p16INK4a and its regulator miR-24 link senescence and chondrocyte terminal differentiation-associated matrix remodeling in osteoarthritis
- Didier Philipot1, 2,
- David Guérit1, 2,
- Daniela Platano3,
- Paul Chuchana1, 2,
- Eleonora Olivotto3,
- Francisco Espinoza1, 2,
- Anne Dorandeu4,
- Yves-Marie Pers1, 2, 5,
- Jacques Piette6,
- Rosa Maria Borzi†3,
- Christian Jorgensen†1, 2, 5,
- Danièle Noel†1, 2 and
- Jean-Marc Brondello†1, 2Email author
© Philipot et al.; licensee BioMed Central Ltd. 2014
Received: 4 March 2013
Accepted: 12 February 2014
Published: 27 February 2014
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).
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.
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.
We disclosed herein a new role of the senescence marker p16INK4a and its regulation by miR-24 during OA and terminal chondrogenesis.
Tissue loss of function and integrity are inherent to aging and age-related disease onset. Because senescent p16INK4a-positive cells accumulate within numerous tissues throughout life , recent strong evidence suggested that these cells contribute to tissue degeneration by sustaining chronic inflammation and extracellular matrix remodeling . Indeed, p16INK4a-positive cells exhibit a specific secretome called SASP (senescence-associated secretory phenotype) including pro-inflammatory cytokines (such as interleukin-6 (IL-6), IL-8, and IL-1β) and matrix remodeling regulatory metalloproteases (such as MMP1 and MMP13) . Remarkably, specific conditional elimination of these cells in a premature aging murine model has revealed their essential role in the onset of several age-related diseases . Interestingly, Ink4a, which encodes an archetypical cyclin-dependent inhibitor (CKI) associated with senescence, is also known to participate in terminal differentiation onset of several cellular lineages [4, 5].
Osteoarthritis (OA) is a chronic degenerative disease characterized by progressive cartilage erosion and lesions in subchondral bone as well as in other joint tissues . The anabolic function of chondrocytes, the major cellular component of articular cartilage, decreases with disease progression. This loss of function is associated mainly with an accumulation of p16INK4a-positive articular chondrocytes  harboring short telomeres  but also features of hypertrophic/terminally differentiated cells [9, 10]. The latter is normally associated with endochondral ossification process during bone development [9, 10]. Although OA regulatory mechanisms remain under investigation, it's now believed that articular mature chondrocytes in response to either inflammatory cytokines or aberrant developmental signals exemplified by Notch activation  are producing matrix remodeling enzymes (MMP1 and MMP13) and inflammatory cytokines (IL-8 and IL-6) [11, 12]. All of these factors are deleterious for cartilage integrity. Therefore, OA is a multi-factorial complex disease in which articular chondrocytes exhibit characteristics of senescent-like and hypertrophic-like cells secreting SASP factors leading to impaired anabolic capacities . Moreover, a reduction of p16INK4a expression by RNA interference in OA chondrocytes was shown to lead to their functional rescue . These results demonstrate a deleterious role for this senescence-associated CKI on articular chondrocytes. It remains to be understood how p16INK4a increased expression occurs and could contribute to OA progression.
MicroRNAs (miRs) are small non-coding RNAs that are part of the miRNA-induced silencing complex (RISC)  and are involved in the regulation of gene expression. MiRs are key regulators of numerous physiological processes that are deregulated in pathological conditions , in particular OA [16, 17]. Among miRs identified in OA, miR-22 targets BMP7, a factor inducing chondrocyte terminal differentiation ; miR-140 targets HDAC4, a histone deacetylase inducer of chondrocyte terminal differentiation [19, 20]; and miR-27b targets MMP13, a key remodeling enzyme in hypertrophic terminally differentiated chondrocyte . So far, none of these miRs has been found to be regulators of p16INK4a-associated senescent phenotypes during OA progression.
