Purpose: In recent years chemicals with endocrine properties, called Endocrine Disrupting Compounds (EDC), have become a topic of scientific and public discussion. In the field of osteoarthritic pathologies, EDC compounds that mimic estrogenic agonists or antagonists are of major concern, because of the central role of estrogens in skeletal developmental processes (ref). EDC are acting on their target genes upon binding the aryl hydrocarbon receptor (AhR). During the ten past years, interactions between estrogen receptor (ER) and some xenobiotic-activated AhR have been well demonstrated. Very few studies have been reported on EDC effects on bone or cartilage. Our laboratory recently studied the effects of low doses and cocktail xenoestrogens on rat cartilage development in vivo, by using a rodent gestational/lactational model (T.A. Auxietre, et al, 2014). We observed transient modifications of caudal vertebral body associated with a decrease in growth plate cartilage thickness with greater impact on the hypertrophic chondrocyte zone. We inferred from these data that the tested compounds could interfere with the dynamic of chondrogenic differentiation and/or maturation processes, possibly by impacting type 2 collagen (Col2), one of the the major components of cartilage that assures cartilage function. The present work was aimed to study the in vitro effects of two xenoestrogens (genistein, G; bisphenol A, BPA) and one anti-androgen (vinclozolin, V) and its metabolite M2 on Col2 expression in two different models of cultured chondrocytes: during the process of chondrogenic induction and in mature chondrocytes. Methods: A murine stem cell line inducible towards chondrogenesis (C1) was used for dynamic studies of differentiation using chondrogenic and non-chondrogenic markers. In parallel, post-natal murine chondrocytes were used for steady-state investigations in differentiated chondrocytes in primary culture (P0) or after dedifferentiation by passages (P3) or FGF2 treatment. Biological markers were studied at the mRNA (qPCR) and protein (western blot) levels. COL2A protein expression was evaluated by western blot and immunocytochemistry by using an antibody which only recognizes the specific COL2A N-terminal propeptide. Fulvestrant and SB 203580 were used respectively as specific inhibitors of ER or p38MEK pathways. Implication of Tia1-splicing was shown after cell transfection with specific anti-Tia1 siRNAs. Results: Vinclozolin was practically inactive. M2, alone or combined with G or BPA, modified the dynamic of Col2A immature isoform of COL2 during chondrogenic induction. These compounds extended the basal expression of COL2A and delayed its replacement by the mature isoform COL2B in C1 cells. This effect was dose dependent with maximum at 10-6M. In post natal chondrocytes, COL2A expression increased with cell dedifferentiation or FGF2 treatment. EDC were inactive on differentiated chondrocytes while COL2A doubled upon M2, G and BPA addition in dedifferentiated cells. Estrogen receptor (ER) and the p38-MEK pathway were involved. As these effects were only partly transcriptional, we investigated and found an effect of FGF2 on Tia-1 splice protein expression. EDC showed no effects on other chondrogenic markers (SOX9, Aggrecan, Col10α1) nor non-chondrogenic markers. Conclusions: These data showed that xenosestrogens as well as the vinclozolin metabolite M2 modified the rate of early chondrogenic differentiation by transiently maintaining COL2A expression. Our hypothesis is that COL2A induced persistence, might provoke matrix weakening in the long term and/or sequester growth factors, eventually modifying the course of cartilage degenerative diseases such as osteoarthritis. EDC also modified COL2A expression in dedifferentiated chondrocytes such as found in aging or osteoarthritic tissues. These data are in accordance with the strong Col2A expression reported in OA cartilage but its role remains to be studied.

