Controlled induction and targeted elimination of p16 INK4a -expressing chondrocytes in cartilage explant culture

• Published in: The FASEB journal 2019-11, Vol.33 (11), p.12364-12373
• Main Authors: Sessions, Garrett A, Copp, Michaela E, Liu, Jie-Yu, Sinkler, Margaret A, D'Costa, Susan, Diekman, Brian O
• Format: Article
• Language: English
• Subjects: Aniline Compounds - pharmacology; Animals; Apoptosis; Cartilage, Articular - cytology; Cell Separation; Cellular Senescence; Chondrocytes - cytology; Cyclin-Dependent Kinase Inhibitor p16 - analysis; Cyclin-Dependent Kinase Inhibitor p16 - genetics; Cyclin-Dependent Kinase Inhibitor p16 - physiology; Mice; Mice, Inbred C57BL; Osteoarthritis - therapy; Sulfonamides - pharmacology; Tissue Culture Techniques
• ISSN: 0892-6638; 1530-6860
• DOI: 10.1096/fj.201900815RR

Cellular senescence is a phenotypic state that contributes to age-related diseases through the secretion of matrix-degrading and inflammatory molecules. An emerging therapeutic strategy for osteoarthritis (OA) is to selectively eliminate senescent cells by initiating apoptosis. This study establishes a cartilage explant model of senescence induction and senolytic clearance using expression as a biomarker of senescence. Growth-factor stimulation of explants increased the expression of p16 at both the mRNA and protein levels. Applying this culture system to cartilage from p16tdTom reporter mice (a knockin allele with tdTomato fluorescent protein regulated by the endogenous p16Ink4a promoter) demonstrated the emergence of a p16-high population that was quantified using flow cytometry for tdTomato. Cell sorting was used to separate chondrocytes based on tdTomato fluorescence and p16-high cells showed higher senescence-associated β-galactosidase activity and increased gene expression of the senescence-associated secretory phenotype as compared with p16-low cells. The potential for effective senolysis within the cartilage extracellular matrix was assessed using navitoclax (ABT-263). Navitoclax treatment reduced the percentage of p16-high cells from 17.9 to 6.1% (mean of 13 matched pairs; < 0.001) and increased cleaved caspase-3 confirmed apoptotic activity. Together, these findings establish a physiologically relevant cartilage explant model for testing the induction and elimination of senescent chondrocytes, which will support investigations of senolytic therapy for OA.-Sessions, G. A., Copp, M. E., Liu, J.-Y., Sinkler, M. A., D'Costa, S., Diekman, B. O. Controlled induction and targeted elimination of p16 -expressing chondrocytes in cartilage explant culture.