In this study, we demonstrate that p16INK4a accumulates not only in response to inflammatory stimuli but also during chondrogenesis. Ink4a participates in cell cycle exit required for chondrocyte terminal differentiation onset during endochondral ossification. Moreover, p16INK4a overexpression is sufficient to trigger MMP1 and MMP13 production in mature chondrocytes. By genome-wide microRNA array, we identify miR-24 as a regulator of p16INK4a in chondrocytes. As expected, miR-24 is repressed in IL-1β-treated chondrocytes, in cartilages of patients with OA but also at the end of chondrogenesis while p16INK4a accumulates. Finally, downregulation of miR-24 by an antagomir approach in primary chondrocytes leads to an increase in p16INK4a expression and MMP1 secretion. Taken together, these data reveal for the first time that the senescent marker p16INK4a and its epigenetic regulator miR-24 are reciprocally involved in both OA and bone developmental-associated matrix remodeling secretomes.
Materials and methods
Cell culture, chondrocytes, mesenchymal stem cells, cartilage samples, and mouse models
Primary human chondrocytes were isolated from cartilage of 11 OA patients (mean age of 62 years) undergoing knee arthroplasty after informed written consent from patients and approval by the local and national ethics committee (‘Cellule de bioéthique de la direction générale pour la recherche et innovation, Ministère de l’Enseignement Supérieur et de la Recherche’; registration number DC-2009-1052) were obtained, as described previously . Cartilages from six healthy adult subjects (mean age of 53 years) were forensic waste from legal medicine with no need of informed consent after consultation with the national ethics committee and in strict agreement with French legislation. OA primary chondrocytes were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum as described . Primary OA chondrocytes (2.5 × 105 cells) were pelleted by centrifugation in 15-mL conical tubes, placed in three-dimensional (3D) setting for 7 days in chondrogenic medium—DMEM supplemented with 0.1 μM dexamethasone (Sigma-Aldrich, St. Louis, MO, USA), 1 mM pyruvate sodium (Invitrogen, Paisley, UK), 0.17 mM ascorbic acid (Sigma-Aldrich), 0.35 mM Proline (Sigma-Aldrich), 1% Insulin Transferin Selenium (Lonza, Basel, Switzerland), 2 mM L-glutamine (Lonza), 100 U/mL penicillin, and 100 μg/mL streptomycin (Lonza)—supplemented with transforming growth factor-beta 3 (TGF-β3) at 10 ng/mL (R&D Systems, Minneapolis, MN, USA). Treatment with recombinant human IL-1β at 10 ng/mL (R&D Systems) was applied for the first 5 days. Wild-type or ink4a knockout mice (1 month old) were obtained as reported . Mice were housed and cared for in accordance with the laboratory animal care guidelines. Approval was obtained from the regional ethics committee on animal experimentation before initiation of the study (approval CEEA-LR-10042). Experiments were performed in accordance with the regional ethics committee on animal research and care.
MicroRNA array analysis
Total RNA was extracted from chondrocytes in micropellet treated (or not) with IL-1β by using a miRvana isolation kit (Ambion, Carlsbad, CA, USA). MiRNA expression profiling was performed by using Miltenyi (Bergisch Gladbach, Germany) microarray facilities. Labeling and hybridization were performed in accordance with the protocol of the manufacturer. Raw data were normalized and additional data analysis was performed as described previously . Microarray data are available in the ArrayExpress database  under accession number E-MTAB-2229.