Purpose:The purpose of this study was to address the question of how subchondral bone osteoblasts (SBOs) and articular cartilage chondrocytes (ACCs) interact with each other with respect to regulation of respective cells' phenotypic properties and in particular the involvement of mitogen activated protein kinase (MAPK) signalling pathways under normal and OA joint condition.We also endeavoured to test the influence of cross-talk between SBOs and ACCs isolated from normal and OA joint on matrix metalloproteinase (MMP) expression.Methods: Tissues from the knees of OA patients and normal controls were collected to isolate SBOs and ACCs.The cellular cross-talk of SBOs and ACCs were studied by means of both direct and indirect co-culture systems, which made it possible to identify the role of both membrane bound and soluble factors.Histology, immunohistochemistry, qRT-PCR, zymography, ELISA and western blotting were some of the techniques applied to distinguish the changes in the co-cultured vs. non co-cultured cells.The MAPK signalling pathways were probed by using targeted MAPK inhibitors, and their activity monitored by western blot analysis using phospho MAPK specific antibodies.Results: Our co-culture studies demonstrated that OA ACCs enhanced the SBOs differentiation compared to normal ACCs.We demonstrated that OA ACCs induced these phenotypic changes in the SBOs via activating an ERK1/2 signalling pathway.The findings from this study thus provided clear evidence that OA ACCs play an integral role in altering the SBO phenotype.In the second study, we tested the influence of normal SBOs and OA SBOs on ACCs phenotype changes.The results showed that OA SBOs increased the hypertrophic gene expression in co-cultured ACCs compared to normal SBOs, a phenotype which is considered as pathological to the health and integrity of articular cartilage.It was observed that these phenotype changes occurred via de-activation of p38 and activation of ERK1/2 signaling pathways.These findings suggest that the pathological interaction of OA SBOs with ACCs is mediated by cross-talking between ERK1/2 and p38 pathways, resulting in ACCs undergoing hypertrophic differentiation.Subsequent experiments to determine the effect on MMP regulation, of SBOs and ACCs cross-talk, revealed that co-culturing OA SBOs with ACCs significantly enhanced the proteolytic activity and expression of MMP-2 and MMP-9.In turn, co-culture of OA ACCs with SBOs led to abundant MMP-2 expression in SBOs.Furthermore, we showed that the addition of ERK1/2 and JNK inhibitors reversed the elevated MMP-2 and MMP-9 production which otherwise resulted from the interactions of OA SBOs-ACCs.Thus, this study has demonstrated that the altered interactions between OA SBOs-ACCs are capable of triggering the pathological pathways leading to degenerative changes seen in the osteoarthritic joint.Conclusions: Our results has given clear in vitro evidence that the altered bi-directional communication of SBOs and ACCs may play a role in OA development and that this process was mediated by MAPK signalling pathways.Targeting these altered interactions by the use of MAPK inhibitors may provide the scientific rationale for the development of novel therapeutic strategies in the treatment and management of OA.

Poster Presentations / Osteoarthritis and Cartilage 18, Supplement 2 (2010) S45-S256 were determined by semi quantitative RT-PCR.In two patients, a biopsy of the repair tissue was taken 6 and 18 months after ACI.Results: In total, 0.81±0.33×10 6cells per gram tissue could be recovered with no difference between dissecate and notch cartilages (cell viability ≥ 90%).Compared with the notch chondrocytes, cells from the dissecate expressed similar levels of collagen type I and -II mRNA including a 100,000fold relative increase of col1 over col2 after cell expansion.Expression of collagen type X mRNA is significantly less in trauma joints compared to OCD cartilages before and after cell culture.The level of collagen type X message is approx.50fold decreased after cell culture, indicating a loss of hypertrophic cells or expression of hypertrophic genes in chondrocytes.Post-implantation biopsies show features of hyaline-like cartilage without signs of hypertrophy or mineralization.Conclusions: The high viability, quality and activity of the extracted cells suggest a still preserved intrinsic repair capacity of the dissecates.The molecular analysis indicates phenotypic modulation of the isolated chondrocytes during cell culture.The similar quality of the cells from both dissecate and notch after cell culture suggests the use of either cartilages as a cell source for ACI.

Objective: To assess the effect of natural chondroitin sulphate (CS) on the ability of neosynthesised sulphated proteoglycans (PGs) to aggregate in cultured chondrocytes treated with interleukin (IL)1β. Methods: Primary cultured rabbit articular chondrocytes were treated or not with IL1β alone or with concentrations of CS for 20 h. Neosynthesised PGs were labelled by incorporation of [35SO4]-sulphate and analysed by chromatography on Sepharose 2B columns. Gelatinolytic activity was measured by zymography, and matrix metalloproteinase (MMP)1 mRNA level in chondrocytes underwent real-time PCR. Expression of ADAMTS (for "a disintegrin and metalloproteinase with thrombospondin motifs") -4 and -5 was analysed by real-time PCR and western blotting. Results: The production of [35SO4]-labelled PGs was significantly increased with 10 μg/ml CS in the cellular pool rather than in the incubation medium. The addition of CS to IL1β-treated cells inhibited in part the disaggregation of sulphated PGs induced by IL1β. This inhibitory effect of CS is associated with a significant decrease in ADAMTS-5 expression at the mRNA and protein levels. No effect of CS was observed on IL1β-induced gelatinolytic activity, MMP1 mRNA expression or ADAMTS-4 expression. Conclusion: CS increases the production of functional sulphated PGs in the direct environment of chondrocytes in vitro. This beneficial effect of CS in IL1β-treated cells is associated with decreased expression of ADAMTS-5.

