Background Xylosyltransferase-1 (XT-1), encoded by xylt1 gene, is an essential anabolic enzyme to catalyze the initial and rate-determining step in glycosaminoglycan chain synthesis. The effect of fibronectin fragments (FN-fs), generated by proteolytic cleavage of FN and known as damage-associated molecular pattern (DAMP) molecules, on cartilage metabolism was poorly characterized.
Objectives In this study we examined 29-kDa amino-terminal fibronectin fragment (29-kDa FN-f)-mediated XT-1 expression mechanism and its signaling pathway, determining the role of 29-kDa FN-f in cartilage matrix synthesis.
Methods Human articular chondrocytes were enzymatically isolated from articular cartilage and cultured in monolayer. In 29-kDa FN-f-stimulated chondrocytes, the relative levels of mRNA and protein for XT-1 were analyzed by real-time quantitative reverse transcription-polymerase chain reaction and Western blot analysis, respectively. In order to investigate the effects of 29-kDa FN-f on XT-1, human chondrocytes were transfected with small interfering RNAs (siRNAs) targeting TLR-2.
Results The level of aggrecan and XT-1 in human osteoarthritis cartilage was significantly decreased compared to normal cartilage. XT-1 expression in cultured primary articluar chondrocytes showed a periodic oscillation in both mRNA and protein level. 29-kDa FN-f significantly suppressed the mRNA and protein levels of XT-1 at 14 h and 24 h, respectively. Knockdown of toll like receptor-2 (TLR-2) using small interference RNA revealed that the decrease of XT-1 expression by 29-kDa FN-f is mediated by TLR-2 signaling pathway. Inhibition of mitogen activated protein kinase (MAPK) and nuclear factor-κB (NF- κB) signaling pathway restored 29-kDa FN-f-inhibited XT-1 expression. In addition, reduction of XT-1 expression by 29-kDa FN-f- was associated with activation of activator protein 1 (AP-1) signaling pathway. Finally, 29-kDa FN-f enhanced the expression of Sp3 and inhibited the expression of Sp1. Knockdown and overexpression experiments confirmed that XT-1 expression was modulated by Sp3 and Sp1, which are ubiquitous transcriptional proteins and compete for the same DNA binding site.
Conclusions These results demonstrated that 29-kDa FN-f plays a detrimental role in the regulation of cartilage extracellular matrix formation including XT-1 expression.
Disclosure of Interest None declared