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 1 nM to 5 μM 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.