Development of a simple osteoarthritis model useful to predict in vitro the anti-hypertrophic action of drugs.

Osteoarthritis (OA) is characterized by cartilage degradation, inflammation, and hypertrophy. Therapies are mainly symptomatic and aim to manage pain. Consequently, medical community is waiting for new treatments able to reduce OA process. This study aims to develop an in vitro simple OA model useful to predict drug ability to reduce cartilage hypertrophy. Human primary OA chondrocytes were incubated with transforming growth factor beta 1 (TGF-ß1). Hypertrophy was evaluated by Runx2, type X collagen, MMP13, and VEGF expression. Cartilage anabolism was investigated by Sox9, aggrecan, type II collagen, and glycosaminoglycan expression. In chondrocytes, TGF-ß1 increased expression of hypertrophic genes and activated canonical WNT pathway, while it decreased dramatically cartilage anabolism, suggesting that this treatment could mimic some OA features in vitro. Additionally, EZH2 inhibition, that has been previously reported to decrease cartilage hypertrophy and reduce OA development in vivo, attenuated COL10A1 and MMP13 upregulation and SOX9 downregulation induced by TGF-ß1 treatment. Similarly, pterosin B (an inhibitor of Sik3), and DMOG (a hypoxia-inducible factor prolyl hydroxylase which mimicks hypoxia), repressed the expression of hypertrophy markers in TGF-ß stimulated chondrocytes. In conclusion, we established an innovative OA model in vitro. This cheap and simple model will be useful to quickly screen new drugs with potential anti-arthritic effects, in complementary to current inflammatory models, and should permit to accelerate development of efficient treatments against OA able to reduce cartilage hypertrophy.

The Antitumoral Effect of the S-Adenosylhomocysteine Hydrolase Inhibitor, 3-Deazaneplanocin A, is Independent of EZH2 but is Correlated with EGFR Downregulation in Chondrosarcomas.

BACKGROUND/AIMS: 3-Deazaneplanocin, DZNep, has been reported to inhibit the EZH2 histone methylase and to induce cell apoptosis in chondrosarcomas (CS). The present study aims to confirm the therapeutic potential of EZH2 inhibitors and investigate the molecular mechanisms of DZNep in chondrosarcomas. METHODS: CS cell lines and primary cultures were used. Apoptosis was investigated using PARP cleavage, caspase 3/7 activity, or Apo2.7 expression. S-adenosylhomocysteine (SAH) and S-adenosylmethionine (SAM) were quantified by UHPLC-MS/MS. Differentially expressed genes in treated-chondrosarcomas and chondrocytes were researched by microarray analysis. RESULTS: DZNep induced apoptosis in chondrosarcomas both in vivo and in vitro. However, this effect was not correlated to EZH2 expression nor activity, and EZH2 knock-down by siRNA did not reduce CS viability. Additionally, the reduction of H3K27me3 induced by GSK126 or tazemetostat (EPZ-6438) did not provoke chondrosarcoma death. However, as expected, DZNep induced SAH accumulation and reduced SAM:SAH ratio. Further, microarray analysis suggests a key role of EGFR in antitumoral effect of DZNep, and pharmacological inhibition of EGFR reduced chondrosarcoma survival. CONCLUSION: EZH2 is not an adequate target for chondrosarcoma treatment. However, DZNep induces apoptosis in chondrosarcomas in vitro and in vivo, by a mechanism likely mediated though EGFR expression. Consequently, it would be worth initiating clinical trials to evaluating efficiency to S-adenosylhomocysteine hydrolase or EGFR inhibitors in patients with chondrosarcomas.

Antiproliferative effect of the histone demethylase inhibitor GSK-J4 in chondrosarcomas.

Chondrosarcoma (CS) is the second most common malignant bone sarcoma. Its treatment remains an issue, because this tumor is radio- and chemo-resistant. In the present study, we investigated the antitumoral potential of GSK-J4, a small molecule described as an inhibitor of histone demethylases UTX and JMJD3 (KDM6A and KDM6B), alone or in combination with cisplatin in CSs. Human CS-derived cell lines were treated with GSK-J4 in the presence or not of cisplatin. Survival curves were established and cell proliferation and cycle were evaluated by flow cytometry using dividing cell tracking technique utilizing carboxyfluorescein succinimidyl ester labeling, or DNA staining by propidium iodide. Apoptosis and senescence were also investigated. GSK-J4 decreased proliferation of CS cells. Additionally, it induced apoptosis in CH2879 and JJ012 cells, but not in SW1353 CSs. In addition, its association with cisplatin decreased cell proliferation more than drugs alone, whereas it did not increase apoptosis compared to cisplatin alone. Interestingly, GSK-J4 alone as well as in association with cisplatin did not affect chondrocyte survival or proliferation. In conclusion, this study suggests that demethylase inhibitors may be useful in improving therapy for CS in reducing its proliferation.

