Periostin interaction with discoidin domain receptor-1 (DDR1) promotes cartilage degeneration

Figures

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  Fig 1. Elevated expression of Postn but not DDR1 in OA cartilage. A) Gene array analysis. Total RNA was isolated from six pools of normal and OA cartilage. For each pool, 5–10 individual cartilage samples (1 g each) were pooled. RNA was hybridized against human U133A arrays. After normalization, DDR1 expression was expressed in arbitrary units. B) qPCR analysis of DDR1 mRNA in 10 normal and 12 OA cartilage specimens from human knee joints. Mean (SD) are shown. The non-parametric Mann-Whitney test determined statistical significance. C) Immunohistochemical analysis of lesional (damaged) and non-lesional (preserved) OA knee cartilage shows cell- and ECM-associated Postn in lesional areas, whereas DDR1 is cell-associated. OA knee cartilage from a total knee replacement was sectioned and stained for Postn and DDR1 with Vectastain reagents (Vector Laboratories, Burlingame, CA, USA).

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  Fig 2. Co-immunoprecipitation of DDR1-Postn complexes in human chondrocytes. A: Western blotting analysis of immunoprecipitates from primary human OA chondrocytes. No antigens are seen in the non-immunoprecipitated cell lysate (lysate) because their concentration is too low. B: Co-immunoprecipitation of DDR1/Postn complexes in human chondrocyte C28/I2 cells. A representative Western blot of immunoprecipitates from chondrocytes is shown. Purified DDR1 and DDR2 proteins were loaded as a positive control.

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  Fig 3. The DDR1 inhibitor 7rh inhibits the Adenovirus-mediated induction of MMP-13 expression of human Postn (Ad-hPostn) in human OA chondrocytes. Human OA chondrocyte cultures were pre-incubated in the presence or absence of DDR1 inhibitor (7rh; 100–1000 nM) for 16 h before Ad-hPostn or AdCon virus (MOI = 100) was added to the cultures. Total RNA was isolated 24 h post-transduction to measure MMP-13, and GAPDH expression (A). The supernatant was used to determine MMP -13 levels by ELISA (. Mean ± SD of triplicate samples are shown. 1-way ANOVA determined significance with the post-Bonferroni multiple comparison test. P <0.05 was considered significant. DDR1 and wnt signaling inhibition reduced MMP-13 expression.

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  Fig 4. DDR1 kinase mediates Postn activation of AKT and β-catenin signaling in human chondrocytes: A: Postn induces DDR1 but not DDR2 phosphorylation. Upper panel: Western blotting analysis of phosphorylated (p) and total (t) DDR1 and DDR2 in human chondrocytes treated with Postn (5 μg/ml) for the indicated times. Total DDR1 and DDR2, are shown as loading controls. A representative Western blot is shown. Lower panel: densitometric analysis of two similar Western blots. The columns represent the densitometric readings of the indicated bands normalized to those of the corresponding loading controls. B: Postn activates AKT and β-catenin signaling. Upper panel: Western blotting analysis of AKT and β-catenin activation in human articular chondrocytes treated with Postn (5 μg/ml) for the indicated times. A representative Western blot of two independent primary human chondrocyte cultures is shown. Lower panel: densitometric analysis of two similar Western blots. The columns represent the densitometric readings of the indicated bands normalized to those of the corresponding loading controls. C: Inhibition of DDR1 kinase blocks Postn induction of AKT and β-catenin signaling. Western blotting…

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  Fig 5. DDR1 mediates Postn induction of MMP-13 expression in mouse chondrocytes. Mouse chondrocytes were isolated from three wt and Ddr1 deficient mice, as described in Materials and Methods. Cells were grown in 12-well plates at a density of 5x104 cells/well. Chondrocytes were transfected with human DDR1 (hDDR1) and also transduced with AdCon or Ad-mPostn in triplicate. Cells were lysed 24 h post- adenovirus transduction in TRIzol for RNA isolation, and the supernatant was collected for MMP-13 activity as described in methods. RNA expression was normalized to GAPDH and expressed as relative fold-change to wt non-transfected and -transduced cells. Mean ± SD of triplicate samples are shown. *: p<0.05 by 1-way ANOVA; vs. wt hDDR1 transfected or AdCon-transduced cells.