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Logo of annrheumdAnnals of the Rheumatic DiseasesCurrent TOCInstructions for authors
Ann Rheum Dis. Jun 2004; 63(6): 636–643.
PMCID: PMC1755017

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-ß1 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.

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Figures and Tables

Figure 1
Representative section of cartilage, showing immunostaining for galectin-3 from normal (A–C) and OA cartilage (D–F). (A and D) Non-immune serum; (B, C, E, and F) polyclonal anti-galectin-3. Original magnification x25; (G) galectin-3 levels ...
Figure 2
Expression of galectin-3 mRNA in human normal and OA cartilage was studied using real time RT-PCR as described in "Materials and methods". The PCR analysis was performed by normalising the PCR products of the galectin-3 to the 18S PCR products. Bars ...
Figure 3
Subcellular OA chondrocyte fractionation was performed as described in "Materials and methods". Proteins were quantified for each fraction, 10 µg were blotted, and immunodetection was performed with either a polyclonal anti-galectin-3 or a monoclonal ...
Figure 4
Immunofluorescence and flow cytometry detection of galectin-3 in OA chondrocytes. Cells were incubated with a specific anti-galectin-3 polyclonal antibody (B, D) or non-immune-serum (A, C) and detected with a fluorescent conjugated secondary antibody. ...
Figure 5
Correlation of galectin-3 presence with integrin-ß1 at the chondrocyte surface. Cell surface fluorescence was measured with either galectin-3 serum or integrin-ß1 antibody on non-permeabilised cells, as explained in "Materials and methods". ...
Figure 6
Proteolytic activity of collagenase-3 (MMP-13) on galectin-3. rh-galectin-3 was incubated in the presence of activated MMPs as described in "Materials and methods". As galectin-3 at high concentration could multimerise,26 two galectin-3 concentrations ...
Figure 7
Schematic representation of galectin-3 sites generated by collagenase-3.

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