CD47 associates with alpha 5 integrin and regulates responses of human articular chondrocytes to mechanical stimulation in an in vitromodelReportar como inadecuado




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Arthritis Research and Therapy

, 10:R4

First Online: 10 January 2008Received: 26 June 2007Revised: 02 December 2007Accepted: 10 January 2008

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.

AbbreviationsBSA= bovine serum albumin

COMP= cartilage oligomeric matrix protein

GAPDH= glyceraldehyde-3-phosphate dehydrogenase

HRP= horseradish peroxidase

IAP= integrin-associated protein

IgV= immunoglobulin variable

OA= osteoarthritis

PCR= polymerase chain reaction

RGD= arginine-glycine-aspartic acid

SIRPα= signal-regulatory protein-alpha

TBST= Tris-buffered saline Tween-20

TSP= thrombospondin.

Electronic supplementary materialThe online version of this article doi:10.1186-ar2350 contains supplementary material, which is available to authorized users.

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Autor: Mahmoud Orazizadeh - Herng Sheng Lee - Bianca Groenendijk - S Jane Millward Sadler - Malcolm O Wright - Frederik P Lind

Fuente: https://link.springer.com/







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