Wnt-signalling in human articular chondrocytes

McCarthy, H.; Owen, S.; Roberts, S.

Abstract

Introduction Aberrant Wnt-signalling in cartilage results inchondrocyte dedifferentiation, hypertrophy, loss of type II collagen, aggrecan and glycosaminoglycans. Articular chondrocytes experience a pO2 gradient from~6% (surface) to~1% (within deeper layers). Hypoxia is known to modulate Wnt-signalling. This study investigates how the Wnt profile alters in human articular chondrocytes (HACs) in respons eto Lithium Chloride (LiCl), a stimulator of canonical Wnt-signalling, during normoxia and hypoxia. Materials and Methods HACs were isolated from macroscopically normal femoral condyle cartilage at the time of joint replacement. HACs were cultures and passaged prior to LiCl exposure at 0, 20, 40 or 80 mM for 3, 8 and 24 h in normoxic or hypoxic conditions (1%, 24 h only). Culture medium was assayed for secreted DKK-1 by ELISA and RNA was extracted. Real-time qPCR was performed to analyse the expression of DKK-1, Wnt3a, Wnt5a, Wnt7b, Wnt10,Wnt11 and Wnt16, using GAPDH as a reference gene. A threshold of two-fold change in expression (either up- or down-regulated) was deemed of biological significance. Localisation of ß-Catenin was analysed by immunocy to chemistry using a specific monoclonal antibody against the active(unphosphorylated) form of ß-Catenin. Results Increasing concentrations of LiCl resulted in down-regulation of DKK-1 and up-regulation of Wnt3a, Wnt5a,Wnt10 and Wnt11. Expression levels reached and maintained the expression threshold or above by 3hrs for DKK-1and Wnt3a, 8 h for Wnt10 and Wnt11 and 24 h for Wnt5a.There was a significant increase in Wnt11 expression at40 mM LiCl under hypoxia versus normoxia. No other effect of hypoxia was observed. Wnt7b and Wnt16 were not detected. Secreted DKK-1 and nuclear translocation of ß-Catenin were decreased and increased respectively in a time-and dose-dependent manner. Discussion LiCl induces canonical Wnt-signalling by increasing nuclear translocation of ß-Catenin and reducing its inhibitor, DKK-1. LiCl also induced the up-regulation of both canonical (Wnt3a and Wnt10) and non-canonical(Wnt5a and Wnt11) signalling ligands. Hypoxia affected the expression of Wnt11 only when compared to normoxia. In vitro modulation of Wnt-signalling lends the opportunity to investigate the effects of increased Wnt-signalling on chondrocyte behaviour and how this may be manipulated for the treatment or prevention of osteoarthritis.

Citation

McCarthy, H., Owen, S., & Roberts, S. Wnt-signalling in human articular chondrocytes

• Presentation Conference Type: Conference Abstract
• Online Publication Date: Apr 17, 2015
• Publication Date: May 28, 2015
• Deposit Date: Jun 13, 2023
• Journal: International Journal of Experimental Pathology
• Print ISSN: 0959-9673
• Electronic ISSN: 1365-2613
• Publisher: Wiley
• Peer Reviewed: Peer Reviewed
• Volume: 96
• Issue: 2
• Pages: A27
• Public URL: https://keele-repository.worktribe.com/output/448984
• Publisher URL: https://onlinelibrary.wiley.com/doi/10.1111/iep.12125