Alterations in the chondrocyte surfaceome in response to pro-inflammatory cytokines

Julia R. Smith,Christopher R. Fellows,David J. Boocock,Susan Liddell,Ali Mobasheri,Csaba Matta,Willem E. van Spil,Floris Lafeber,Bernadette Jeremiasse

doi:10.1186/s12860-020-00288-9

Abstract

BackgroundChondrocytes are exposed to an inflammatory micro-environment in the extracellular matrix (ECM) of articular cartilage in joint diseases such as osteoarthritis (OA) and rheumatoid arthritis (RA). In OA, degenerative changes and low-grade inflammation within the joint transform the behaviour and metabolism of chondrocytes, disturb the balance between ECM synthesis and degradation, and alter the osmolality and ionic composition of the micro-environment. We hypothesize that chondrocytes adjust their physiology to the inflammatory microenvironment by modulating the expression of cell surface proteins, collectively referred to as the ‘surfaceome’. Therefore, the aim of this study was to characterize the surfaceome of primary equine chondrocytes isolated from healthy joints following exposure to the pro-inflammatory cytokines interleukin-1-beta (IL-1β) and tumour necrosis factor-alpha (TNF-α). We employed combined methodology that we recently developed for investigating the surfaceome in stem cells. Membrane proteins were isolated using an aminooxy-biotinylation technique and analysed by mass spectrometry using high throughput shotgun proteomics. Selected proteins were validated by western blotting.ResultsAmongst the 431 unique cell surface proteins identified, a high percentage of low-abundance proteins, such as ion channels, receptors and transporter molecules were detected. Data are available via ProteomeXchange with identifier PXD014773. A high number of proteins exhibited different expression patterns following chondrocyte stimulation with pro-inflammatory cytokines. Low density lipoprotein related protein 1 (LPR-1), thrombospondin-1 (TSP-1), voltage dependent anion channel (VDAC) 1–2 and annexin A1 were considered to be of special interest and were analysed further by western blotting.ConclusionsOur results provide, for the first time, a repository for proteomic data on differentially expressed low-abundance membrane proteins on the surface of chondrocytes in response to pro-inflammatory stimuli.

Highlights

Chondrocytes are exposed to an inflammatory micro-environment in the extracellular matrix (ECM) of articular cartilage in joint diseases such as osteoarthritis (OA) and rheumatoid arthritis (RA)
Validation of the in vitro inflammatory chondrocyte monolayer model by western blotting Western blotting using the secretome of control and cytokine-treated chondrocytes demonstrated a protein band around 53 kDa, corresponding to the predicted molecular mass of matrix metalloproteinase 1 (MMP-1) (Fig. 2a)
A substantial number of the cell surface proteins identified showed differential expression pattern following cytokine exposure. This is in agreement with published data suggesting that several genes encoding ion channels that are involved in the regulation of mechanotransduction, cell volume, resting membrane potential (RMP) and apoptosis are differentially expressed in OA chondrocytes [20]

Summary

Introduction

Chondrocytes are exposed to an inflammatory micro-environment in the extracellular matrix (ECM) of articular cartilage in joint diseases such as osteoarthritis (OA) and rheumatoid arthritis (RA). In OA, degenerative changes and low-grade inflammation within the joint transform the behaviour and metabolism of chondrocytes, disturb the balance between ECM synthesis and degradation, and alter the osmolality and ionic composition of the micro-environment. The aim of this study was to characterize the surfaceome of primary equine chondrocytes isolated from healthy joints following exposure to the pro-inflammatory cytokines interleukin-1-beta (IL-1β) and tumour necrosis factor-alpha (TNF-α). OA is characterized by articular cartilage loss, osteophyte development, subchondral bone changes, and synovial inflammation. The elevated concentration of these mediators during joint inflammation stimulate the gradual deterioration of cartilage, synovial membrane and subchondral bone [3]

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