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Antisense oligonucleotides to the integrin receptor subunit alpha(5) decrease fibronectin fragment mediated cartilage
G A Homandberg1, V Costa, V Ummadi
1Department of Biochemistry, Rush Medical College at Rush-Presbyterian-St Luke's Medical Center, 1653 West Congress Parkway, Chicago, Illinois 60612-3864, USA. ghomandb@rush.edu
Objective:
To investigate involvement of the integrin alpha(5) 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 alpha(5) subunit. The effects of the oligonucleotides on mRNA and protein expression of the alpha(5) 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 alpha(5) 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 microM 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 alpha(5) subunit to enhance PG degradation, but were ineffective in blocking endotoxin or IL-1beta enhanced degradation.
Conclusions:
These data implicate the alpha(5) integrin subunit in Fn-f mediated activities, consistent with a role for the alpha(5)beta(1) integrin in this pathway.
Insights
Antisense oligonucleotide targeting the integrin alpha(5) subunit inhibited fibronectin fragment-induced cartilage degradation and suppressed proteoglycan synthesis. This suggests alpha(5)beta(1) integrin plays a role in chondrolytic activities.
Area of Science:
- Biochemistry
- Cell Biology
- Integrin Signaling
Background:
- Fibronectin fragments (Fn-f) contribute to cartilage degradation.
- The classical fibronectin receptor, integrin alpha(5)beta(1), is a potential mediator of these effects.
Purpose of the Study:
- To investigate the role of the integrin alpha(5) subunit in fibronectin fragment-induced chondrolytic activities.
- To determine if blocking alpha(5) subunit expression affects cartilage catabolism.
Main Methods:
- Bovine chondrocytes and cartilage explants were treated with antisense oligonucleotide (ASO) targeting the alpha(5) subunit.
- mRNA and protein expression of alpha(5) were analyzed using rtPCR and Western blotting.
- Effects on proteoglycan degradation, synthesis, and protein synthesis suppression by Fn-f were assessed.
Main Results:
- ASO significantly reduced alpha(5) mRNA and protein expression.
- ASO partially reversed Fn-f-mediated suppression of proteoglycan and protein synthesis.
- ASO suppressed Fn-f-induced proteoglycan degradation in a dose-dependent and reversible manner.
Conclusions:
- The alpha(5) integrin subunit is implicated in fibronectin fragment-mediated cartilage degradation.
- These findings support a role for the alpha(5)beta(1) integrin in chondrolytic pathways.