Rheumatoid arthritis (RA) is a connective tissue disease characterized by destruction of the joint cartilage and subsequently of the underlying bone. Cartilage destruction is due to proteolysis by enzymes called metalloproteinases (MMPs), whose production and expression are regulated by numerous local mediators such as cytokines, growth factors, prostaglandins, oxygen species, and neuropeptides. MMP activation is largely due to a stimulatory effect of cytokines including IL-1beta and TNFalpha. When these cytokines bind to their membrane receptor, they set off signaling cascades, with activation of TGFbeta-activating kinase (TAK-1), of NF-kappaB by Ikappa-B kinase, of mitogen-activated protein kinases (MAP kinases), and finally of activator protein-1 (AP-1). Tissue inhibitors of MMPs (TIMPs) specifically inhibit MMPs. The interrelations between joint inflammation and joint destruction remain poorly understood. Experimental data suggest that IL-1 may be involved chiefly in joint destruction and TNF in joint inflammation. However, TNF antagonists are potent inhibitors of joint destruction in clinical practice. These results suggest that the mediators function as a network and that inhibition of a single mediator can affect the entire web. Insights gained into the innermost mechanisms of cartilage breakdown in patients with RA have led to major therapeutic breakthroughs. Thus, TNF antagonists have proved highly effective in RA. Future progress will no doubt stem from new knowledge about the extracellular mediators and intracellular signaling pathways that lead to the production and activation of enzymes responsible for cartilage degradation.

The cartilage tissue has a limited self-regenerative capacity. Tissue-engineering represents a promising trend for cartilage repair. The present study was aimed to develop a biomaterial formulation by combining fragments of chitosan hydrogel with isolated rabbit or human chondrocytes. We first reported the properties of the constructs elaborated with rabbit chondrocytes and pure chitosan physical hydrogels with defined molecular weight, acetylation degree and polymer concentration. Morphological data showed that chondrocytes were not penetrating the hydrogels but tightly bound to the surface of the fragments and spontaneously formed aggregates of combined cell/chitosan. A significant amount of neo-formed cartilage-like extracellular matrix (ECM) was first accumulated in-between cells and hydrogel fragments and furthermore was widely distributed within the neo-construct. The optimal biological response was obtained with hydrogel fragments concentrated at 1.5% (w/w) of polymer made from a chitosan with a degree of acetylation between 30 and 40%. Such hydrogels were then mixed with human chondrocytes. The phenotype of the cells was analyzed by using chondrocytic (mRNA expression of mature type II collagen and aggrecan as well as secretion of proteoglycans of high molecular weight) and non chondrocytic (mRNA expression of immature type II collagen and type I collagen) molecular markers. As compared with human chondrocytes cultured without chitosan hydrogel which rapidly dedifferentiated in primary culture, cells mixed with chitosan rapidly loose the expression of type I and immature type II collagen while they expressed mature type II collagen and aggrecan. In these conditions, chondrocytes maintained their phenotype for as long as 45 days, thus forming cartilage-like nodules. Taken together, these data suggest that a chitosan hydrogel does not work as a scaffold, but could be considered as a decoy of cartilage ECM components, thus favoring the binding of chondrocytes to chitosan. Such a biological response could be described by the concept of reverse encapsulation.

Antiinflammatory effects by peroxisome proliferator-activated receptor gamma (PPARgamma) agonists have been previously reported. However, PPARgamma dependency and the molecular mechanism involved in these effects require more investigation to clearly demonstrate whether PPARgamma is a key modulator of the antiinflammatory process. This would permit the design of more specific agonists or antagonists able to address the gamma subtype without cross reactions with other transcription factors, thus preventing undesirable side effects. However, several hurdles need to be taken into consideration, such as the coexpression of several PPAR isotypes in the same cell type. As PPARgamma and -alpha seem to play equal antiinflammatory roles, determining the subset of specific PPAR subtype target genes appears to be crucial. The work described here is our current understanding of the modulations of interleukin-1 target gene expression by PPARgamma and its ligands.