Reverse transcription, microRNA reverse transcription, and quantitative polymerase chain reaction
One microgram of Trizol-extracted total RNAs including microRNAs from the different samples were poly(A)-tailed with poly(A) polymerase (NEB M0276L). Then the polyadenylated RNA samples were reverse-transcribed as previously described using 50 units M-MLV Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) and either random primers or dTmiR adapter . For microRNA and mRNA quantitative analysis, cDNA was mixed with Sybr Green Master Mix (Roche Diagnostics, Indianapolis, IN, USA) in 96-well plates containing specific primers for hsa-miR-24 (universal reverse + specific primer), interest genes or the ribosomal subunit protein-9, housekeeping gene (hRSP9). Quantitative polymerase chain reaction (qPCR) conditions as described  used the following primer-probe combinations: for hRSP9 sense 5′-GATTACATCCTGGGCCTGAA antisense 5′-ATGAAGGACGGGATGTTCAC; for Aggrecan (hACAN) sense 5′- TCGAGGACAGCGAGGCC anti-sense 5′-TCGAGGGTGTAGCGTGTAGAGA; for hCOL2A1, variant 2 (hCol2A1) sense 5′-CAGACGCTGGTGCTGCT anti-sense 5′-TCCTGGTTGCCGGA CAT; For hMMP13 sense 5′-TAAGGAGCATGGCGACTTCT anti-sense 5′-GTCTGGCGTTTTTGGATGTT; for hp16INK4a sense 5′- GAAGGTCCCTCAGACATCCCC anti-sense 5′-CCCTGTAGGACCTTCGGTGAC; for hsa-miR-24 sense 5′-TGGCTCAGTTCAGCAGGAACAG Universal Reverse 5′- GCGAGCACAGAATTATACGACT.
cDNA constructs and luciferase reporter assay
Plasmids encoding for miR-24-2 promoter (−2041 base-pair) Luciferase and CMV β-galactosidase were provided by Charles Lecellier . Empty vector or p16INK4a encoding vector were purchased from Addgene . For promoter activity assay, OA human primary chondrocytes were transfected at day 0, placed in pellet culture conditions and treated with IL-1β during 48 hours. Cells were then lysed according to the dual luciferase/βgal kit (Promega, Charbonnières-les-Bains, France). Firefly Luciferase and β-galactosidase activities were detected using specific substrates with MultiScan FC (Thermo Scientific, Loughborough, UK). Luciferase activity was normalized to β-galactosidase activity.
In vitro differentiation of human bone marrow-mesenchymal stromal cells to chondrocytes
Human bone marrow-mesenchymal stromal cell (hBM-MSC) culture were established from bone marrow of patients undergoing Hip replacement surgery, after patient informed written consent and approval by the local and national ethics committee (“Cellule de bioéthique de la direction générale pour la recherche et innovation, Ministère de l’Enseignement supérieur et de la Recherche”; registration number DC-2009-1052). Human mesenchymal stromal cells (hMSCs) were isolated and amplified by using a complete alpha-minimum essential medium supplemented with 10% fetal bovine serum + 1 ng/mL of basic fibroblast growth factor. hBM-MSCs were positive for CD44, CD73, CD90, and CD105 but negative for CD14, CD34, and CD45. Chondrogenic differentiation of BM-MSCs was induced by 21-day culture in micropellet . Chondrogenesis was monitored by measuring the expression of chondrocyte-specific markers by reverse transcription-qPCR (RT-qPCR) as described .
Human chondrocytes (75 × 104) were transfected with 15 μg of plasmid for 24 hours by using Transit-LT1 Reagent (Euromedex, Souffelweyersheim, France). Chondrocytes were transfected with 100 nM of AntagomiR control or AntagomiR-24 (purchased from Ambion) by using oligofectamine (Invitrogen, USA). After transfection, cells were trypsinized and pelleted in chondrogenic medium and cultured for 7 days.
Western Blot and enzyme-linked immunosorbent assay
For Western blotting, chondrocytes in pellet cultures were lysed in RIPA-Benzonase buffer . After the addition of the lysis buffer, the samples were left on ice for 15 minutes with vortexing every 5 minutes for 10 seconds. Lysate protein samples were then sonicated for 5 minutes, followed by centrifugation at 7000 g for 15 minutes. The protein quantity loaded on Western blot gel corresponded to 25 × 104 cells. Primary antibodies and dilutions were anti-CDKN2A/p16INK4a (ab54210; Abcam; 1:1,000) and anti-β actin (Sigma-Aldrich A228; 1:8,000). Secondary antibody used for Western blot analysis was goat anti-mouse IgG HRP conjugate (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA; 115-035-003; 1:80,000). Enzyme-linked immunosorbent assays (ELISAs) were performed by using kits (IL-6, IL-8, pro-MMP13, and MMP1) from R&D Systems on supernatants stored at −20°C until analysis. Data were normalized and expressed as picograms per milliliter.
