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THU0018 Regulation of Xylosyltransferase-1 Expression by Fibronectin Fragment in Human Articular Chondrocytes
  1. C.M. Lee1,
  2. G.H. Park1,
  3. S.Y. Lee2,
  4. K.M. Son3,
  5. H.S. Lee4,
  6. M.H. Lee5,
  7. H.S. Hwang1,
  8. H.A. Kim1
  1. 1Division of Rheumatology, Department of Internal Medicine, Hallym University Sacred Heart Hospital, Kyunggi, 431–070
  2. 2Division of Rheumatology, Department of Internal Medicine, Hallym University Sacred Heart Hospital, Anyang
  3. 3Division of Rheumatology, Department of Internal Medicine, Hallym University Chuncheon Sacred Heart Hospital
  4. 4Division of Rheumatology, Department of Internal Medicine, Hallym University Chuncheon Sacred Heart Hospital, Chuncheon
  5. 5Division of Rheumatology, Department of Internal Medicine, Hallym University Sacred Heart Hospital, Kyunggi, 431–070, Korea, Republic Of

Abstract

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

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