CD47 associates with alpha 5 integrin and regulates responses of human articular chondrocytes to mechanical stimulation in an in vitro model

Abstract

Background: Recent 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.

Methods: Chondrocytes, 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.

Results: Human articular chondrocytes were shown to express CD47/IAP, predominantly the type 2 isoform. Immunoprecipitation showed association of CD47/IAP with alpha5 integrin and thrombospondin but not SIRPalpha (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.

Conclusion: CD47/IAP is necessary for chondrocyte mechanotransduction. Through interactions with alpha5beta1 integrin and thrombospondin, CD47/IAP may modulate chondrocyte responses to mechanical signals.

Figure 1

Expression of CD47/IAP in human articular cartilage. Sections of human articular cartilage were stained with the anti-CD47 antibody Bric 126, resulting in strong labelling of resident chondrocytes. (a) Normal articular cartilage (original magnification ×50). Inset: high-power view (original magnification ×400). (b) Osteoarthritic articular cartilage (original magnification ×50). Inset: negative control non-immune mouse immunoglobulin (original magnification ×100). IAP, integrin-associated protein.

Figure 2

Expression of CD47/IAP by human articular chondrocytes. (a) Western blotting of protein extracts from human articular chondrocytes with three different anti-CD47/IAP antibodies (monoclonal antibodies 2B7, miap 400.1, and a goat polyclonal anti-CD47/IAP) showing a predominant band of approximately 50 kDa in protein extracts from chondrocytes extracted from both normal and osteoarthritic chondrocytes. (b) Reverse transcription-polymerase chain reaction on RNA extracted from primary cultures of chondrocytes from normal and osteoarthritic chondrocytes undertaken with specific primers for CD47/IAP showing predominant expression of the type 2 isoform. IAP, integrin-associated protein; OA, osteoarthritic human articular chondrocytes; OV10-315, CD47 negative ovarian carcinoma cell line transfected with human CD47 type 2 isoform; N, normal human articular chondrocytes; Neg, negative control (reverse transcription step omitted).

Figure 3

Expression of thrombospondin-1 (TSP-1) and signal-regulatory protein-alpha (SIRPα) by human articular chondrocytes and molecular associations of CD47/IAP. (a) Protein extracts from primary cultures of human articular chondrocytes were separated by SDS-PAGE and immunoblotted with antibodies against TSP-1 and SIRPα. For TSP-1, a 6% gel under reducing conditions was used, whereas for SIRPα, an 8% gel under non-reducing conditions was used. (b) Protein extracts from primary cultures of human articular chondrocytes were immunoprecipitated with antibodies against CD47/IAP and immunoblotted for TSP-1, SIRPα, or α5 integrin. TSP-1, SIRPα, and α5 integrin are identified in non-immunoprecipitated extracts, but only TSP-1 and α5 integrin co-immunoprecipitate with CD47/IAP. IP, immunoprecipitated protein extracts; MW, molecular weight; N, normal human articular chondrocytes; Non IP, non-immunoprecipitated protein extracts; OA, osteoarthritic human articular chondrocytes.

Figure 4

Effect of anti-CD47/IAP antibody on chondrocyte responses to mechanical stimulation. (a) Cell lysates from normal human articular chondrocytes at rest or mechanically stimulated at 0.33 Hz for 1 minute in the presence of either non-immune mouse immunoglobulin (Ig) or the function-blocking CD47/IAP antibody Bric 126 were immunoprecipitated with anti-phosphotyrosine and immunoblotted with mouse monoclonal antibody anti-phosphotyrosine-horseradish peroxidase, PY-20, at 1:1,000. Identical amounts of whole-cell lysates were used for immunoprecipitation of phosphotyrosinated proteins from stimulated and unstimulated cells. Tyrosine phosphorylation of a 125-kDa protein is increased after 1 minute of mechanical stimulation but this response is diminished by the presence of the anti-CD47/IAP antibody Bric 126. (b) Following 0.33-Hz mechanical stimulation for 20 minutes in the presence of control non-immune mouse Ig or anti-CD47/IAP antibody Bric 126, relative levels of aggrecan mRNA were assessed in cells immediately and 1 and 3 hours after incubation at 37°C. Consistent with previous results [7], mechanical stimulation of normal articular chondrocytes in the presence of non-immune mouse Ig results in an increase in relative levels of aggrecan mRNA. This response is inhibited by the presence of the anti-CD47/IAP antibody Bric 126. *p < 0.05, 1 hour after mechanical stimulation versus immediate post stimulation. **p < 0.05, 1 hour after stimulation in the presence of Bric 126 versus 1 hour after stimulation in the presence of non-immune mouse Ig. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IAP, integrin-associated protein; MS, mechanical stimulation.