Antisense oligonucleotides to the integrin receptor subunit alpha5 decrease fibronectin fragment mediated cartilage chondrolysis

Abstract

Objective To investigate involvement of the integrin alpha5 subunit of the classical fibronectin receptor in cartilage chondrolytic activities of fibronectin fragments (Fn-f).Design Bovine chondrocytes and cartilage explants were cultured in the presence of antisense oligonucleotide (ASO), or sense (SO) or scrambled sequence oligonucleotide (SCO) corresponding to the bovine alpha5 subunit. The effects of the oligonucleotides on mRNA and protein expression of the alpha5 subunit were analysed by rtPCR and Western blotting, respectively. To test effects on Fn-f activities, three different Fn-f were first added to serum or serum-free cultures, followed by addition of oligonucleotides and the effects on Fn-f mediated proteoglycan (PG) degradation, cartilage PG depletion and PG and general protein synthesis suppression were tested.Results The ASO decreased alpha5 mRNA and protein expression to 69% and 55%, respectively, in monolayer cultures and decreased protein expression 67% in cartilage explants, while SO and SCO were ineffective. The ASO partially reversed the ability of the Fn-fs to suppress PG and general protein synthesis in cartilage explant and high density chondrocyte cultures. Concentrations of ASO from 1nM to 5μM effectively suppressed Fn-f activities in particular assays and the effects were reversible, while SO and SCO were not significantly effective. ASO also suppressed, in a dose-dependent and reversible fashion, the ability of the Fn-fs to enhance degradation and release of PG from cartilage explants. The ASO were also effective in suppressing the ability of an antibody to the alpha5 subunit to enhance PG degradation, but were ineffective in blocking endotoxin or IL-1β enhanced degradation.Conclusions These data implicate the alpha5 integrin subunit in Fn-f mediated activities, consistent with a role for the alpha5beta1 integrin in this pathway. Copyright 2002 OsteoArthritis Research Society International. Published by Elsevier Science Ltd. All rights reserved.