Research ArticlePhysiology

A dual role for NOTCH signaling in joint cartilage maintenance and osteoarthritis

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Science Signaling  21 Jul 2015:
Vol. 8, Issue 386, pp. ra71
DOI: 10.1126/scisignal.aaa3792
  • Fig. 1 Sustained activation of NOTCH1 signaling in postnatal chondrocytes results in a progressive, OA-like pathology.

    (A and B) Wild-type (WT) mice and Col2a1Cre; tetO-NICD1; Rosa-rtTAf/+ mice (sGOF NICD1) were subjected to high-dose administration of DOX (100 μg/g), and then sGOF NICD1 and WT samples were analyzed at the indicated times by (A) immunofluorescence staining for NOTCH1 and (B) real-time qPCR assay to determine the relative abundances of NICD1 and Hes1 mRNAs. All mRNA abundances were normalized to that of the gene encoding β-actin (Actb) and then were normalized to the controls. Data are means ± SD of at least three independent experiments. *P < 0.05 by two-tailed Student’s t test. Images are representative of at least three independent experiments. Scale bars, 50 μm. DAPI, 4′,6-diamidino-2-phenylindole. (C) ABH/OG staining and immunohistochemical analysis of COL2A1, SOX9, COL10A1, COL3A1, and MMP13 in knee sections from 2-month-old WT and sGOF NICD1 mice. High magnification of a centralized region of articular cartilage is shown in the yellow boxes. White arrows indicate cell clusters. Scale bars, 50 μm. Data are representative of at least three independent experiments. (D) Histomorphometry analysis of articular cartilage thickness, area, and chondrocyte number was performed on knee sections from 2-month-old WT and sGOF NICD1 mice. All results were normalized to WT controls, which were set at 1. Data are means ± SD of at least three independent experiments. *P < 0.05 by two-tailed Student’s t test. (E) Real-time qPCR analysis was performed to compare the relative expression of the indicated genes in articular chondrocytes isolated from 2-month-old WT and sGOF NICD1 mice. All mRNA abundances were normalized to that of Actb mRNA and then were normalized to the controls. Data are means ± SD of at least three independent experiments. *P < 0.05 by two-tailed Student’s t test.

  • Fig. 2 Transient activation of NOTCH1 signaling in postnatal chondrocytes results in increased synthesis of cartilage ECM and joint maintenance.

    (A and B) WT mice and Col2a1Cre; tetO-NICD1; Rosa-rtTAf/+ mice (tGOF NICD1) were subjected to low-dose administration of DOX (1 μg/g), and then tGOF NICD1 and WT samples were analyzed at the indicated times by (A) immunofluorescence staining for NOTCH1 and (B) real-time qPCR assay to determine the relative abundances of NICD1 and Hes1 mRNAs. All mRNA abundances were normalized to that of Actb and then normalized to the controls. Data are means ± SD of at least three independent experiments. *P < 0.05 by two-tailed Student’s t test. Images are representative of at least three independent experiments. Scale bars, 50 μm. (C) ABH/OG staining and immunohistochemical analysis of COL2A1, SOX9, COL10A1, COL3A1, and MMP13 in knee sections of 2-month-old WT and tGOF NICD1 mice. High magnification of a centralized domain of articular cartilage is shown in the yellow boxes. Scale bars, 50 μm. Data are representative of at least three independent experiments. (D) Histomorphometry analysis of articular cartilage thickness, area, and chondrocyte number was performed on knee sections from 2-month-old WT and tGOF NICD1 mice. Data are means ± SD of at least three independent experiments. *P < 0.05 by two-tailed Student’s t test. (E) Real-time qPCR analysis was performed to compare the relative expression of the indicated genes in articular chondrocytes isolated from 2-month-old WT and sGOF NICD1 mice. All mRNA abundances were normalized to that of Actb mRNA and then were normalized to the controls. Data are means ± SD of at least three independent experiments. *P < 0.05 by two-tailed Student’s t test.