Modulation of Skin Inflammatory Response by Active Components of Silymarin.

In this study, we compared selected silymarin components, such as quercetin (QE), 2,3-dehydrosilybin (DHS) and silybin (SB), with the anti-inflammatory drug indomethacin (IND) in terms of their wound healing potential. In view of the fact that pathological cutaneous wound healing is associated with persistent inflammation, we studied their anti-inflammatory activity against inflammation induced by bacterial lipopolysaccharide (LPS). We investigated the regulation of crucial pro-inflammatory transcription factors-nuclear factor kappa-B (NF-κB) and activator protein 1 (AP-1)-as well as the expression of downstream inflammatory targets by Western blotting, real-time PCR (RT-PCR), electrophoretic mobility shift assay (EMSA), and/or enzyme-linked immunosorbent assay (ELISA) in vitro using primary normal human dermal fibroblasts (NHDF). We demonstrated the greater ability of DHS to modulate the pro-inflammatory cytokines production via the NF-κB and AP-1 signaling pathways when compared to other tested substances. The prolonged exposure of LPS-challenged human dermal fibroblasts to DHS had both beneficial and detrimental consequences. DHS diminished interleukin-6 (IL-6) and interleukin-8 (IL-8) secretion but induced the significant upregulation of IL-8 mRNA associated with NF-κB and AP-1 activation. The observed conflicting results may compromise the main expected benefit, which is the acceleration of the healing of the wound via a diminished inflammation.

Identification of an easy to use 3D culture model to investigate invasion and anticancer drug response in chondrosarcomas.

BACKGROUND: Cytotoxic efficacy of anticancer drugs has been widely studied with monolayer-cultured cancer cells. However, the efficacy of drugs under two-dimensional (2D) culture condition usually differs from that of three-dimensional (3D) one. In the present study, an in vitro tumor tissue model was constructed using alginate hydrogel, and in vitro cytotoxic efficacy of two anticancer drugs (cisplatin and DZNep) was investigated in chondrosarcomas, and compared to in vivo response. METHODS: Three cell lines derived from human chondrosarcomas, CH2879, JJ012 and SW1353, were embedded in alginate hydrogel. Proliferation and survival were assayed by ATP measurement using Cell Titer-Glo luminescent cell viability assay kit, and by counting viable cells in beads. Collagen and COMP expression was determined by RT-PCR. Invasion/migration was estimated by counting cells leaving alginate beads and adhering to culture dish. Then, chondrosarcoma response to cisplatin and DZNep was compared between cells cultured in monolayer or embedded in alginate, and using chondrosarcoma xenografts in nude mice. RESULTS: Chondrosarcomas survived at least for 8 weeks, after embedment in alginate. However, only CH2879 cells could proliferate. Also, this cell line is more invasive than SW1353 and JJ012, which was coherent with the grade of their respective primary tumors. Furthermore, the expression of type II collagen was higher in chondrosarcomas cultured in 3D than in 2D. Interestingly, this 3D culture system allows to validate the absence of response of chondrosarcomas to cisplatin, and to predict the efficiency of DZNep to reduce chondrosarcoma growth in vivo. CONCLUSIONS: This study validates alginate beads as a relevant 3D model to study cancer biology and tumor responses to biological treatments.

Hypoxia inducible factor 1 alpha down-regulates type i collagen through Sp3 transcription factor in human chondrocytes.

