Background/Purpose: Notch1 to Notch4 are transmembrane receptors that determine cell differentiation and function.  Interactions of Notch with its ligands result in the cleavage of the Notch intracellular domain (NICD), which translocates to the nucleus to induce gene expression.  Notch suppresses collagen type II a1 (Col2a1) and induces collagen type X a1 (Col10a1) expression in murine chondrocytes.  Activation of Notch signaling was observed in human osteoarthritic chondrocytes, although it was not reported whether Notch plays a role in the progression of osteoarthritis.  Matrix metalloproteinase (Mmp)13 is a collagen-degrading enzyme expressed by osteoarthritic chondrocytes, and overexpression of Mmp13 in murine articular chondrocytes causes joint degeneration, demonstrating that Mmp13 contributes to cartilage matrix degradation.  Interleukin (Il6) is a secreted inflammatory molecule that suppresses Col2a1 and induces Mmp13 expression in chondrocytes.  Il6 is expressed by osteoarthritic chondrocytes, and Notch induces Il6 in synoviocytes, but it was not reported whether Notch regulates Il6 expression in chondrocytes.  To understand whether Notch regulates the expression of gene markers of osteoarthritis, we investigated the effects of Notch on Mmp13 and Il6 expression in primary murine chondrocytes, and tested whether Il6 mediates the effects of Notch.

Methods: Notch was induced in chondrocytes from Rosa^Notch mice, where the Rosa26 promoter is followed by a STOP cassette flanked by LoxP sites, and the NICD coding sequence.  Primary chondrocytes from 3 to 4 day old Rosa^Notch mice were infected with an adenoviral vector expressing Cre recombinase, which excises the STOP cassette and induces expression of NICD directed by the Rosa26 promoter.  As controls, parallel cultures of Rosa^Notch chondrocytes were infected with an adenoviral vector expressing green fluorescent protein (GFP).

To document Notch activation, Rosa^Notch chondrocytes were transfected with Notch reporter constructs.  To assess the effects of Notch on gene expression, changes in mRNA levels were analyzed by quantitative reverse transcription PCR.  To investigate whether Il6 mediates the effects of Notch in Rosa^Notch chondrocytes, cells were cultured in the absence of serum and exposed to an inhibitory monoclonal murine antibody against Il6, or to a murine immunoglobulin G, either under basal conditions or in the context of Notch activation.

Results: NICD overexpression in Rosa^Notch chondrocytes transactivated Notch reporter constructs and induced Notch target genes, demonstrating activation of Notch signaling.  The effects of Notch on the expression of Col2a1 and Col10a1 were confirmed.  NICD induced Mmp13 and Il6 mRNA levels, and exposure of Rosa^Notch chondrocytes to an inhibitory Il6 antibody opposed the induction of Mmp13 by Notch, whereas it did not modify the effects of Notch on Col2a1 and Col10a1 expression.

Conclusion: Activation of Notch signaling in primary chondrocytes induces expression of gene markers of osteoarthritis.  Induction of Mmp13 by Notch is mediated by Il6, whereas the effects of Notch on the expression of chondrocyte gene markers are determined by alternate mechanisms.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/notch-promotes-matrix-metalloproteinase-13-expression-by-inducing-interleukin-6-in-primary-murine-chondrocytes/