  • Fig. 3 Large-scale temporal gene expression profiling reveals potential NOTCH1 target genes responsible for cartilage fibrosis and degradation.

    P2 costal chondrocytes isolated from control and Col2a1Cre; tetO-NICD1f/+; Rosa-rtTAf/+ mice were treated with DOX (10 μg/ml) for 6 or 48 hours in culture, after which RNA was collected from the chondrocytes for RNA-seq experiments. Each group contained three biological repeats. The log2 of the fold change in gene expression (Log2 FC) in the Col2a1Cre; tetO-NICD1f/+; Rosa-rtTAf/+ mice relative to that in the control mice of the indicated genes of interest at the indicated times are shown, including Notch pathway genes, typical chondrogenic and catabolic genes, genes encoding fibrous collagens, as well as genes whose products are involved in inflammatory signaling, tyrosine kinase signaling, GPCR signaling, and NO signaling. *P < 0.05 after Benjamini-Hochberg correction for multiple testing (Cufflinks version 2.0.2). Negative values represent a reduction in gene expression as compared to controls at each time indicated.

  • Fig. 4 Sustained NOTCH1 signaling activates the IL-6–STAT3 pathway in cultured cells and OA cartilages.

    (A) WT and Col2a1Cre; tetO-NICD1f/+; Rosa-rtTAf/+ mice (sGOF NICD1) were treated with DOX (10 μg/ml) for 6 or 48 hours. RNA was then collected from P2 costal chondrocyte cultures (top) or P21 articular chondrocyte cultures (bottom) isolated from the mice, which was subjected to real-time qPCR analysis to determine the relative abundance of Il6 mRNA. All mRNA abundances were normalized to that of Actb and then were normalized to the controls. Data are means ± SD of three independent experiments. *P < 0.05 by two-tailed Student’s t test. (B) ATDC5 cells were transfected with FLAG control plasmid or with plasmid encoding FLAG-NICD1 (top) or were separately treated with control diluents or rIL-6 protein (1 ng/ml) for 24 hours (bottom), and then the relative abundances of the indicated mRNAs were determined by real-time qPCR analysis. All mRNA abundances were normalized to that of Actb and then were normalized to the controls. Data are means ± SD of three independent experiments. *P < 0.05 by two-tailed Student’s t test. (C) ATDC5 cells that were left untreated or were treated with rIL-6 (100 ng/ml) for 30 or 60 min (top) and R26-NICD1f/+ primary chondrocytes that were infected with adenoviruses expressing either green fluorescent protein (Ad-GFP) or Cre (Ad-CRE) (bottom) were subjected to Western blotting analysis with antibodies specific for the indicated proteins. Western blots are representative of three independent experiments. (D) ABH/OG staining and immunohistochemical analysis of IL-6 and pSTAT3 in knee sections from 2-month-old WT and sGOF NICD1 mice. Red arrowheads indicate pSTAT3-positive cells. Scale bars, 50 μm. Data are representative of at least three independent experiments. (E) ATDC5 cells that were transfected with FLAG control plasmid or that were transfected with plasmid encoding FLAG-NICD1 and then were either left untreated or treated with IL-6–neutralizing antibody (anti–IL-6) for 24 hours were subjected to real-time qPCR analysis to determine the relative abundances of the indicated mRNAs. All mRNA abundances were normalized to that of Actb and then were normalized to the controls, which were set at 1. Data are means ± SD of three independent experiments. *P < 0.05 by one-way analysis of variance (ANOVA), followed by the Bonferroni method.

  • Fig. 5 The IL-6–STAT3 pathway is activated in a trauma-induced mouse model of OA.

    ABH/OG staining and immunohistochemical analysis of IL-6 and pSTAT3 in knee sections from 5-month-old WT mice 12 weeks after they were subjected to sham treatment or MLI surgery. Red arrowheads indicate pSTAT3-positive cells. Scale bars, 50 μm. Data are representative of at least five independent experiments.