Cartilage engineering is one challenging issue in regenerative medicine. Low oxygen tension or hypoxia inducible factor-1 (HIF-1α) gene therapy are promising strategies in the field of cartilage repair. Previously, we showed that hypoxia and its mediator HIF-1 regulate matrix genes expression (collagens and aggrecan). Here, we investigated the molecular mechanism involved in the regulation of type I collagen (COL1A1) by HIF-1 in human articular chondrocytes. We show that HIF-1α reduces COL1A1 transcription, through a distal promoter (-2300 to -1816 bp upstream transcription initiation site), containing two GC boxes that bind Sp transcription factors (Sp1/Sp3). Sp1 acts as a positive regulator but is not induced by HIF-1. COL1A1 inhibition caused by HIF-1 implies only Sp3, which accumulates and competes Sp1 binding on COL1A1 promoter. Additionally, Sp3 ectopic expression inhibits COL1A1, while Sp3 knockdown counteracts the downregulation of COL1A1 induced by HIF-1. In conclusion, we established a new regulatory model of COL1A1 regulation by HIF-1, and bring out its relationship with Sp3 transcription factor. In a fundamental level, these findings give insights in the mechanisms controlling COL1A1 gene expression. This may be helpful to improve strategies to impair type I collagen expression during chondrocyte differentiation for cartilage engineering. © 2016 IUBMB Life, 68(9):756-763, 2016.

TOL19-001 reduces inflammation and MMP expression in monolayer cultures of tendon cells.

BACKGROUND: Tendinopathies are tendon conditions associated with degeneration and disorganization of the matrix collagen fibers, tendon cells apoptosis and inflammation through up-regulation of proinflammatory cytokines, matrix metalloproteinase (MMP) expression, and prostaglandin E2 (PGE2) production. Currently, the pharmacological treatment is mainly based on non-steroidal anti-inflammatory drugs (NSAIDs) use and corticosteroid injections, which both can lead to numerous side effects for patients. TOL19-001 is a diet supplementary composed mostly of spirulina and glucosamine sulfate whose antioxidant properties could be helpful to treat tendinopathies while avoiding taking NSAIDs. In this study we developed an in vitro model of tendinopathy in order to evaluate the therapeutic potential of TOL19-001. METHODS: Tendon cells were cultured on monolayer and treated with interleukin-1ß (IL-1ß) or ciprofloxacin (CIP), and then, MMPs, PGE2 and collagen expression was evaluated by RT-PCR or Elisa. In addition, a cotreatment with increased doses of TOL19-001 was done. Toxicity of TOL19-001 was evaluated using a metabolic activity assay. RESULTS: This study demonstrates that IL-1ß mimics some aspects of tendinopathies with PGE2 induction, MMP expression (mostly MMP1 and MMP3), and increases of type III/I collagen ratio. CIP, meanwhile, leads to an increase of MMP2 and p65 mRNA, whereas it reduces TIMP1 expression. Scleraxis expression was also increased by CIP whereas it was decreased by IL-1ß treatment. Besides, TOL19-001 cotreatment suppresses tendon cell inflammation in vitro, marked by the downregulation of PGE2, MMPs and type III collagen in IL-1ß stimulated-cells. TOL19-001 also represses CIP induced-changes. CONCLUSIONS: These findings indicate that TOL19-001 exerts anti-inflammatory effects on tendon cells, which might explain why TOL19-001 diet may improve tendon function in patients with tendon injury. Future research is required to determine TOL19-001 effect on injured or overused tendons in vivo.

The p65 subunit of NF-κB inhibits COL1A1 gene transcription in human dermal and scleroderma fibroblasts through its recruitment on promoter by protein interaction with transcriptional activators (c-Krox, Sp1, and Sp3).

Transcriptional mechanisms regulating type I collagen genes expression in physiopathological situations are not completely known. In this study, we have investigated the role of nuclear factor-κB (NF-κB) transcription factor on type I collagen expression in adult normal human (ANF) and scleroderma (SF) fibroblasts. We demonstrated that NF-κB, a master transcription factor playing a major role in immune response/apoptosis, down-regulates COL1A1 expression by a transcriptional control involving the -112/-61 bp sequence. This 51-bp region mediates the action of two zinc fingers, Sp1 (specific protein-1) and Sp3, acting as trans-activators of type I collagen expression in ANF and SF. Knockdown of each one of these trans factors by siRNA confirmed the trans-activating effect of Sp1/Sp3 and the p65 subunit of NF-κB trans-inhibiting effect on COL1A1 expression. Despite no existing κB consensus sequence in the COL1A1 promoter, we found that Sp1/Sp3/c-Krox and NF-κB bind and/or are recruited on the proximal promoter in chromatin immunoprecipitation (ChIP) assays. Attempts to elucidate whether interactions between Sp1/Sp3/c-Krox and p65 are necessary to mediate the NF-κB inhibitory effect on COL1A1 in ANF and SF were carried out; in this regard, immunoprecipitation assays revealed that they interact, and this was validated by re-ChIP. Finally, the knockdown of Sp1/Sp3/c-Krox prevents the p65 inhibitory effect on COL1A1 transcription in ANF, whereas only the siRNAs targeting Sp3 and c-Krox provoked the same effect in SF, suggesting that particular interactions are characteristic of the scleroderma phenotype. In conclusion, our findings highlight a new mechanism for COL1A1 transcriptional regulation by NF-κB, and these data could allow the development of new antifibrotic strategies.

