IntroductionRecent 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).MethodsWe 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.Resultsp16INK4a 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.ConclusionsWe disclosed herein a new role of the senescence marker p16INK4a and its regulation by miR-24 during OA and terminal chondrogenesis.
Skeletal development and cartilage formation require stringent regulation of gene expression for mesenchymal stem cells (MSCs) to progress through stages of differentiation. Since microRNAs (miRNAs) regulate biological processes, the objective of the present study was to identify novel miRNAs involved in the modulation of chondrogenesis. We performed miRNA profiling and identify miR-29a as being one of the most down-regulated miRNAs during the chondrogenesis. Using chromatin immunoprecipitation, we showed that SOX9 down-regulates its transcription. Moreover, the over-expression of miR-29a strongly inhibited the expression of chondrocyte-specific markers during in vitro chondrogenic differentiation of MSCs. We identified FOXO3A as a direct target of miR-29a and showed a down- and up-regulation of FOXO3a protein levels after transfection of, respectively, premiR- and antagomiR-29a oligonucleotides. Finally, we showed that using the siRNA or premiR approach, chondrogenic differentiation was inhibited to a similar extent. Together, we demonstrate that the down-regulation of miR-29a, concomitantly with FOXO3A up-regulation, is essential for the differentiation of MSCs into chondrocytes and in vivo cartilage/bone formation. The delivery of miRNAs that modulate MSC chondrogenesis may be applicable for cartilage regeneration and deserves further investigation.
The aim of this study was to identify new microRNAs (miRNAs) that are modulated during the differentiation of mesenchymal stem cells (MSCs) toward chondrocytes. Using large scale miRNA arrays, we compared the expression of miRNAs in MSCs (day 0) and at early time points (day 0.5 and 3) after chondrogenesis induction. Transfection of premiRNA or antagomiRNA was performed on MSCs before chondrogenesis induction and expression of miRNAs and chondrocyte markers was evaluated at different time points during differentiation by RT-qPCR. Among miRNAs that were modulated during chondrogenesis, we identified miR-574-3p as an early up-regulated miRNA. We found that miR-574-3p up-regulation is mediated via direct binding of Sox9 to its promoter region and demonstrated by reporter assay that retinoid X receptor (RXR)α is one gene specifically targeted by the miRNA. In vitro transfection of MSCs with premiR-574-3p resulted in the inhibition of chondrogenesis demonstrating its role during the commitment of MSCs towards chondrocytes. In vivo, however, both up- and down-regulation of miR-574-3p expression inhibited differentiation toward cartilage and bone in a model of heterotopic ossification. In conclusion, we demonstrated that Sox9-dependent up-regulation of miR-574-3p results in RXRα down-regulation. Manipulating miR-574-3p levels both in vitro and in vivo inhibited chondrogenesis suggesting that miR-574-3p might be required for chondrocyte lineage maintenance but also that of MSC multipotency.
Objective:This study aims at highlighting the common signature between cartilaginous tissue in osteoarthritis (OA) and preneoplasic tissues preceding neoplasia and tumour formation and, second, focusing on the molecular mechanisms at the aetiology of both pathologies.Results:Because age is the highest risk factor common for both OA and cancer development, it is tempting to compare the molecular mechanisms occurring at the onset of OA and preneoplasic lesions. Indeed, cellular senescence seems to be a common characteristic. Cellular senescence represents a natural barrier to suppress the unscheduled proliferation of damaged cells acting as a strong tumour suppressor pathway and in OA, it also occurs prematurely in chondrocytes. In this study, we review a number of molecular factors associated with the senescent phenotype.Conclusion:Whereas accumulation of senescent cells in preneoplasic-like lesions leads to tissue degeneration and potentially tumour development; in OA, senescent cells accumulate in a slowly proliferative tissue. This is likely contributing at reducing the risk of cell transformation.
