Agrin expression is downregulated in OA and is required for chondrocyte differentiation

Abstract

Purpose: Background: Agrin is a large basement membrane heparan sulphate proteoglycan best known for its function at the neuromuscular junction. It is responsible for synapse formation via binding to the MuSK and LRP4 receptor complex, leading to Acetylcholine receptor aggregation. Mice deficient of Agrin die perinatally as a result of respiratory failure. Agrin is also expressed in other cell types including chondrocytes of the developing growth plate, and agrin-deficient embryos in which agrin expression had been rescued at the neuromuscular junction develop skeletal abnormalities 1. Therefore it is possible that Agrin may play a role in cartilage homeostasis. Aims: The aim of this project is to determine if agrin plays a functional role in the homeostasis of the articular cartilage and in osteoarthritis. Methods: Human adult articular cartilage explants were obtained from preserved or damaged cartilage from individuals undergoing joint replacement for osteoarthritis. Experimental osteoarthritis was induced in 10 week old mice by destabilization of the medial meniscus2. Sham-operated controlateral knees were used as controls. Knees were collected 8weeks after surgery, decalcified and embedded in paraffin. Bovine primary articular chondrocytes (BPAC) were isolated by pronase/collagenase digestion from the articular cartilage of the metatarso-phalangeal joints of 18 month old calves from a local slaughterhouse. The BPAC and the human chondrogenic cell line C28/I2 were cultured in micromass to promote differentiation and extracellular matrix production. For gain- and loss-of-function experiments, chondrocytes were transfected with a mammalian expression vector encoding for full-length Agrin (FL-Agrin) or with Agrin siRNA's respectively. Accumulation of cartilage-specific extracellular matrix production, rich in highly sulphated glycosaminoglycans (GAGs), was quantified by alcian blue staining at pH 0.2 and alizarin red staining. Chondrogenic potential was measured using pellet analysis as previously described3, BPAC pellets were cultured for 14 days, weighed, sectioned and stained with alizarin red and safranin o. Gene expression analysis of Agrin, Col2A1, Aggrecan, Sox9 was performed by real time PCR. Immunofluorescence was used to determine the expression levels of Agrin at protein level in chondrocytes cultured in monoloayer and in paraffin-embedded tissue sections. Results: Agrin was expressed in healthy adult human and bovine articular cartilage. Agrin was signifcantly downregulated in human osteoarthritic cartilage. This downregulation was also replicated in experimental murine osteoarthritis, indicating that it is a consequence rather than a cause of osteoarthritis. Agrin expression was partially retained in the articular cartilage of sham operated knees, indicating that inflammation alone (present both in DMM and in sham) is not sufficient to induce Agrin downregulation, but cartilage damage is necessary. Endogenous expression of Agrin was confirmed in C28/I2 and in BPAC. Overexpression of FL-Agrin resulted in enhanced differentiation as demonstrated by upregulated the cartilage key transcription factor Sox9. . Knock-down of Agrin by siRNA resulted in reduced GAG production in C28/I2 and chondrocyte de-differentiation as documented by decreased expression Sox9, Col2A1 and Aggrecan mRNA. Conclusions: Agrin expressed in healthy adult articular cartilage and is downregulated in osteoarthritis; - Agrin is anabolic in cultured articular chondrocytes; - Agrin is required for chondrocyte differentiation in vitro.