Regulatory mechanism of transforming growth factor beta receptor type II degradation by interleukin-1 in primary chondrocytes.

Interleukin-1ß (IL-1ß), a key-cytokine in osteoarthritis, impairs TGFß signaling through TßRII down-regulation by increasing its degradation. Here, we investigated the molecular mechanism that controls TßRII fate in IL-1ß treated cells. Chondrocytes were treated with IL-1ß in the presence of different inhibitors. TßRII and Cav-1 expression were assayed by Western blot and RT-PCR. We showed that IL-1ß-induced degradation of TßRII is dependent on proteasome and on its internalization in caveolae. In addition, IL-1ß enhances Cav-1 expression, a major constituent of lipid raft. In conclusion, we enlighten a new mechanism by which IL-1ß antagonizes TGFß pathway and propose a model of TßRII turnover regulation upon IL-1ß treatment.

Hypoxia Differentially Affects Chondrogenic Differentiation of Progenitor Cells from Different Origins.

Background and Objectives: Ear cartilage malformations are commonly encountered problems in reconstructive surgery, since cartilage has low self-regenerating capacity. Malformations that impose psychological and social burden on one's life are currently treated using ear prosthesis, synthetic implants or autologous flaps from rib cartilage. These approaches are challenging because not only they request high surgical expertise, but also they lack flexibility and induce severe donor-site morbidity. Through the last decade, tissue engineering gained attention where it aims at regenerating human tissues or organs in order to restore normal functions. This technique consists of three main elements, cells, growth factors, and above all, a scaffold that supports cells and guides their behavior. Several studies have investigated different scaffolds prepared from both synthetic or natural materials and their effects on cellular differentiation and behavior. Methods and Results: In this study, we investigated a natural scaffold (alginate) as tridimensional hydrogel seeded with progenitors from different origins such as bone marrow, perichondrium and dental pulp. In contact with the scaffold, these cells remained viable and were able to differentiate into chondrocytes when cultured in vitro. Quantitative and qualitative results show the presence of different chondrogenic markers as well as elastic ones for the purpose of ear cartilage, upon different culture conditions. Conclusions: We confirmed that auricular perichondrial cells outperform other cells to produce chondrogenic tissue in normal oxygen levels and we report for the first time the effect of hypoxia on these cells. Our results provide updates for cartilage engineering for future clinical applications.

Asporin expression is highly regulated in human chondrocytes.

A significant association between a polymorphism in the D repeat of the gene encoding asporin and osteoarthritis, the most frequent of articular diseases, has been recently reported. The goal of the present study was to investigate the expression of this new class I small leucine-rich proteoglycan (SLRP) in human articular chondrocytes. First, we studied the modulation of asporin (ASPN) expression by cytokines by Western blot and reverse transcription-polymerase chain reaction. Interleukin-1ß and tumor necrosis factor-α downregulated ASPN, whereas transforming growth factor-ß1 (when incubated in a serum-free medium) upregulated it. Similarly to proinflammatory cytokines, chondrocyte dedifferentiation induced by a successive passages of cells was accompanied by a decreased asporin expression, whereas their redifferentiation by three-dimensional culture restored its expression. Finally, we found an important role of the transcription factor Sp1 in the regulation of ASPN expression. Sp1 ectopic expression increased ASPN mRNA level and promoter activity. In addition, using gene reporter assay and electrophoretic mobility shift assay, we showed that Sp1 mediated its effect through a region located between -473 and -140 bp upstream of the transcription start site in ASPN gene. In conclusion, this report is the first study on the regulation of asporin expression by different cytokines in human articular chondrocytes. Our data indicate that the expression of this gene is finely regulated in cartilage and suggest a major role of Sp1.