Aryl hydrocarbon receptor (AhR) ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or benzo(a)pyrene interfere with hormonal regulatory pathways, leading to endocrine disruption. Notably, the activated AhR exerts complex effects on estrogens and retinoids at both levels of their metabolism and regulation of cognate genes. Our current investigation of these AhR effects revealed the TCDD-dependent activation of a subset of retinoid-dependent genes (tissue-transglutaminase, IGF binding protein-3, AhR) in MCF-7 breast cancer cells. A collection of in vitro hormone-dependent reporter gene models showed that AhR activation by TCDD stimulated transactivation by several class I heteromeric receptors (retinoic and thyroid hormone receptors) while it antagonized homodimeric nuclear receptors (estrogen and progesterone receptors, ER and PR). TCDD exerted a dose-dependent effect on a retinoic acid-dependent reporter gene expressed in MCF-7 cells. AhR was shown to be involved in a mutual antagonism with RARα corepressor SMRT (silencing mediator of retinoid and thyroid receptors). This, and the documented physical interaction between AhR and SMRT suggested that SMRT sequestration by AhR might activate RARα in the absence of ligand. Immunocytochemical studies of AhR and SMRT strongly suggested they colocalized in nuclear bodies during this sequestration. Concurring with this interpretation, we observed an interaction in vitro between AhR and the PML protein, the core component of nuclear bodies. This ability of AhR to elicit spurious activation of retinoid receptors expands the scope of AhR ligands influence beyond ER antagonism and specific Dioxin-responsive genes. Unknown AhR endogenous ligands may also elicit gene transactivation by class I receptors, while being inactive on classic xenobiotic-responsive genes.

On propose une méthode de détermination du cadmium, de l'indium et du tellure par polarographie impulsionnelle différentielle. L'électrolyte support requis est KI 0,1 M dans l'acide tartrique saturé et doit être préparé sous azote. Dans ce milieu les pics de Cd, In et Te sont bien séparés et se prêtent au dosage sans séparation préalable. Le cadmium et le tellure présentent des interférences mutuelles mais le dosage reste précis. L'indium n'interfére pas. Une application au composé CdIn2Te4 est décrite.Cadmium, indium and tellurium can be determined without preliminary separation by differential pulse polarography in a specially prepared supporting electrolyte of 0.1 M KI saturated with tartaric acid. In this medium the three peaks are well separated. There is no interference for Cd—In and In—Te mixtures, but Cd—Te mixtures can prove difficult. However, the peak intensity of tellurium is over ten times that of cadmium for the same concentration, and it is possible to determine tellurium near the limit of detection. Cadmium is determined afterwards in more concentrated solution. Indium does not interfere. An application to CdIn2Te4 is reported.

Interleukin-1β (IL-1β) induces degradation via hyperexpression of an array of genes, including metalloproteinases (MMP), in cartilage cells during articular degenerative diseases. In contrast, natural ligands for peroxisome proliferator-activated receptors (PPARs) display protective anti-cytokine effects in these cells. We used the PPAR agonist rosiglitazone (Rtz) to investigate PPAR-γ isotype on IL-1β-target genes. Immunocytochemistry, electrophoretic mobility shift, and transient transfection assays revealed a functional PPAR-γ in chondrocytes in vitro. Rtz displayed significant inhibition of IL-1β effects in chondrocytes. Low Rtz concentrations (close to Kd values for PPAR-γ, 0.1 to 1 μm) inhibited the effects of IL-1β on 35S-sulfated proteoglycan production and gelatinolytic activities and downregulated MMP1 expression at mRNA and protein levels. We have investigated the mechanism of action of Rtz against IL-1β-mediated MMP1 gene hyperexpression. Rtz effect occurs at the transcriptional level of the MMP1 promoter, as observed in transiently transfected cells with pMMP1-luciferase vector. Transient expression of wild type PPAR-γ enhanced Rtz inhibitory effect in chondrocytes, whereas a mutated dominant negative PPAR-γ abolished it, supporting the role of PPAR-γ in this effect. MMP1 gene promoter analysis revealed the involvement of a cis-acting element located at -83 to -77, shown to be a composite PPRE/AP1 site. Gel mobility and supershift assays demonstrated that PPAR-γ and c-Fos/c-Jun proteins bind this cis-acting element in a mutually exclusive way. Our data highlight a new PPAR-γ-dependent inhibitory mechanism on IL-1β-mediated cartilage degradation occurring through DNA binding competition on the composite PPRE/AP1 site in the MMP1 promoter.

