Expression of p16INK4a is a biomarker of chondrocyte aging but does not cause osteoarthritis

Abstract

Cellular senescence drives a functional decline of numerous tissues with aging by limiting regenerative proliferation and/or by producing pro-inflammatory molecules known as the senescence-associated secretory phenotype (SASP). The senescence biomarker p16^INK4a is a potent inhibitor of the cell cycle but is not essential for SASP production. Thus, it is unclear whether p16^INK4a identifies senescence in hyporeplicative cells such as articular chondrocytes and whether p16^INK4a contributes to pathologic characteristics of cartilage aging. To address these questions, we examined the role of p16^INK4a in murine and human models of chondrocyte aging. We observed that p16^INK4a mRNA expression was significantly upregulated with chronological aging in murine cartilage (~50-fold from 4 to 18 months of age) and in primary human chondrocytes from 57 cadaveric donors (r² = .27, p < .0001). Human chondrocytes exhibited substantial replicative potential in vitro that depended on the activity of cyclin-dependent kinases 4 or 6 (CDK4/6), and proliferation was reduced in cells from older donors with increased p16^INK4a expression. Moreover, increased chondrocyte p16^INK4a expression correlated with several SASP transcripts. Despite the relationship between p16^INK4a expression and these features of senescence, somatic inactivation of p16^INK4a in chondrocytes of adult mice did not mitigate SASP expression and did not alter the rate of osteoarthritis (OA) with physiological aging or after destabilization of the medial meniscus. These results establish that p16^INK4a expression is a biomarker of dysfunctional chondrocytes, but that the effects of chondrocyte senescence on OA are more likely driven by production of SASP molecules than by loss of chondrocyte replicative function.

Keywords: Ink4a; aging; cellular senescence; chondrocyte; geroscience; mouse models; osteoarthritis; p16.

Figure 1

Gene expression in murine and human cartilage with age. (a) p16 ^{INK 4a} (left) and p19 ^ARF (right) gene expression of hip cartilage from wild‐type mice. n ≥ 6 per group; mean ± SEM; ^# p < .05 to 4 months by ANOVA, Tukey's post hoc. (b) Gene expression in primary human chondrocytes from 57 cadaveric donors. R ² and p values from linear regression analysis shown

Figure 2

Effect of cyclin‐dependent kinase (CDK) inhibition on human chondrocyte proliferation. (a) A set of young and older (24.25 ± 2.6 vs. 64 ± 2.1 years old) donors were analyzed for p16 ^{INK 4a} gene expression (left) and the percentage of cells in S phase during monolayer culture (right). p Value shown by t test. (b) S phase percentage in chondrocytes from five donors treated with vehicle control, 1 μm Palbociclib, or 50 nm Dinaciclib. p Value shown by ANOVA with Tukey's post hoc. (c) Representative flow cytometry plots for percentage S phase calculation after a 4‐hr pulse of 5‐ethynyl‐2′‐deoxyuridine (EdU)

Figure 3

Gene expression in primary human chondrocytes. Data from 57 donors aged 17–72 years old are presented as a function of (a) age or (b) p16 ^{INK 4a} gene expression. The lowest value for each plot was set to 1. R ² and p values from linear regression analysis shown. ACAN, Aggrecan; IGFBP3, insulin‐like growth factor binding protein 3; MMP, matrix metalloproteinase. (c) The effect of scrambled control or siRNA targeting p16 ^{INK 4a} and p14 ^ARF on gene expression. (d) The effect of Palbociclib treatment on gene expression. In panels (c) and (d), data were normalized to control and all comparisons by paired t test were not significant (p > .05)

Figure 4

Effect of p16 ^{INK 4a} loss on spontaneous age‐related OA. (a) Histological sections of hindlimbs from 18‐month‐old mice were stained with Safranin‐O (red, glycosaminoglycans) and Fast Green (green, collagen). Representative images of mice with mild, moderate, and high total joint Safranin‐O scores from both p16 ^{INK 4a} intact and p16 ^{INK 4a} loss groups are shown. Scale bars = 200 μm. Sections were scored by a blinded observer for (b) the degree of Safranin‐O staining loss (high = OA, max score = 48) and (c) the size of osteophytes (high = OA, max score = 12). Analysis by Mann–Whitney test showed no significant difference between groups (p > .05) for either measure

Figure 5

Effect of p16 ^{INK 4a} loss on injury‐induced OA. (a) Histological sections of hindlimbs from mice 8 weeks after destabilization of the medial meniscus (DMM) surgery were stained with Safranin‐O (red, glycosaminoglycans) and Fast Green (green, collagen). Representative images of the medial side of DMM hindlimbs from mice with mild, moderate, and high total joint Safranin‐O scores are shown. Scale bars = 100 μm. Sections were scored by a blinded observer for (b) the degree of Safranin‐O staining loss (high = OA, max score = 48) and (c) the size of osteophytes (high = OA, max score = 12). Analysis by Mann–Whitney test showed no significant difference between groups (p > .05) for either measure