A proteomic approach for identification and localization of the pericellular components of chondrocytes

Abstract

Although the pericellular matrix (PCM) plays a central role in the communication between chondrocytes and extracellular matrix, its composition is largely unknown. In this study, the PCM was investigated with a proteomic approach using chondrons, which are enzymatically isolated constructs including the chondrocyte and its surrounding PCM. Chondrons and chondrocytes alone were isolated from human articular cartilage. Proteins extracted from chondrons and chondrocytes were used for two-dimensional electrophoresis. Protein spots were quantitatively compared between chondron and chondrocyte gels. Cellular proteins, which had similar density between chondron and chondrocyte gels, did not proceed for analysis. Since chondrons only differ from chondrocytes in association of the PCM, protein spots in the chondron gels that had higher quantity than that in the chondrocyte gels were selected as candidates of the PCM components and processed for mass spectrometry. Among 15 identified peptides, several were fragments of the three type VI collagen chains (α-1, α-2, and α-3). Other identified PCM proteins included triosephosphate isomerase, transforming growth factor-β induced protein, peroxiredoxin-4, ADAM (A disintegrin and metalloproteinases) 28, and latent-transforming growth factor beta-binding protein-2. These PCM components were verified with immunohisto(cyto)chemistry for localization in the PCM region of articular cartilage. The abundance of type VI collagen in the PCM emphasizes its importance to the microenvironment of chondrocytes. Several proteins were localized in the PCM of chondrocytes for the first time and that warrants further investigation for their functions in cartilage biology.

Roles of type VI collagen and decorin in human mesenchymal stem cell biophysics during chondrogenic differentiation

“…DCN is a ubiquitous proteoglycan, consisting of a core protein with a single chondroitin sulphate (Li et al, 2010) or dermatan sulphate (Knudson et al, 1996) side chain. It interacts with multiple collagens to create functional bridges between the PCM and surrounding ECM, and is involved in controlling fibrillogenesis and growth factor bioavailability (Chen et al, 2009;Lewis et al, 2010;Zhang et al, 2011). This study shows that these two specific proteins, ColVI and DCN, have profound influence over PCM composition and biomechanical behaviour surrounding hMSCs undergoing chondrogenesis.…”

“…Our data are in agreement with chondrogenic stage specific gene expression (Xu et al, 2008), with col6a1 being significantly greater in the first seven days of chondrogenesis in non-infected and GFP-transduced cells, which decreases in expression by 28 d of culture. DCN is involved in the sequestration of TGF-β3, FGF-2, TNF-α, PDGF, and IGF-1 (Ferdous et al, 2007;Chen et al, 2009;Zhang et al, 2011) and has been associated with cell proliferation signalling (Chen et al, 2009), causing the cell to cease proliferation and enhance matrix synthesis.…”

“…During chondrogenesis, hMSCs develop a pericellular matrix (PCM) that is rich in type VI collagen (ColVI) (Poole et al, 1992;Wiberg et al, 2003;Zhang et al, 2011), fibronectin (Wiberg et al, 2003;DiMicco et al, 2007;Connelly et al, 2008), hyaluronan (Knight et al, 1998;Zhang et al, 2011), and proteoglycans (PGs) such as aggrecan, decorin (DCN), and biglycan (BGN) (Wiberg et al, 2003;Connelly et al, 2008). This thin 2-6 µm layer of matrix acts as both a biomechanical buffer (Knight et al, 1998;Lee et al, 2010), controlling the amount of deformation applied to the cell (Knight et al, 2001;Choi et al, 2007;Vigfusdottir et al, 2010), as well as a biochemical conductor, presenting the cell with growth factors and signalling molecules (Chen et al, 2009;Vigfusdottir et al, 2010;Zhang et al, 2011). The PCM is believed to modulate the mechanoresponsiveness of chondrocytes and chondrogenic hMSCs, but the exact roles of the individual PCM proteins has yet to be determined.…”

Chondrocyte responses to neurovascular peptides, cytokines, and a 3D environment: focus on ADAMs

“…23 More recently, a proteomic approach found ADAM28 in the pericellular matrix of chondrocytes in articular cartilage. 50 Since ADAM28 can degrade IGFBP-3, 51 it is possible that this proteinase plays a role in chondrocyte proliferation, as has been demonstrated for breast cancer cells. 52 Another function of ADAM28 appears to be in the shedding of TNFα from the cell surface, demonstrated in monocytes and which may be of relevance in arthritic conditions.…”

“…We were particularly interested to explore further the regulation of ADAM28 since this proteinase has recently been found in the pericellular matrix of chondrocytes. 50 We pursued its expression alongside that of IL6, since this cytokine was upregulated by several stimuli. We exposed cells in 3D culture to cytokines or neuropeptides both alone and in combination with cytokines under normoxic and hypoxic conditions.…”