OBJECTIVE:To determine whether 17beta-oestradiol (E2) modulates interleukin (IL) 1beta-induced proteoglycan degradation in chondrocytes, and to analyse the part played by metalloproteinases (MMPs) in this process.METHODS:Primary cultured rabbit articular chondrocytes were prepared and treated with 10 ng/ml IL1beta combined or not with 0.1-10 nM E2. Neosynthesised proteoglycans (PGs) were evaluated after incorporation of [(35)SO(4)]sulphate and further analysed after chromatography on a Sepharose 2B column. Chondrocyte mRNA levels of aggrecan, MMP-1, -3, -13, and tissue inhibitor of metalloproteinase-1 (TIMP-1) were studied by northern blot. MMP-1 activity was measured by zymography. MMP-1 gene transcription was studied by transient transfection of chondrocytes with an MMP-1-luciferase construct.RESULTS:E2 modulated the IL1beta-induced total sulphated PGs in rabbit articular chondrocytes, which decreased as the E2 concentration was increased. At a low concentration (0.1 nmol/l) E2 counteracts the IL1beta-induced decrease in sulphated PG, while at high concentration (10 nmol/l) E2 enhances the IL1beta effects. A biphasic E2 effect was also observed on IL1beta-induced disaggregation of PG, 53-58 kDa gelatinolytic activity, and MMP-1, -3, and -13 mRNA levels. In contrast, E2 did not modify the level of aggrecan mRNA and had no effect on TIMP-1 mRNA expression. Finally, simultaneous addition of IL1beta and E2 (0.1-10 nmol/l) did not modify IL1beta-induced MMP-1-luciferase activity, suggesting that E2 effects probably occur at the post-transcriptional level of MMP gene expression.CONCLUSION:Oestrogen concentration may have an inverse effect on IL1beta stimulated proteoglycan degradation and MMP production by chondrocytes.

Matrix Gla protein (MGP) is a member of the vitamin K-dependent gamma carboxylase protein family expressed in cartilage. Insulin-like growth factor I (IGF1) stimulates chondrocyte differentiation, whereas basic fibroblast growth factor (FGF2) acts in an opposite manner. We explored the differential expression and regulation by IGF1 and FGF2 of the MGP gene during chondrocyte differentiation. We used a primary culture system of rabbit epiphyseal chondrocytes to show that MGP mRNA is mainly expressed during serum-induced proliferation. Much lower MGP mRNA content is observed in post-mitotic chondrocytes, which newly express alpha 1X procollagen mRNA, a marker of late-differentiated cells. From studies of a series of growth factors, it was shown that IGF1 decreased chondrocyte MGP transcripts, whereas FGF2 had the opposite effect. FGF2 stimulated chondrocyte MGP production in a dose- and time-dependent manner at the mRNA and protein levels. FGF2 acted in a dose- and time-dependent manner, reaching a maximum at 10 ng/ml at 20 h. The protein synthesis inhibitor cycloheximide did not modify FGF2 action, in agreement with a direct effect. Actinomycin D abolished FGF2-induced stimulation, strongly suggesting that FGF2 modulated MGP gene transcription. We transiently transfected chondrocytes with a construct containing the mouse MGP promoter from -5000 to -168 base pairs, relative to the transcription start site of the gene linked to the luciferase gene (MGP-Luc). In transfected cells, FGF2 stimulated luciferase activity up to sevenfold while IGF1 had no effect. Hence, FGF2 induces transcription of the MGP gene via the 5'-flanking region of the gene. Using a series of deleted MGP-Luc constructs, we identified a sequence of 748 base pairs which was sufficient for transcriptional activation by FGF2. These results led us to postulate that the inhibitory chondrogenic action of FGF2 involves a mechanism whereby MGP gene transcription and protein are induced.