p16INK4a and its regulator miR-24 link senescence and chondrocyte terminal differentiation-associated matrix remodeling in osteoarthritis

Abstract

Introduction: Recent evidence suggests that tissue accumulation of senescent p16INK4a-positive cells during the life span would be deleterious for tissue functions and could be the consequence of inherent age-associated disorders. Osteoarthritis (OA) is characterized by the accumulation of chondrocytes expressing p16INK4a and markers of the senescence-associated secretory phenotype (SASP), including the matrix remodeling metalloproteases MMP1/MMP13 and pro-inflammatory cytokines interleukin-8 (IL-8) and IL-6. Here, we evaluated the role of p16INK4a in the OA-induced SASP and its regulation by microRNAs (miRs).

Methods: We used IL-1-beta-treated primary OA chondrocytes cultured in three-dimensional setting or mesenchymal stem cells differentiated into chondrocyte to follow p16INK4a expression. By transient transfection experiments and the use of knockout mice, we validate p16INK4a function in chondrocytes and its regulation by one miR identified by means of a genome-wide miR-array analysis.

Results: p16INK4a is induced upon IL-1-beta treatment and also during in vitro chondrogenesis. In the mouse model, Ink4a locus favors in vivo the proportion of terminally differentiated chondrocytes. When overexpressed in chondrocytes, p16INK4a is sufficient to induce the production of the two matrix remodeling enzymes, MMP1 and MMP13, thus linking senescence with OA pathogenesis and bone development. We identified miR-24 as a negative regulator of p16INK4a. Accordingly, p16INK4a expression increased while miR-24 level was repressed upon IL-1-beta addition, in OA cartilage and during in vitro terminal chondrogenesis.

Conclusions: We disclosed herein a new role of the senescence marker p16INK4a and its regulation by miR-24 during OA and terminal chondrogenesis.

Figure 1

Interleukin-1-beta (IL-1β) induces both p16^INK4a expression and a senescence-associated secretory phenotype (SASP) in mature chondrocytes. Osteoarthritis (OA) human primary chondrocytes were placed in pellet culture and treated with IL-1β (10 ng/mL) for 5 days. (A) Aggrecan mRNA (Acan) expression level was evaluated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) (n = 3). (B) p16^INK4a protein expression level was measured by Western blotting. (C) P-p38^MAPK protein level was detected by immunohistochemistry (IHC) on a section of paraffin-embedded pellets. Images were taken with a ×20 objective. (D-G) Matrix metalloprotease 1 (MMP1), MMP13, IL-6, and IL-8 secretion were measured by enzyme-linked immunosorbent assay (ELISA) (n = 4). Data are shown as mean ± standard deviation (SD) of fold changes compared with control. *P <0.05, **P <0.01.

Figure 2

MiR-24 and p16^INK4a expressions during chondrogenesis and role of p16^INK4a in chondrocyte cell cycle arrest. Human primary mesenchymal stromal cells (MSCs) were placed in three-dimensional (3D) culture conditions for 21 days. RNAs were harvested at indicated time points. (A-D) Gene expression analysis was performed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) for Collagen 2b, matrix metalloprotease 13 (MMP13), p16^INK4a, and miR-24. Data are shown as mean ± standard deviation (SD) (n = 3) normalized to D21. Immunohistochemistry (IHC) was performed on sections of formalin fixed paraffin-embedded long bones of transgenic mice deficient in p16^INK4a or wild-type at the age of 1 month. (E,F) Growth plate was marked by Safranin-O staining and proliferating cell nuclear antigen (PCNA) (P, proliferative zone; H, pre-hypertrophic/hypertrophic zone). Images were taken with ×20 objective. (G) Quantification of the percentage of hypertrophic non-proliferative terminally differentiated cells on total cells within the growth plate in transgenic and wild-type mice (n = 4) was carried out by using ImageJ software. Data were normalized to 1 for wild-type and are shown as mean ± SD. **P <0.01, ***P <0.001.

Figure 3

Cyclin-dependent kinase inhibitor p16^INK4a participates in matrix metalloproteinase 1 (MMP1) and MMP13 expression in mature chondrocytes. Osteoarthritis (OA) human primary chondrocytes were transfected with vector encoding for p16^INK4a or empty vector (EV) for 24 hours and placed in pellet for 7 days. (A) Gene expression analysis was performed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) for p16^INK4a (n = 5). (B) p16^INK4a protein expression was detected by immunohistochemistry (IHC) on pellet paraffin sections. Images were taken with a ×20 objective. (C-F) MMP1, MMP13, interleukin-6 (IL-6), and IL-8 secretions were measured by enzyme-linked immunosorbent assay (ELISA) (n = 5). Data are shown as mean ± standard deviation (SD). *P <0.05.

Figure 4

MiR-24 repression correlates with p16^INK4a induction in interleukin-1-beta (IL-1β)-treated chondrocytes and osteoarthritis (OA) cartilage. OA human primary chondrocytes are placed in pellet and treated with IL-1β at 10 ng/mL for 5 days. (A) List of microRNAs downregulated by IL-1β. (B,C) Gene expression analysis was performed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) for miR-24 and p16^INK4a (n = 3). (D) Luciferase/β-gal activities of promoter miR-24-2 cluster after IL-1β treatment (n = 3). Data are shown as mean ± standard deviation (SD). MiR-24 repression in OA cartilage samples is shown. (E,F) MiR-24 repression in OA cartilage: gene expression for miR-24 and p16^INK4a on OA (n = 5) and healthy (n = 6) cartilage samples. *P <0.05, **P <0.01, ***P <0.001.

Figure 5

MiR-24 downregulation is sufficient to trigger p16^INK4a expression and matrix metalloproteinase 1 (MMP1) production in mature chondrocytes. Osteoarthritis (OA) human primary chondrocytes were transfected with irrelevant antagomiR (Ctrl) and antagomiR-24 (A24) at a concentration of 100 nM. They were placed in pellet culture conditions for 7 days. (A,B) Gene expression analysis was performed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) for miR-24 and p16^INK4a (n = 4). (C) p16^INK4a protein expression by immunohistochemistry (IHC) on pellet paraffin-embedded sections. Images were taken with a ×20 objective. (D-G) MMP1, MMP13, interleukin-6 (IL-6), and IL-8 secretion were measured by enzyme-linked immunosorbent assay (ELISA) (n = 4). Data are shown as mean ± standard deviation (SD). *P <0.05.