Knockdown of sirtuin3 in cartilage protects male mice against high-fat diet-induced osteoarthritis

Abstract

Purpose: Cellular senescence is a phenotypic state that emerges in response to stress after injury and during aging. Senescent cells are characterized by increased expression of the cell cycle inhibitor p16 INK4a (p16), high lysosomal activity, and the production of pro-inflammatory and matrix-degrading proteins known as the senescence associated secretory phenotype (SASP). Expression of p16 increases in both murine and human chondrocytes with age and high p16 expression correlates with SASP factor production. Furthermore, treatment with "senolytic" compounds that specifically target senescent cells for apoptosis was shown to mitigate post-traumatic OA in a murine model. The aim of this study was to establish a cartilage explant model system to explore the functional effects of selectively eliminating p16-expressing (senescent) chondrocytes. Methods: A knock-in murine genetics approach was used to replace the coding region of p16 with the fluorescent molecule tdTomato in order to quantify p16 expression on a single-cell basis with flow cytometry. Femoral cap cartilage explants were isolated from mice with one intact copy of p16 and one copy of the p16 tdTomato allele. Explants were cultured for three weeks either in control media or senescence induction media containing 1 ng/ml transforming growth factor beta-1 and 5 ng/ ml basic fibroblastic growth factor. Explants were digested and the percentage of p16 tdTomato -high cells was determined by flow cytometry. Cell sorting was used to separate chondrocytes into p16 tdTomato -high and p16 tdTomato -low fractions and these populations were analyzed for senescence features such as senescence-associated beta galactosidase (SABG) activity. Senescent cells upregulate anti-apoptotic pathways to maintain survival despite significant cellular damage. The ability to selectively eliminate p16-high cells with a drug that inhibits this apoptotic block is both a potential treatment and a way to further confirm the senescent nature of the p16 tdTomato -high cell fraction. In this study, senolytic treatment was performed using navitoclax, a BCL pathway inhibitor, at a concentration of 5 mM for 72 hours. As an initial assessment of how senolytic clearance affects cartilage health, the glycosaminoglycans (GAGs) content was measured in femoral cap explants that were cultured for an additional week following control or navitoclax treatment. Results: The treatment of murine cartilage explants with growth factors for three weeks induces a significant fraction of p16 tdTomato -high chondrocytes (Figure 1). This p16 tdTomato -high cell population demonstrates higher SABG activity as compared to the p16 tdTomato -low fraction (n¼4 sorts, mean ¼ 39.1% vs. 20.5% positive, p¼0.03 by paired t-test). Following treatment with the senolytic navitoclax, explants showed a significant reduction in the percentage of p16 tdTomato -high cells (Figure 2). As compared to control treatment, clearing senescent cells with navitoclax shows a trend towards increasing the GAG content per cell in cartilage exp...