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

Abstract

Background: 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. 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. Results: Amongst 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. Conclusions: Our 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.

Ex Vivo Equine Cartilage Explant Osteoarthritis Model: A Metabolomics and Proteomics Study

“… 17 − 19 TNF-α and IL-1β have therefore become established experimental treatments for modeling OA pathology within in vitro and ex vivo studies, having been used both independently and as a combined treatment. 20 − 28 …”

Physiological Effects of the Electrogenic Current Generated by the Na+/K+Pump in Mammalian Articular Chondrocytes

“…[43][44][45] Very recent proteomic studies have taken an agnostic and unbiased molecular discovery approach to exploring the ''surfaceome'' of chondrocytes, confirming the presence of multiple Na + /K + pump isoforms in these cells. 24 These findings considered in the context of the extensive literature on Na + /K + pump cell physiology 46,47 make it very likely that in the chondrocyte the classical ''coupled stoichiometry'' that is characteristic of this enzyme is 3 Na + pumped out of the cell, coupled with 2 K + pumped into the chondrocyte cytoplasm (Fig. 1A).…”

“…17,18 Within the last decade, a number of different ion selective channels and exchangers have been identified and shown to play essential roles in chondrocyte physiology. 16,[19][20][21][22][23][24] Any such ion channel being functional in chondrocytes may seem somewhat surprising to researchers working on excitable cells, since the chondrocyte is considered to be a nonexcitable cell. However, despite chondrocytes being nonexcitable cells, their dynamic membrane potential serves many cellular roles, 25 is metabolically active, 16 especially during hypertrophy, 26 and is capable of responses to commonly used drugs for treating hypertension.…”

Physiological Effects of the Electrogenic Current Generated by the Na /K Pump in Mammalian Articular Chondrocytes

“…Within the last decade, a number of different ion selective channels and exchangers have been identi ed and shown to play essential roles in chondrocyte physiology 16,[19][20][21][22][23][24] . Any such ion channel being functional in chondrocytes may seem somewhat surprising to researchers working on excitable cells, since the chondrocyte is considered to be a non-excitable cell.…”

“…This dataset, based on demonstration of relatively high a nity, saturable binding sites for ouabain (a classical sodium potassium pump antagonist); combined with more recent molecular studies that have identi ed transcripts and proteins of each of the known subunits (a, b and g) of this integral membrane protein 42 , strongly suggest that in the mammalian chondrocyte the electrochemical gradients for sodium and potassium are established and maintained by this active or ATP-requiring pump mechanism [43][44][45] . Very recent proteomic studies have taken an agnostic and unbiased molecular discovery approach to exploring the "surfaceome" of chondrocytes, con rming the presence of multiple Na + /K + pump isoforms in these cells 24 . These ndings considered in the context of the extensive literature on Na + /K + pump cell physiology 46,47 make it very likely that in the chondrocyte the classical 'coupled stoichiometry' that is characteristic of this enzyme is 3 Na + pumped out of the cell, coupled with 2 K + pumped into the chondrocyte cytoplasm ( Figure 1A).…”