The Expression of Thrombospondin-4 Correlates with Disease Severity in Osteoarthritic Knee Cartilage

Abstract

Osteoarthritis (OA) is a progressive joint disease characterized by a continuous degradation of the cartilage extracellular matrix (ECM). The expression of the extracellular glycoprotein thrombospondin-4 (TSP-4) is known to be increased in injured tissues and involved in matrix remodeling, but its role in articular cartilage and, in particular, in OA remains elusive. In the present study, we analyzed the expression and localization of TSP-4 in healthy and OA knee cartilage by reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry, and immunoblot. We found that TSP-4 protein expression is increased in OA and that expression levels correlate with OA severity. TSP-4 was not regulated at the transcriptional level but we detected changes in the anchorage of TSP-4 in the altered ECM using sequential protein extraction. We were also able to detect pentameric and fragmented TSP-4 in the serum of both healthy controls and OA patients. Here, the total protein amount was not significantly different but we identified specific degradation products that were more abundant in sera of OA patients. Future studies will reveal if these fragments have the potential to serve as OA-specific biomarkers.

Osteoarthritis and Cartilage Regeneration: Focus on Pathophysiology and Molecular Mechanisms

“…The thrombospondin (TSP) family of large ECM glycoproteins composed of five members (TSP1-TSP5), and among them, mutations in TSP-5 (or cartilage oligomeric protein, COMP) cause human pseudoachondroplasia and multiple epiphyseal dysplasia associated with early onset OA [26]. As the roles of other TSPs for articular cartilage homeostasis are less known, Maly and colleagues investigated the expression and localization of TSP-4 in healthy and osteoarthritic human knee articular cartilage [27]. Immunohistochemistry and immunoblotting revealed that TSP-4 is present at very low level in normal articular cartilage but its ECM deposition dramatically increases in OA tissues correlating well with OA severity.…”

COMP and TSP-4: Functional Roles in Articular Cartilage and Relevance in Osteoarthritis

“…Osteochondral cylinders were generated and processed as described before [39]. Samples were fixed in 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) in PBS, pH = 7.4, overnight at 4 • C. After decalcification in 10% ethylenediaminetetraacetic acid (EDTA; VWR, Osterode am Harz, Germany), samples were embedded in paraffin.…”

“…Proteins from human cartilage samples were extracted as described previously [39]. Briefly, cartilage from areas showing different severity grades were scraped off and cut into pieces (1-3 mm 3 ), and proteins were extracted with an extraction buffer (4 M guanidine hydrochloride, 50 mM Tris, 10 mM EDTA (pH = 7.4)) overnight at 4 • C. After precipitating the proteins with 96% ethanol for 24 h at -20 • C, the protein pellet was washed and resuspended in Laemmli buffer (250 mM Tris-HCl, pH 6.8, 40% glycerol, 0.04% bromophenol blue, 8% SDS).…”

“…The ECM protein COMP is a target of degradation in early OA, but is also re-expressed in later stages [ 15 ]. In contrast, its close family member, thrombospondin-4 (TSP-4), is hardly detectable in healthy cartilage, but its expression increases dramatically in OA cartilage and correlates with disease severity [ 39 ]. Both proteins have similar structures and binding partners [ 40 , 41 , 42 , 43 , 44 , 45 , 46 ], suggesting similar or additive roles in articular cartilage.…”

“…It has also been reported that COMP binds TGF-β1, thereby promoting its transcriptional capacity [ 47 ], while this has not yet been shown for TSP-4. The re-expression of COMP and the new synthesis of TSP-4 in OA was interpreted as an attempt to restore the integrity of the cartilage ECM [ 15 , 39 ]. However, the effect of these ECM proteins on the chondrocyte phenotype and behavior, as well as their contribution to OA relevant processes and ECM repair, have not yet been investigated in detail.…”