  • Fig. 6 Sustained NOTCH1 signaling activates the ERK and p38 pathways in cultured cells and OA cartilages, leading to selected effects on the expression of catabolic, chondrogenic, and fibrous genes.

    (A) ATDC5 cells that were left untreated or were treated with rIL-6 (100 ng/ml) for 30 or 60 min (left) and R26-NICD1f/+ primary chondrocytes that were infected with Ad-GFP or Ad-Cre viruses (right) were subjected to Western blotting analysis with antibodies specific for the indicated proteins. Western blots are representative of three independent experiments. (B) Immunohistochemical analysis of pERK in knee sections from 2-month-old WT and sGOF NICD1 mice. Red arrowheads indicate pERK-positive cells. Scale bars, 50 μm. Data are representative of three independent experiments. (C and D) Primary chondrocytes from R26-NICD1f/+ mice were infected with Ad-GFP virus or with Ad-Cre virus alone or in the presence of (C) the MAPK kinase (MEK) inhibitor U0126 or (D) the p28 MAPK inhibitor SB202190. Seventy-two hours later, RNA was isolated from the cells and was analyzed by real-time qPCR analysis to determine the relative abundances of the indicated mRNAs. All mRNA abundances were normalized to that of Actb and then were normalized to the control cells (Ad-GFP–infected cells; set at 1). Data are means ± SD of three independent experiments. *P < 0.05 by one-way ANOVA followed by the Bonferroni method.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/8/386/ra71/DC1

    Fig. S1. Development of a NOTCH1 activation model with the combined Tet-On/Cre system.

    Fig. S2. Sustained activation of NOTCH1 signaling in postnatal chondrocytes results in increased chondrocyte apoptosis.

    Fig. S3. Sustained activation of NOTCH1 signaling in postnatal chondrocytes results in a progressive, OA-like pathology.

    Fig. S4. Transient activation of NOTCH1 signaling in postnatal chondrocytes results in increased synthesis of cartilage ECM and joint maintenance for as long as 3 months after injection with DOX, but does not protect from cartilage degradation after MLI surgery.

    Fig. S5. Suppression of tyrosine kinase signaling leads to selected effects on NOTCH-regulated genes.

    Fig. S6. Suppression of GPCR signaling leads to selected effects on NOTCH-regulated genes.

    Fig. S7. Suppression of NO signaling leads to selected effects on NOTCH-regulated genes.

  • Supplementary Materials for:

    A dual role for NOTCH signaling in joint cartilage maintenance and osteoarthritis

    Zhaoyang Liu, Jianquan Chen, Anthony J. Mirando, Cuicui Wang, Michael J. Zuscik, Regis J. O'Keefe, Matthew J. Hilton*

    *Corresponding author. E-mail: matthew.hilton{at}dm.duke.edu

    This PDF file includes:

    • Fig. S1. Development of a NOTCH1 activation model with the combined Tet-On/Cre system.
    • Fig. S2. Sustained activation of NOTCH1 signaling in postnatal chondrocytes results in increased chondrocyte apoptosis.
    • Fig. S3. Sustained activation of NOTCH1 signaling in postnatal chondrocytes results in a progressive, OA-like pathology.
    • Fig. S4. Transient activation of NOTCH1 signaling in postnatal chondrocytes results in increased synthesis of cartilage ECM and joint maintenance for as long as 3
      months after injection with DOX, but does not protect from cartilage degradation after MLI surgery.
    • Fig. S5. Suppression of tyrosine kinase signaling leads to selected effects on NOTCH-regulated genes.
    • Fig. S6. Suppression of GPCR signaling leads to selected effects on NOTCH-regulated genes.
    • Fig. S7. Suppression of NO signaling leads to selected effects on NOTCH-regulated genes.

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    Citation: Z. Liu, J. Chen, A. J. Mirando, C. Wang, M. J. Zuscik, R. J. O'Keefe, M. J. Hilton, A dual role for NOTCH signaling in joint cartilage maintenance and osteoarthritis. Sci. Signal. 8, ra71 (2015).

    © 2015 American Association for the Advancement of Science

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