Immunohistochemistry and staining
Samples were fixed in 3.7% paraformaldehyde for 24 hours, washed in phosphate-buffered saline (PBS), and processed for routine histology. Paraffin-embedded sample sections (5 μm) were rehydrated through a gradient of xylene and ethanol. Samples were first incubated for 20 minutes at room temperature with pepsin solution (EmergoEurope, The Hague, The Netherlands) for antigen retrieval. Endogenous peroxidase blocking was done with 1% H2O2 for 20 minutes at room temperature. Samples were pre-incubated with blocking solution (PBS + 10% goat serum + 0.1% Triton) for 30 minutes at room temperature. Endogenous biotins were blocked by using a Streptavidin/Biotin blocking kit (Vector SP-2002) for 30 minutes. Primary antibody anti-CDKN2A/p16INK4a monoclonal mouse antibody (1:200; Abcam ab54210) was incubated for 72 hours at 4°C. Incubation with Biotin-coupled secondary antibody, IgG (1:200; ABC kit Vector PK6100) was done for 1 hour at room temperature. Incubation with Avidin/Biotin complex (ABC kit Vector PK6100) was done for 30 minutes at room temperature. Immunolocalized antigens were detected by means of a DAB revelation kit (Sigma-Aldrich). Safranin-O staining was performed as described . Quantification of proliferating cell nuclear antigen (PCNA)-positive or PCNA-negative hypertrophic chondrocytes was performed on four different sections of long bones of four mice of each genotype by using ImageJ software (D-0426) in accordance with the instructions of the manufacturer.
Experiments were performed with at least three independent individual chondrocyte or MSC samples. Comparisons of two conditions were done by using a paired Student t test. Unpaired Mann-Whitney test was applied for cartilage samples by using GraphPad Prism Software (GraphPad Software, Inc., La Jolla, CA, USA). Differences were considered significant when P values were less than 0.05.
Results and discussion
p16INK4a accumulates with other senescence-associated secretory phenotype factors in interleukin-1-beta-treated mature chondrocytes
The senescence marker p16INK4a is expressed during in vitro chondrogenesis and participates in the terminal differentiation-dependent cell cycle exit during endochondral ossification
Expression of p16INK4a is sufficient for MMP1 and MMP13 secretion by mature chondrocytes
p16INK4a induction correlates with miR-24 repression in interleukin-1-beta-treated chondrocytes, osteoarthritic cartilage, and the end of an in vitro chondrogenesis
Since inhibition of miR-processing enzymes such as Dicer induces senescence-associated phenotypes in primary cells  and promotes chondrocyte terminal differentiation in animal models , we focused our attention on miRs that were repressed by IL-1β (Figure 4A). We found miR-24, a known negative regulator of p16INK4a, through the presence of two binding sites for this miR within its encoding and 3′ untranslated region (UTR) . We next confirmed, on three independent chondrocyte samples placed in 3D, that IL-1β significantly repressed miR-24 expression (Figure 4B) with a concomitant expected induction of p16INK4a mRNA (Figure 4C).
miR-24 is encoded by two genes: miR-24-1 and miR-24-2 . These genes are organized in a cluster including three different miRs (miR-23a or b/27a or b/24). Each cluster is regulated by one promoter common for the three miRs of the cluster . Our array analysis revealed that, upon IL-1β stimulation, chondrocytes show a reduced expression of several members of these two clusters (Figure 4A), suggesting a global repression of the transcription of the clusters. In keeping with this hypothesis, we confirmed the transcriptional repression of miR-24-2 promoter, upon IL-1β addition (Figure 4D), by using a reporter luciferase assay (Figure 4D) previously described .