> La sénescence cellulaire entraîne l'arrêt irré-versible de la prolifération en réponse à divers stress génotoxiques ou stimulus inappropriés. Cet arrêt est sous le contrôle d'un réseau complexe de signalisation inhibant certains régulateurs du cycle cellulaire, comme les kinases dépendantes des cyclines (CDK). La pérennisation de cet arrêt et l'induction de la sénescence requièrent l'action de plusieurs suppresseurs de tumeurs, dont p53, pRb et p16 Ink4a. Outre d'importants changements morphologiques et métaboliques, des altérations de la chromatine et de l'expression génique caractérisent également la sénescence. Par ailleurs, les cellules sénescentes synthétisent un ensemble de cytokines et chimiokines désigné sous le terme de senescence-associated secretory phenotype (SASP), qui favorise l'inflammation et peut modifier de façon drastique le tissu environnant. Bien qu'elle favorise la cicatrisation et s'érige en barrière oncosuppressive, la sénes-cence, à l'image du dieu romain Janus, offre un second visage, moins bénéfique, puisqu'elle contribue au vieillissement et aux pathologies qui lui sont associées, comme le cancer. < associé à d'importants changements morphologiques et physiologiques [2]. Suivant une bonne intuition, L. Hayflick a associé la notion de sénescence à celles de cancer et de vieillissement : notion de cancer car, pour échapper à la sénescence, les cellules doivent acquérir certaines caractéristiques des cellules cancéreuses ; notion de vieillissement car l'accumulation de cellules sénescentes contribue au vieillissement global de l'organisme [1]. Ses hypothèses étaient fondamentalement justes, néanmoins la nature du « compteur mitotique » et des méca-nismes moléculaires qui expliquent les liens entre sénescence, cancer et vieillissement ne furent découverts que récemment. Très tôt, sur la base de considérations théoriques, A. Olovnikov avait proposé que le « compteur » suggéré par L. Hayflick pouvait être le raccourcissement des télomères, structures nucléoprotéiques proté-geant les extrémités des chromosomes [3]. En effet, au fil des divisions et de la réplication des chromosomes, les télomères des cellules somatiques raccourcissent de plus en plus. Il a donc été proposé que cette érosion des télomères, reconnue par les cellules comme de l'ADN endommagé, pourrait activer la réponse au stress génotoxique, qui est identique à celle qu'induisent les rayonnements ionisants et conduit à l'arrêt de la division cellulaire, d'où le nom de « sénescence réplicative » [4]. Cette hypothèse a été confirmée par des travaux ultérieurs [5]. La sénescence peut également être déclenchée par des lésions irréparables de l'ADN, par le stress oxydatif qui survient dans des conditions de culture cellulaire non adaptées, les dysfonctions mitochondriales (voir Encadré 1), ainsi que par l'expression La sénescence cellulaire a été originellement décrite par L. Hayflick qui a découvert que les cellules somatiques cessent de proliférer après un nombre précis de divisions en culture, comptabilisées selon lui par une ...
under Good Laboratory Practise (GLP) conditions, after local intraarticular (IA) injection in mice. Methods: Clinical grade ASC were isolated from subcutaneous abdominal fat, expanded in a-MEM medium with 10% platelet lysate and used at passage 1. ASC were injected either IA (106 cells/knee joint) or systemically (IV) via the tail vein (106 cells) of SCID mice. At different time points (day 11, 28, 90 186), 10 mice were euthanasized and several organs/tissues recovered. After DNA extraction, qPCR was performed using primers specific for human Alu or murine actine sequences. Results: Quantification of hASC engraftment was performed through detection of human-specific Alu sequences and normalization using murine actine sequences on DNA extracted from 14 different organs or tissues. Using serial ten-fold dilutions of hASCs in murine MSCs, a linear correlation curve between the number of ASC and Alu signal was established. This curve was comparable between different cell samples and the detection limit was 0.005% hASCs. After IA injection, hASCs were detected in 10/10 mice at the different time points. They were in the joint in more than 90% of mice for the first 3 months and were still observed in 60% of the mice after 6 months. At day 11 and 28, approximately 15% of the injected cells were recovered in various organs. Cells were predominantly observed in the knee joints, bone marrow and fat. After 3 and 6 months, 1.5-4% of infused hASC were still detected in the joint, bone marrow, fat and muscle. After systemic injection, hASC were detected in 5 and 4/12 mice in one or two organs at day 11 and 28, respectively. Only 1.4 and 0.8% of the total infused cells were detected predominantly in lung, intestine, stomach, liver and brain.Conclusions: Contrary to a systemic route, the IA injection of hASC allowed their survival on the long-term. Cells were predominantly localized at the site of injection but also, in the stem cell reservoirs, bone marrow and fat tissue.
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