Galectin-3 surface expression on human adult chondrocytes: a potential substrate for collagenase-3

Abstract

Background: Galectin-3 is a lectin detected in mature and early hypertrophic chondrocytes; osteoarthritic (OA) chondrocytes can re-express hypertrophic markers. Objective: To investigate the synthesis and subcellular localisation of galectin-3 in adult chondrocytes as well as the possibility of cleavage of galectin-3 by collagenase-1 and -3. Methods: Galectin-3 was assessed by immunohistochemistry and real time polymerase chain reaction (PCR) in normal and OA cartilage. Its localisation was investigated by subcellular fractionation, immunocytology, and flow cytometry. Proteolysis of galectin-3 by collagenase-1 and -3 was determined by in vitro assay. Results: Galectin-3 expression was increased 2.4-fold as measured by reverse transcriptase (RT)-PCR (p,0.05, n = 5) and threefold by immunohistochemistry (p,0.003 n = 6) in OA cartilage compared with normal cartilage. In adult chondrocytes, galectin-3 was found in the cytosol and membrane enriched fractions. Both immunocytology and flow cytometry confirmed the presence of galectin-3 at the surface of chondrocytes. A strong correlation was found between integrin-b1 and galectin-3 expression at the surface of chondrocytes. Moreover, collagenase-3 cleaved galectin-3 with a higher activity than collagenase-1. The proteolysed sites generated were identical to those produced by gelatinases A and B. Conclusion: Galectin-3 may play a part in OA, having two roles, one intracellular and not yet identified, and another at the cell surface, possibly related to the interaction of chondrocytes and the cartilage matrix.

Estrogen receptor–related receptor α regulation by interleukin‐1β in prostaglandin E2– and cAMP‐dependent pathways in osteoarthritic chondrocytes

“…Chondrocytes were released from the articular cartilage by sequential enzymatic digestion at 37°C, as previously described (21,22). OA chondrocytes obtained at first passage were seeded at 1 ϫ 10 5 cells/cm 2 in 24-or 12-well plates in Dulbecco's modified Eagle's medium (DMEM; Invitrogen) containing 10% fetal bovine serum (FBS; Invitrogen) and an antibiotic mixture (100 units/ml of penicillin, 100 g/ml of streptomycin; Invitrogen) and were cultured for 48 hours at 37°C in a humidified atmosphere of 5% CO 2 /95% air.…”

“…Cell proteins were extracted in 0.5% sodium dodecyl sulfate (SDS), separated in 4-12% SDSpolyacrylamide gel electrophoresis gels, then transferred to nitrocellulose membranes (Bio-Rad) according to established methods (23). Immunodetection was performed as previously described (21), with a rabbit monoclonal antibody against ERR␣ (Epitomics) at a dilution of 1:1,000 and the secondary antibody (horseradish peroxidase [HRP]-conjugated goat antirabbit IgG) at a dilution of 1:20,000 (Pierce). For evaluating protein loading, a rabbit polyclonal antibody against GAPDH (Sigma-Aldrich) was used at a dilution of 1:20,000; secondary antibody was used at a dilution of 1:20,000 (Pierce).…”

ADAMTS-5: The story so far

“…Adenovirus infection of mouse synovium with connective tissue growth factor induces ADAMTS-5 expression and synovial fibrosis (Blaney Davidson et al, 2006). Galectin-3, a soluble lectin increased in human OA cartilage (Guevremont et al, 2004), weakly stimulates ADAMTS-5 expression in human OA chondrocytes (Janelle-Montcalm et al, 2007). Conversely, nutraceuticals such as glucosamine and chondroitin sulphate at biologically relevant concentrations decrease IL-1-induced ADAMTS-5 expression in bovine cartilage explants (Chan et al, 2006).…”

Galectins: Structures, Binding Properties and Function in Cell Adhesion

“…Moreover phosphorylation of Ser6 seems to regulate affinity for different ligands and thereby cellular activity of galectin-3 (Dumic et al, 2006;Mazurek et al, 2000;Szabo et al, 2009;Yoshii et al, 2002). Galectin-3 can be cleaved by different proteases such as metalloproteinases-2 and -9 (gelatinases A and B respectively), metalloproteinase-13 (collagenase-3) and with low activity metalloproteinase-1 (collagenase-1) separating the fulllength CRD from the N-terminal extension (Guévremont et al, 2004;Ochieng et al, 1994). The main cleavage position is located between Ala62 and Tyr63 while other cleaving sites are only recognised by some specific proteases to lesser extend (Dumic et al, 2006;Guévremont et al, 2004;Ochieng et al, 1994).…”

“…Galectin-3 can be cleaved by different proteases such as metalloproteinases-2 and -9 (gelatinases A and B respectively), metalloproteinase-13 (collagenase-3) and with low activity metalloproteinase-1 (collagenase-1) separating the fulllength CRD from the N-terminal extension (Guévremont et al, 2004;Ochieng et al, 1994). The main cleavage position is located between Ala62 and Tyr63 while other cleaving sites are only recognised by some specific proteases to lesser extend (Dumic et al, 2006;Guévremont et al, 2004;Ochieng et al, 1994). The single CRD is mainly described to have an increased affinity for different carbohydrates such as N-acetyllactosamine, the glycoprotein asialofetuin or glycans presented on endothelial cells but to have less biological activity as it looses the ability to form oligomers.…”