Characterization of distinct mesenchymal-like cell populations from human skeletal muscle in situ and in vitro.

Human skeletal muscle is an essential source of various cellular progenitors with potential therapeutic perspectives. We first used extracellular markers to identify in situ the main cell types located in a satellite position or in the endomysium of the skeletal muscle. Immunohistology revealed labeling of cells by markers of mesenchymal (CD13, CD29, CD44, CD47, CD49, CD62, CD73, CD90, CD105, CD146, and CD15 in this study), myogenic (CD56), angiogenic (CD31, CD34, CD106, CD146), hematopoietic (CD10, CD15, CD34) lineages. We then analysed cell phenotypes and fates in short- and long-term cultures of dissociated muscle biopsies in a proliferation medium favouring the expansion of myogenic cells. While CD56(+) cells grew rapidly, a population of CD15(+) cells emerged, partly from CD56(+) cells, and became individualized. Both populations expressed mesenchymal markers similar to that harboured by human bone marrow-derived mesenchymal stem cells. In differentiation media, both CD56(+) and CD15(+) cells shared osteogenic and chondrogenic abilities, while CD56(+) cells presented a myogenic capacity and CD15(+) cells presented an adipogenic capacity. An important proportion of cells expressed the CD34 antigen in situ and immediately after muscle dissociation. However, CD34 antigen did not persist in culture and this initial population gave rise to adipogenic cells. These results underline the diversity of human muscle cells, and the shared or restricted commitment abilities of the main lineages under defined conditions.

Co-Treatment with the Epigenetic Drug, 3-Deazaneplanocin A (DZNep) and Cisplatin after DZNep Priming Enhances the Response to Platinum-Based Therapy in Chondrosarcomas.

BACKGROUND: We have previously shown that 3-Deazaneplanocin A (DZNep) induces apoptosis in chondrosarcomas. Herein, we tested whether the combination of this epigenetic drug to a standard anticancer therapy may enhance the response to each drug in these bone tumors. METHODS: Two chondrosarcoma cell lines (SW1353 and JJ012) were cultured in the presence of DZNep and/or cisplatin. Cell growth was evaluated by counting viable cells, and apoptosis was determined by Apo2.7 expression by flow cytometry. In vivo, the antitumoral effect of the DZNep/cisplatin combination was assessed through measurements of tumor volume of JJ012 xenografts in nude mice. RESULTS: In vitro, the DZNep/cisplatin combination reduced cell survival and increased apoptosis compared to each drug alone in chondrosarcomas, but not in normal cells (chondrocytes). This enhancement of the antitumoral effect of the DZNep/cisplatin combination required a priming incubation with DZNep before the co-treatment with DZNep/cisplatin. Furthermore, in the chondrosarcoma xenograft mice model, the combination of both drugs more strongly reduced tumor growth and induced more apoptosis in tumoral cells than each of the drugs alone. CONCLUSION: Our results show that DZNep exposure can presensitize chondrosarcoma cells to a standard anticancer drug, emphasizing the promising clinical utilities of epigenetic-chemotherapeutic drug combinations in the future treatment of chondrosarcomas.

The benefit of combining curcumin, bromelain and harpagophytum to reduce inflammation in osteoarthritic synovial cells.

