostosin-1 (EXT1) gene encodes a glycosyltransferase that is required for heparan sulfate (HS) chain elongation, which is a key step in HS-proteoglycan biosynthesis. Importantly, HS chains serve as binding partners for key signaling proteins such as hedgehogs and BMPs, affecting their distribution, activity and target selection, and thus influencing their roles in joint development and growth. The Wnt signaling pathway has emerged as a critical regulator of skeletal cell differentiation processes. However, how EXT1 activity and subsequent HS levels affect the activation of the Wnt signaling during endochondral ossification remains poorly understood. In this study, we aimed to define the function of EXT1 and HS-proteoglycan levels in determining the strength of Wnt signaling during chondrogenesis. Methods: Ext1 was stably knocked down or overexpressed in ATDC5 chondrogenic cells cultured as micromasses. HS content was determined using ELISA. Gene expression of chondrogenic markers (Col2a1, Acan andSox9) and direct Wnt target genes (Axin2 andLef1) was measured by RT q-PCR, at day 1, 7 and 14 of the chondrogenic differentiation process. Total proteoglycan content was evaluated by Alcian blue staining and DMMB assay. Activation of canonical Wnt signaling was assessed by Western blot of active β-catenin protein compared to total β-catenin levels, and by TOP/FOP luciferase activity assay. To evaluate whether Wnt signaling reciprocally regulates Ext1 expression, wild-type ATDC5 micromasses were treated with Wnt activators (recombinant WNT3A protein and CHIR99021) and a pharmacological Wnt antagonist (XAV939). Results: Ext1 was efficiently knocked down and overexpressed in ATDC5 cells during the whole chondrogenic differentiation process. Ext1 knockdown cells, which exhibited reduced HS content, showed increased expression of chondrogenic markers Col2a and Acan compared to control cells, and this paralleled an increase in total proteoglycan content. These results indicate that Ext1 knockdown facilitates the chondrogenic differentiation process. Next, Ext1 knockdown led to a reduced active Wnt signaling, as it resulted in a lower gene expression of Axin2, lower ratio of active β-catenin/totalβ-catenin protein levels, and lower luciferase activity compared to control cells. Contrarily, Ext1 overexpressing cells, which had a higher HS content, showed decreased chondrogenic differentiation potential and enhanced Wnt signaling activation compared to corresponding control cells. Interestingly, activation of Wnt signaling led to a downregulation of Ext1 expression in a concentration-dependent manner, and a specific Wnt antagonist upregulated Ext1 expression. Conclusions: Changes in EXT1 function, which has an impact on HS-proteoglycan content, affect the chondrogenic differentiation potential of precursor cells, and these effects are in part attributable to modifications in the strength of the Wnt signaling pathway. Simultaneously, Wnt signaling controls the expression of Ext1. Collectively, these results suggest the existence of a regulatory loop between Ext1 and the Wnt signaling during chondrogenesis. These insights may also have implications in osteoarthritis, since excessive Wnt signaling in articular cartilage is strongly linked to disease development and progression.