ADAMTS5-mediated aggrecanolysis in murine epiphyseal chondrocyte cultures.

摘要

OBJECTIVE: Aggrecan degradation by aggrecanases [a disintegrin and metalloproteinase with thrombospondin-like motifs (ADAMTS) 1, 4, 5, 8, 9, 15] is considered to initiate much of the cartilage pathology seen in human arthritis, however, the proteinase responsible and its mode of control is unclear. The present work was done to examine mechanisms of aggrecanase control in a novel murine epiphyseal cell system and to determine whether ADAMTS5 alone is responsible for aggrecanolysis by these cells. METHODS: Epiphyseal cells from 4-day-old mice (wild type, TS-5 (-/-), CD44(-/-), syndecan-1(-/-), membrane type-4 matrix metalloproteinase [MT4MMP(-/-)]) were maintained in non-adherent aggregate cultures and aggrecanolysis studied by biochemical and histochemical methods. Confocal immunolocalization analyses were done with specific probes for ADAMTS5, hyaluronan (HA) and aggrecanase-generated fragments of aggrecan. RESULTS: Aggrecanolysis by these cells was specifically aggrecanase-mediated and it occurred spontaneously without the need for addition of catabolic stimulators. Chondrocytes from ADAMTS5-null mice were aggrecanase-inactive whereas all other mutant cells behaved as wild type in this regard suggesting that ADAMTS5 activity is not controlled by CD44, syndecan-1 or MT4MMP in this system. Immunohistochemical analysis supported the central role for ADAMTS5 in the degradative pathway and indicated that aggrecanolysis occurs primarily in the HA-poor pericellular region in these cultures. CONCLUSION: These findings are consistent with published in vivo studies showing that single-gene ADAMTS5 ablation confers significant protection on cartilage in murine arthritis. We propose that this culture system and the analytical approaches described provide a valuable framework to further delineate the expression, activity and control of ADAMTS-mediated aggrecanolysis in human arthritis.