An increase in expression of p16INK4a has been demonstrated in cartilage from patients with OA . We then checked whether miR-24 expression could be reversely correlated with that of p16INK4a in OA cartilage compared with healthy cartilage. By RT-qPCR on mRNA from OA (n = 5) versus healthy (n = 6) human cartilage samples, we revealed a significant miR-24 downregulation in OA cartilage (Figure 4E) while p16INK4a is increased (Figure 4F). These results were confirmed at the protein level on serial sections of OA cartilage samples by using p16INK4a immunohistochemistry and miR-24 in situ hybridization (Additional file 1).
Figure 2 shows that p16INK4a mRNA accumulates throughout the time course of an in vitro chondrogenesis from days 7 to 21 (Figure 2C). We therefore evaluated, during chondrogenesis, whether the expression of miR-24 could also be reciprocal to that of p16INK4a expression. By RT-qPCR, we revealed that, compared with days 0 to 7, miR-24 level is decreased at day 14 and significantly at day 21 (Figure 2D) in parallel with an increase in expression of the terminal differentiation marker, MMP13 (Figure 2B), while p16INK4a remains elevated (Figure 2C). Taken together, these results demonstrate that the expressions of Ink4a and its epigenetic regulator are mutually exclusive in both in vitro and in vivo OA models and during the end of the chondrogenesis. Moreover, miR-24 downregulation seems to follow and sustain a high level of p16INK4a rather than initiate p16INK4a accumulation.
MiR-24 downregulation is sufficient to trigger p16INK4a expression and MMP1 production in mature chondrocytes
Determining the role and the regulatory pathways controlling p16INK4a expression in chondrocytes during OA progression is essential for future innovative long-term therapeutic approaches. In the present work, we demonstrated that the senescence CKI, p16INK4A, is also associated with chondrocyte terminal differentiation and can regulate the expression of matrix remodeling metalloproteases MMP1 and MMP13. We further showed that miR-24 expression plays a role as a negative regulator of the p16INK4a/MMP1 axis.
We propose that—during OA progression, in response to IL-1β, or during endochondral-induced terminal chondrogenesis—a repression of miR-24- and miR-24-encoding clusters takes place. This is likely to trigger p16INK4a, MMP1, MMP13, and Runx2 expression, thereby pushing chondrocytes toward a senescent-like phenotype resembling that of terminally differentiated chondrocytes . Accumulation of p16INK4a-positive chondrocytes within articular cartilage could thus be deleterious not only for tissue regeneration by blocking cell proliferation and replacement but also for tissue integrity through MMPs secretion [7, 55–57]. On the other hand, p16INK4a accumulation within the growth plate will favor bone development. One therapeutic strategy for OA treatment could be to restore/maintain the expression level of miR-24-encoding clusters in order to prevent p16INK4a-dependent pathways in articular chondrocytes.
bone marrow-mesenchymal stromal cell
cyclin-dependent kinase inhibitor
Dulbecco’s modified Eagle’s medium
human bone marrow-mesenchymal stromal cell
proliferating cell nuclear antigen
reverse transcription-quantitative polymerase chain reaction
senescence-associated secretory phenotype
transforming growth factor-beta 3.
We thank P Canovas, of the Service Chirugie Orthopédique, for OA cartilage samples (CHU La Peyronie, Montpellier, France); Marc Mathieu, Charles Lecellier, and Manuel Serrano, Chantal Ripoll and Stephanie Venteo for providing plasmids technical help, a mouse model and discussions. And the Montpellier Histology Facility (RHEM) and the-Montpellier animal facility network (RAM).
This work was supported by INSERM and by Fondation pour la Recherche Médicale (FRM) that was awarded to DN. DPh was a recipient of PhD program from “Région Languedoc Roussillon-Université de Montpellier 1”.
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