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. 2010 Aug;62(8):2370-81.
doi: 10.1002/art.27512.

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

Rosa Maria Borzí  1 , Eleonora Olivotto, Stefania Pagani, Roberta Vitellozzi, Simona Neri, Michela Battistelli, Elisabetta Falcieri, Annalisa Facchini, Flavio Flamigni, Marianna Penzo, Daniela Platano, Spartaco Santi, Andrea Facchini, Kenneth B Marcu
Affiliations

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

Rosa Maria Borzí et al. Arthritis Rheum. 2010 Aug.

Abstract

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.

Conclusion: MMP-13 loss leads to a breakdown in primary human articular chondrocyte differentiation by altering the expression of multiple regulatory factors.

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Figures

Figure 1
Figure 1. Stable knock-down (KD) of MMP-13 expression by retroviral transduction
A. Real time PCR verification of MMP-13 KD by shOligos targeted to different MMP-13 exons. The loss of MMP-13 expression (mean percentage KD±SEM) were as follows: OE7 (3 patients) 91%±3.8; OE16 (6 patients): 83.1%±10,5. B. One representative experiment out of three different patients’chondrocytes tested in triplicate wells, where KD efficiencies were quantified by MMP-13 ELISA assay of cell supernatants in unstimulated or stimulated conditions with 100 nM chemokines (BCA, GROα, SDF) or 100 units/ml IL-1β for 72 hours. C. 3 week micromasses were left unstimulated or stimulated with 100 nM chemokines (BCA, SDF, Larc, GROα) or 100 units/ml IL-1β for 72 hours. MMP-13 KD statistically decreased MMP-13 release from IL-1 stimulated OE16 micromasses (p=0.046). Results were obtained from duplicate micromass cultures of 6 different patients.
Figure 2
Figure 2. Effects of MMP-13 ablation on ECM remodeling, chondrocyte terminal differentiation and mineralization
A. Col2 IHC analysis across a micromass maturation time course of 1–3 weeks (bar = 50 μm); B. Left side: ELISA assays for release of type II collagen C1,2C neo-epitopes into 3 week micromass supernatants in response to MMP-13 inductive stimuli. Results were obtained from two different patients (mean+/−SEM); B. Right side: IHC staining and western blotting of C1,2C neo-epitopes in control vs. MMP-13 KD micromasses (1 w); C. Left side: Example of toluidine blue staining for GAG/proteoglycan in control vs. MMP-13 KD (OE16 and OE7) micromasses (3 w) (bar = 50 μm); C. Right side: Cumulative data from 5 different patients of quantitative DMMB assay for sulphated GAG accumulation in controls vs. MMP-13 KD micromasses (3 w). MMP-13 KD micromasses had statistically higher GAG (μg) content compared to control micromasses (p= 0.012). D. Alizarin red staining for calcium deposition in control vs. MMP-13 KD (OE16 and OE7) micromasses (3 w).
Figure 3
Figure 3. Effects of MMP-13 loss on VEGF and multiple transcriptional regulators of chondrocyte diffentiation in maturing (1–3 week) micromasses
A. Left side: VEGF IHCs of GL2 control vs. MMP13-KD micromass cultures (bar = 50 μm). A. Right side: levels (mean±SEM) of VEGF mRNA, relative to matched GL2 control mRNA, in high density monolayer (5 patients, with p=0.043) and 1-week micromasses (4 patients, empty boxes); VEGF western blot of high density monolayer and micromasses (3 w); B. Left side: Sox9 IHC of GL2 and MMP-13 KD micromass (bar = 50 μm). B. Right side: levels (mean±SEM) of Sox9 mRNA, relative to matched GL2 control mRNA, in high density monolayer (6 patients, significant difference at p=0.028) and 1-week micromasses (4 patients); Sox-9 western blots of control and MMP-13 KD high density monolayer and micromasses (1 w); C. Left side: Runx2 IHC of GL2 and MMP13-KD micromasses (bar = 50μM). C. Right side: levels (mean±SEM) of Runx2 mRNA, in MMP-13 KD samples relative to matched GL2 controls, in high density monolayer (5 patients, significant difference at p=0.043) and 1-week micromasses (2 patients); Runx2 immunoblots of control and MMP-13 KD high density monolayer and micromasses at 3 weeks.
Figure 4
Figure 4. Effects of MMP-13 ablation on β catenin expression and activation, hypertrophy and cell viability
A. (Left): β-catenin IHCs of GL2 and MMP-13 in differentiating micromasses (bar = 50 μm); A (Right): β-catenin mRNA levels (mean±SEM) in MMP-13 KD vs. matched GL2 controls, in high density monolayer (5 patients, p=0.043) and 1 week (w) micromasses (5 patients, p=0.043). Immunoblots of β-catenin in control vs. MMP-13 KD high density monolayer and micromasses (2w); B (Left): Activated β-catenin in GL2, MMP-13 KD and IKKα KD 3w micromasses (bar =50 μm). Higher magnification GL2 images (bar=12.5 μm) reveal nuclear activated β-catenin and hypertrophic chondrocyte morphology (larger size and lower nuclear/cytoplasmic index); B (Right): Immunoblots of active β-catenin in GL2 control vs. MMP-13 KD and IKKα KD micromasses (3w); C (Left): Collagen X IHC (3w, bar = 18.7 μm), TUNEL fluorescence (2w) and cell morphology by TEM (3w, bar = 2 μm) in GL2 vs. MMP-13 KD micromasses; C (Right): TEM analysis of nuclear morphology and integrity in MMP-13 KD, IKKα KD and IKKβ KD micromasses (5 patients each). MMP-13 KD significantly increases chondrocyte viability over GL2 control micromasses (p= 0.043).
Figure 5
Figure 5. Sox9 subcellular localization in micromasses with and without MMP-13 and IKKα expression and in cartilage tissues with scant vs. more intact ECM
Confocal microscopy analysis of Sox9 subcellular distribution in 1 week micromasses: Sybr Green nuclear staining, Sox9 staining, and merge. Areas identified by the white square are shown at higher magnification on the right (original images were acquired with a 40× objective in the left panels, and with a 60× in the right panels). Upper row: GL2 control micromasses; middle row: MMP-13 KD micromasses; lower row: IKKα KD micromasses. B and C: Confocal analysis of Sox9 subcellular distribution in the middle zone chondrocytes of degraded cartilage (B) and conserved cartilage (C) (40× and 60× magnifications are shown). The areas of cartilage used for confocal acquisition of scant or conserved cartilage are shown in panels b1 and c1 (sybr green staining only), respectively. Panels b2 and c2 show safranin-O staining in consecutive sections of the same samples used for confocal analysis.
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