BACKGROUND: Osteoarthritis (OA) is the most common form of arthritis, affecting millions of people worldwide and characterised by joint pain and inflammation. It is a complex disease involving inflammatory factors and affecting the whole joint, including the synovial membrane. Since drug combination is widely used to treat chronic inflammatory diseases, a similar strategy of designing plant-derived natural products to reduce inflammation in OA joints may be of interest. In this study, we characterised the response of OA synovial cells to lipopolysaccharide (LPS) and investigated the biological action of the combination of curcumin, bromelain and harpagophytum in this original in vitro model of osteoarthritis. METHODS: Firstly, human synovial cells from OA patients were stimulated with LPS and proteomic analysis was performed. Bioinformatics analyses were performed using Cytoscape App and SkeletalVis databases. Additionally, cells were treated with curcumin, bromelain and harpagophytum alone or with the three vegetal compounds together. The gene expression involved in inflammation, pain or catabolism was determined by RT-PCR. The release of the encoded proteins by these genes and of prostaglandin E2 (PGE2) were also assayed by ELISA. RESULTS: Proteomic analysis demonstrated that LPS induces the expression of numerous proteins involved in the OA process in human OA synovial cells. In particular, it stimulates inflammation through the production of pro-inflammatory cytokines (Interleukin-6, IL-6), catabolism through an increase of metalloproteases (MMP-1, MMP-3, MMP-13), and the production of pain-mediating neurotrophins (Nerve Growth Factor, NGF). These increases were observed in terms of mRNA levels and protein release. LPS also increases the amount of PGE2, another inflammation and pain mediator. At the doses tested, vegetal extracts had little effect: only curcumin slightly counteracted the effects of LPS on NGF and MMP-13 mRNA, and PGE2, IL-6 and MMP-13 release. In contrast, the combination of curcumin with bromelain and harpagophytum reversed lots of effects of LPS in human OA synovial cells. It significantly reduced the gene expression and/or the release of proteins involved in catabolism (MMP-3 and -13), inflammation (IL-6) and pain (PGE2 and NGF). CONCLUSION: We have shown that the stimulation of human OA synovial cells with LPS can induce protein changes similar to inflamed OA synovial tissues. In addition, using this model, we demonstrated that the combination of three vegetal compounds, namely curcumin, bromelain and harpagophytum, have anti-inflammatory and anti-catabolic effects in synovial cells and may thus reduce OA progression and related pain.

Description of Joint Alterations Observed in a Family Carrying p.Asn453Ser COMP Variant: Clinical Phenotypes, In Silico Prediction of Functional Impact on COMP Protein and Stability, and Review of the Literature.

The role of genetics in the development of osteoarthritis is well established but the molecular bases are not fully understood. Here, we describe a family carrying a germline mutation in COMP (Cartilage Oligomeric Matrix Protein) associated with three distinct phenotypes. The index case was enrolled for a familial form of idiopathic early-onset osteoarthritis. By screening potential causal genes for osteoarthritis, we identified a heterozygous missense mutation of COMP (c.1358C>T, p.Asn453Ser), absent from genome databases, located on a highly conserved residue and predicted to be deleterious. Molecular dynamics simulation suggests that the mutation destabilizes the overall COMP protein structure and consequently the calcium releases from neighboring calcium binding sites. This mutation was once reported in the literature as causal for severe multiple epiphyseal dysplasia (MED). However, no sign of dysplasia was present in the index case. The mutation was also identified in one of her brothers diagnosed with MED and secondary osteoarthritis, and in her sister affected by an atypical syndrome including peripheral inflammatory arthritis of unknown cause, without osteoarthritis nor dysplasia. This article suggests that this mutation of COMP is not only causal for idiopathic early-onset osteoarthritis or severe MED, but can also be associated to a broad phenotypic variability with always joint alterations.

Heterogeneity of chondrosarcomas response to irradiations with X-rays and carbon ions: A comparative study on five cell lines.

OBJECTIVES: Chondrosarcomas are malignant bone tumors considered as resistant to radiotherapy. To unravel mechanisms of resistance, we compared biological responses of several chondrosarcomas to X-ray irradiations in normoxia and hypoxia. Since hadrontherapy with Carbon-ions gave interesting clinical outcomes, we also investigated this treatment in vitro. METHODS: Five human chondrosarcoma cell lines were used and cultured in normoxia or hypoxia. Their sensitivities to irradiations were determined by carrying out survival curves. DNA damage was monitored by γH2AX expression. Apoptosis was assessed by cell cycle analysis and Apo2.7 expression, and by evaluating PARP cleavage. Senescence was evaluated using SA ß-galactosidase assay. Necrosis, and autophagy, were evaluated by RIP1 and beclin-1 expression, respectively. Mutations in relevant biological pathways were screened by whole-exome sequencing. RESULTS: X-ray radiations induced death in some chondrosarcomas by both apoptosis and senescence (CH2879), or by either of them (SW1353 and JJ012), whereas no death was observed in other cell lines (FS090 and 105KC). Molecularly, p21 was overexpressed when senescence was elicited. Genetic analysis allowed to identify putative genes (such as TBX3, CDK2A, HMGA2) permitting to predict cell response to irradiations. Unexpectedly, chronic hypoxia did not favor radioresistance in chondrosarcomas, and even increased the radiosensitivity of JJ012 line. Finally, we show that carbon ions triggered more DNA damages and death than X-rays. CONCLUSIONS: Chondrosarcomas have different response to irradiation, possibly due to their strong genetic heterogeneity. p21 expression is suggested as predictive of X-ray-induced senescence. Surprisingly, hypoxia does not increase the radioresistance of chondrosarcomas, but as expected Carbon ion beams are more effective that X-rays in normoxia, whereas their efficiency was also variable depending on cell lines.

