OBJECTIVE: To demonstrate the activation of the Notch signaling pathway during changes in the phenotype of chondrocytes in vitro, and to assess the influence of Notch on the production of chondrocyte markers. METHODS: Serial monolayer primary cultures of murine articular chondrocytes (MACs), as a model of chondrocyte dedifferentiation, were prepared. MACs were cultured with or without a Notch inhibitor and transfected with different Notch-expressing vectors. The Notch pathway and chondrocyte marker profiles were assessed by quantitative reverse transcription-polymerase chain reaction, immunoblotting, and immunocytochemistry. RESULTS: Successive passages of MACs resulted in a loss of type II collagen and aggrecan (chondrocyte differentiation markers), an increase in type I collagen (dedifferentiation marker), an increase in Notch ligands, and augmented target gene activity. The Notch inhibitor decreased the type II collagen protein content but had no effect on Col2a1 messenger RNA, while transfection with the constitutive active forms of the Notch1 receptor led to a decrease in type II collagen in transfected cells. In assays to investigate the mechanism of type II collagen breakdown, matrix metalloproteinase 13 (MMP-13) synthesis was regulated in a Notch-dependent manner, whereas MMP-2 synthesis was unchanged. CONCLUSION: The Notch signaling pathway is associated with decreased type II collagen production during the dedifferentiation of MACs in vitro. This may be correlated with the increase in MMP-13 production linked to activation of Notch.
OBJECTIVE: To demonstrate the activation of the Notch signaling pathway during changes in the phenotype of chondrocytes in vitro, and to assess the influence of Notch on the production of chondrocyte markers. METHODS: Serial monolayer primary cultures of murine articular chondrocytes (MACs), as a model of chondrocyte dedifferentiation, were prepared. MACs were cultured with or without a Notch inhibitor and transfected with different Notch-expressing vectors. The Notch pathway and chondrocyte marker profiles were assessed by quantitative reverse transcription-polymerase chain reaction, immunoblotting, and immunocytochemistry. RESULTS: Successive passages of MACs resulted in a loss of type II collagen and aggrecan (chondrocyte differentiation markers), an increase in type I collagen (dedifferentiation marker), an increase in Notch ligands, and augmented target gene activity. The Notch inhibitor decreased the type II collagen protein content but had no effect on Col2a1 messenger RNA, while transfection with the constitutive active forms of the Notch1 receptor led to a decrease in type II collagen in transfected cells. In assays to investigate the mechanism of type II collagen breakdown, matrix metalloproteinase 13 (MMP-13) synthesis was regulated in a Notch-dependent manner, whereas MMP-2 synthesis was unchanged. CONCLUSION: The Notch signaling pathway is associated with decreased type II collagen production during the dedifferentiation of MACs in vitro. This may be correlated with the increase in MMP-13 production linked to activation of Notch.
Authors: Carina Strell; Janna Paulsson; Shao-Bo Jin; Nicholas P Tobin; Artur Mezheyeuski; Pernilla Roswall; Ceren Mutgan; Nicholas Mitsios; Hemming Johansson; Sarah Marie Wickberg; Jessica Svedlund; Mats Nilsson; Per Hall; Jan Mulder; Derek C Radisky; Kristian Pietras; Jonas Bergh; Urban Lendahl; Fredrik Wärnberg; Arne Östman Journal: J Natl Cancer Inst Date: 2019-09-01 Impact factor: 13.506
Authors: Anthony J Mirando; Zhaoyang Liu; Tyler Moore; Alexandra Lang; Anat Kohn; Alana M Osinski; Regis J O'Keefe; Robert A Mooney; Michael J Zuscik; Matthew J Hilton Journal: Arthritis Rheum Date: 2013-10
Authors: Hua Wang; Ye Tian; Jianru Wang; Kate L E Phillips; Abbie L A Binch; Sara Dunn; Alison Cross; Neil Chiverton; Zhaomin Zheng; Irving M Shapiro; Christine L Le Maitre; Makarand V Risbud Journal: J Biol Chem Date: 2013-04-15 Impact factor: 5.157