Abstract
BackgroundRecent studies provide evidence of roles for integrins in mechanical signalling in bone and cartilage. Integrin signalling is modulated by various mechanisms, including interaction with other transmembrane proteins. We aimed to identify whether one such protein, integrin-associated protein (CD47/IAP), is expressed by chondrocytes and whether it may regulate integrin-dependent mechanotransduction.MethodsChondrocytes, isolated from macroscopically normal and osteoarthritic articular cartilage of human knee joints, were studied in a resting state or following mechanical stimulation at 0.33 Hz. CD47/IAP expression and associations were confirmed by immunohistology, reverse transcription-polymerase chain reaction, Western blotting, and immunoprecipitation. Roles in mechanotransduction were studied by assessing effects of function-blocking antibodies on a range of electrophysiological, cellular, and molecular responses of primary chondrocytes and responses of CD47/IAP-null cell lines transfected with CD47/IAP.ResultsHuman articular chondrocytes were shown to express CD47/IAP, predominantly the type 2 isoform. Immunoprecipitation showed association of CD47/IAP with α5 integrin and thrombospondin but not SIRPα (signal-regulatory protein-alpha). The function-blocking anti-CD47/IAP antibody Bric 126 inhibited changes in membrane potential, tyrosine phosphorylation, and elevation of relative levels of aggrecan mRNA induced by mechanical stimulation, whereas in the presence of B6H12, an antibody that has partial agonist activity, a membrane depolarisation rather than a membrane hyperpolarisation response was induced by mechanical stimulation. CD47-null cell lines did not show changes in cell membrane potential following mechanical stimulation. Changes in cell membrane potential following mechanical stimulation were seen when CD47-null cells were transfected with CD47/IAP expression vectors but were not seen following mechanical stimulation of cells transfected with vectors for the extracellular immunoglobulin variable (IgV) domain of CD47/IAP in the absence of the transmembrane and intracellular domains.ConclusionCD47/IAP is necessary for chondrocyte mechanotransduction. Through interactions with α5β1 integrin and thrombospondin, CD47/IAP may modulate chondrocyte responses to mechanical signals.
Highlights
Structural integrity of articular cartilage is dependent on physical loading and joint movement
Changes in cell membrane potential following mechanical stimulation were seen when CD47-null cells were transfected with CD47/ IAP expression vectors but were not seen following mechanical stimulation of cells transfected with vectors for the extracellular immunoglobulin variable (IgV) domain of CD47/IAP in the absence of the transmembrane and intracellular domains
No difference in expression of CD47/IAP was seen when protein extracts from primary monolayer cultures of chondrocytes derived from normal and osteoarthritic cartilage were immunoblotted with a panel of three different anti-CD47/IAP antibodies
Summary
Structural integrity of articular cartilage is dependent on physical loading and joint movement. The mechanisms by which mechanical forces regulate chondrocyte function are beginning to be defined and appear to involve the activation of a variety of intracellular signalling pathways, at least some of which require integrin-mediated events. In normal human articular chondrocytes, cyclical mechanical stimulation at 0.33 Hz (2 seconds on, 1 second off) in vitro results in activation of a mechanotransduction pathway that leads to upregulation of aggrecan gene expression and downregulation of matrix metalloproteinase-3 gene expression [4]. Mechanical stimulation activates α5β1-mediated signalling events, chondrocytes from osteoarthritic cartilage do not show alteration in levels of aggrecan or matrix metalloproteinase-3 mRNA following 0.33Hz mechanical stimulation [4,8]. We aimed to identify whether one such protein, integrin-associated protein (CD47/IAP), is expressed by chondrocytes and whether it may regulate integrin-dependent mechanotransduction
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