EZH2 inhibition reduces cartilage loss and functional impairment related to osteoarthritis.

Histone methyltransferase EZH2 is upregulated during osteoarthritis (OA), which is the most widespread rheumatic disease worldwide, and a leading cause of disability. This study aimed to assess the impact of EZH2 inhibition on cartilage degradation, inflammation and functional disability. In vitro, gain and loss of EZH2 function were performed in human articular OA chondrocytes stimulated with IL-1ß. In vivo, the effects of EZH2 inhibition were investigated on medial meniscectomy (MMX) OA mouse model. The tissue alterations were assayed by histology and the functional disabilities of the mice by actimetry and running wheel. In vitro, EZH2 overexpression exacerbated the action of IL-1ß in chondrocytes increasing the expression of genes involved in inflammation, pain (NO, PGE2, IL6, NGF) and catabolism (MMPs), whereas EZH2 inhibition by a pharmacological inhibitor, EPZ-6438, reduced IL-1ß effects. Ex vivo, EZH2 inhibition decreased IL-1ß-induced degradation of cartilage. In vivo, intra-articular injections of the EZH2 inhibitor reduced cartilage degradation and improved motor functions of OA mice. This study demonstrates that the pharmacological inhibition of the histone methyl-transferase EZH2 slows the progression of osteoarthritis and improves motor functions in an experimental OA model, suggesting that EZH2 could be an effective target for the treatment of OA by reducing catabolism, inflammation and pain.

Comparative proteomic analysis of human mesenchymal and embryonic stem cells: towards the definition of a mesenchymal stem cell proteomic signature.

Mesenchymal stem cells (MSC) are adult multipotential progenitors which have a high potential in regenerative medicine. They can be isolated from different tissues throughout the body and their homogeneity in terms of phenotype and differentiation capacities is a real concern. To address this issue, we conducted a 2-DE gel analysis of mesenchymal stem cells isolated from bone marrow (BM), adipose tissue, synovial membrane and umbilical vein wall. We confirmed that BM and adipose tissue derived cells were very similar, which argue for their interchangeable use for cell therapy. We also compared human mesenchymal to embryonic stem cells and showed that umbilical vein wall stem cells, a neo-natal cell type, were closer to BM cells than to embryonic stem cells. Based on these proteomic data, we could propose a panel of proteins which were the basis for the definition of a mesenchymal stem cell proteomic signature.

Epigenetic dynamic during endochondral ossification and articular cartilage development.

Within the last decade epigenetics has emerged as fundamental regulator of numerous cellular processes, including those orchestrating embryonic and fetal development. As such, epigenetic factors play especially crucial roles in endochondral ossification, the process by which bone tissue is created, as well during articular cartilage formation. In this review, we summarize the recent discoveries that characterize how DNA methylation, histone post-translational modifications and non-coding RNA (e.g., miRNA and lcnRNA) epigenetically regulate endochondral ossification and chondrogenesis.

Interleukin-1 and transforming growth factor-beta 1 as crucial factors in osteoarthritic cartilage metabolism.

In osteoarthritis (OA), interleukin-1 (IL-1) stimulates the expression of metalloproteinases and aggrecanases, which induce cartilage degradation. IL-1 is also capable of reducing the production of cartilage-specific macromolecules, including type II collagen, through modulation of the transcription factors Sp1 and Sp3. Conversely, Transforming growth factor-beta (TGF-beta) counteracts with most of the IL-1 deleterious effects and contributes to cartilage homeostasis. However, OA chondrocytes progressively loose the expression of TGF-beta type II receptor and become insensitive to the factor. This downregulation is also driven by IL-1. This review provides insights into the molecular mechanisms that underly the interplay between IL-1 and TGF-beta in OA cartilage metabolism and enlightens the central role of Sp1 and Sp3 transcription factors in the matrix pathological alterations.