Matrix metalloproteinase 13 loss associated with impaired extracellular matrix remodeling disrupts chondrocyte differentiation by concerted effects on multiple regulatory factors.

Purpose—To link MMP-13 activity and ECM remodeling to alterations in regulatory factors leading to a disruption in chondrocyte homeostasis. Methods—Matrix-metalloproteinase-13 (MMP-13) expression was ablated in primary human chondrocytes by stable retrotransduction of short-hairpin RNAs. The effects of MMP-13 KD on key regulators of chondrocyte differentiation (Sox9, Runx2 and β-catenin), and angiogenesis (VEGF) were scored at the protein (immunohistochemistry or western blot) and RNA (real time PCR) levels in high density monolayer and micromass cultures under mineralizing conditions. Effects on cellular viability in conjunction with chondrocyte progression towards a hypertrophiclike state were assessed in micromass cultures. Alterations in Sox9 subcellular distribution were assessed by confocal microscopy in micromass cultures and also in OA cartilage. Results—Differentiation of control chondrocyte micromasses progressed up to a terminal phase, with calcium deposition in conjunction with reduced cell viability and scant ECM. MMP-13 knock-down (KD) impaired ECM remodeling and suppressed differentiation in conjunction with reduced levels of Runx2, β-catenin and VEGF. MMP-13 levels in vitro and ECM remodeling in vitro and in vivo were linked to changes in Sox9 sub-cellular localization. Sox9 was largely excluded from the nuclei of chondrocytes with MMP-13 remodeled or degraded ECM, and exhibited an intranuclear staining pattern in chondrocytes with impaired MMP-13 activity in vitro or with more intact ECM in vivo. Conclusions—MMP-13 loss leads to a break-down in primary human articular chondrocyte differentiation by altering the expression of multiple regulatory factors.