Laboratory of Tissue Homeostasis and Disease, Skeletal Biology and Engineering Research Center, KU Leuven, Leuven, Belgium.
Laboratory of Tissue Homeostasis and Disease, Skeletal Biology and Engineering Research Center, KU Leuven, Leuven, Belgium; Division of Rheumatology, University Hospitals Leuven, Leuven, Belgium.
Osteoarthritis Cartilage. 2019 Nov;27(11):1702-1710. doi: 10.1016/j.joca.2019.07.007. Epub 2019 Jul 19.
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.
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.
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.
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.
外切聚糖-1（Ext1）编码一种糖基转移酶，该酶在硫酸乙酰肝素（HS）蛋白聚糖生物合成中延长 HS 链必不可少。HS 链作为信号蛋白的结合伴侣，影响其分布和活性。Wnt/β-连环蛋白途径已成为软骨生成的关键调节剂。然而，EXT1 和 HS 如何在软骨生成过程中影响 Wnt/β-连环蛋白信号仍未可知。
在培养的微团中，稳定敲低或过表达 ATDC5 软骨细胞中的 Ext1。通过 ELISA 测定 HS 含量。通过 RT-qPCR 测定 Sox9、Col2a1、聚集蛋白聚糖和 Wnt 直接靶基因 Axin2 的软骨生成标志物。通过 Alcian 蓝和 DMMB 测定法评估蛋白聚糖含量，通过β-连环蛋白 Western blot 和 TOP/FOP 测定法评估经典 Wnt 信号激活。用 Wnt 激活剂 CHIR99021 和重组 WNT3A 处理 ATDC5 细胞和人关节软骨细胞。
Ext1 敲低降低了 HS，并增加了软骨生成标志物和蛋白聚糖的积累。Ext1 敲低减少了活性 Wnt/β-连环蛋白信号。相反，具有更高 HS 含量的 Ext1 过表达细胞显示出较低的软骨分化和增强的 Wnt/β-连环蛋白信号。Wnt/β-连环蛋白信号激活导致 ATDC5 细胞和人关节软骨细胞中 Ext1 表达下调。
EXT1 通过改变 Wnt/β-连环蛋白信号的活性影响前体细胞的软骨分化。Wnt/β-连环蛋白信号控制 Ext1 的表达，提示在软骨生成过程中 EXT1 和 Wnt/β-连环蛋白信号之间存在调节环。
