Matrix metalloproteinase 13 loss associated with impaired extracellular matrix remodeling disrupts chondrocyte differentiation by concerted effects on multiple regulatory factors.
OBJECTIVE
To link matrix metalloproteinase 13 (MMP-13) activity and extracellular matrix (ECM) remodeling to alterations in regulatory factors leading to a disruption in chondrocyte homeostasis.


METHODS
MMP-13 expression was ablated in primary human chondrocytes by stable retrotransduction of short hairpin RNA. The effects of MMP-13 knockdown on key regulators of chondrocyte differentiation (SOX9, runt-related transcription factor 2 [RUNX-2], and beta-catenin) and angiogenesis (vascular endothelial growth factor [VEGF]) were scored at the protein level (by immunohistochemical or Western blot analysis) and RNA level (by real-time polymerase chain reaction) in high-density monolayer and micromass cultures under mineralizing conditions. Effects on cellular viability in conjunction with chondrocyte progression toward a hypertrophic-like state were assessed in micromass cultures. Alterations in SOX9 subcellular distribution were assessed using confocal microscopy in micromass cultures and also in osteoarthritic 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 knockdown impaired ECM remodeling and suppressed differentiation in conjunction with reduced levels of RUNX-2, beta-catenin, and VEGF. MMP-13 levels in vitro and ECM remodeling in vitro and in vivo were linked to changes in SOX9 subcellular 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 breakdown in primary human articular chondrocyte differentiation by altering the expression of multiple regulatory factors.
Borzí
Rosa Maria
RM
Istituti Ortopedici Rizzoli, Bologna, Italy. rosamaria.borzi@ior.it
Olivotto
Eleonora
E
Pagani
Stefania
S
Vitellozzi
Roberta
R
Neri
Simona
S
Battistelli
Michela
M
Falcieri
Elisabetta
E
Facchini
Annalisa
A
Flamigni
Flavio
F
Penzo
Marianna
M
Platano
Daniela
D
Santi
Spartaco
S
Facchini
Andrea
A
Marcu
Kenneth B
KB
D015153:Blotting, Western; D002358:Cartilage, Articular / Q000166:cytology / Q000378:metabolism*; D002454:Cell Differentiation / Q000502:physiology*; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology / Q000378:metabolism*; D020219:Chondrogenesis / Q000502:physiology; D024043:Collagen Type II / Q000378:metabolism; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics / Q000378:metabolism; D005109:Extracellular Matrix / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D053509:Matrix Metalloproteinase 13 / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D046529:Microscopy, Electron, Transmission; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055758:SOX9 Transcription Factor / Q000235:genetics / Q000378:metabolism; D018709:Statistics, Nonparametric; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism; D051176:beta Catenin / Q000235:genetics / Q000378:metabolism
D024043:Collagen Type II; D050659:Core Binding Factor Alpha 1 Subunit; C493709:RUNX2 protein, human; D055758:SOX9 Transcription Factor; C527932:SOX9 protein, human; D042461:Vascular Endothelial Growth Factor A; D051176:beta Catenin; D053509:Matrix Metalloproteinase 13