Search

Objectives: To investigate the effects of interleukin 1beta (IL1beta) treatment on the Notch1/Hes1 pathway in chondrocytes in vitro. Methods: Mouse articular chondrocytes in primary culture were challenged with IL1beta, alone or combined with Notch1 and IL1beta pathway inhibitors. Notch1 and Hes1 expressions were investigated by immunocytochemistry, western blot and real-time quantitative (q)PCR. IL1beta-responsive genes were assessed by real-time qPCR and a specific siRNA against Hes1 was used to identify Hes1 target genes. Results: Notch1 labelling remained nuclear and stable in intensity irrespective of treatment, suggesting a steady state activation of this pathway in our model. IL1beta transiently increased Hes1 mRNA (2.5-fold) and protein expression in treated versus naive chondrocytes. Hes1 mRNA level then decreased below control and its cyclic pattern of expression was lost. This was associated with nuclear translocation of the cytoplasmic Hes1 protein. IL1beta induced increase in Hes1 mRNA was transcriptional, occurred through nuclear factor (NF)kappaB activation and appeared to be associated with downregulation by its own protein. Hes1 induction was insensitive to the gamma-secretase inhibitor N-(N-(3,5-difluorophenacetyl)-l-alanyl)-S-phenylglycine t-butyl ester (DAPT), which suggested its independence from novel Notch1 activation. Hes1 expression was efficiently silenced by a specific siRNA. This experiment revealed that Hes1 did not mediate IL1beta-induced downregulation of Sox9, type II collagen and aggrecan transcription but mediated IL1beta induction of matrix metalloproteinase (MMP)13 and ADAM metallopeptidase with thrombospondin type 1 motif, 5 (ADAMTS5). The Hes1-related repressor Hey1 was expressed at a very low level and was not inducible by IL1beta. Conclusion: Hes1 is a novel IL1beta target gene in chondrocytes which influences a discrete subset of genes linked to cartilage matrix remodelling and/or degradation. [ABSTRACT FROM AUTHOR]

Several endocrine disrupting compounds (EDC) elicit skeletal dysgenesis at pharmacological doses. We have investigated the impact of doses below the "No Observed Adverse Effect" (NOAEL) for vinclozolin (V), an anti-androgenic fungicide, alone or associated with xenoestrogens (Genistein, G and bisphenol-A, BPA). V, G, BPA and their combinations were administered orally to female Wistar rats during gestation and lactation. F1 and F2 offspring were investigated for skeletal anomalies at post-natal days 30, 110 (d30, d110). Skeletal development was monitored by measuring caudal vertebrae and long bones dimensions by X-ray micro-CT-scan. A significant increase in Inter Transverse Apophysis (ITA) distance at the upper head of caudal vertebrae, associated with a reduction in vertebral body height was observed in treated F1 females, but not males. Histometrical analysis of vertebral body growth plate cartilage was performed on serial sections of caudal vertebrae. F1 females but not males showed a diminution in growth plate thickness, with greater impact on the hypertrophic zone. All effects were maximal at d30. Effects on ITA width persisted until d110 while effects on growth plate disappeared. These effects were essentially vinclozolin or BPA-dependent. F2 animals were not affected. Our data suggest that vinclozolin and xenoestrogens act as cartilage developmental disruptors. We suggest that present NOAEL values for these compounds, and EDC at large, might be reconsidered using gestational exposure models. Finally, micro CT-scan appears a valuable non-invasive technique to detect EDC effects on live fauna., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

Objectives: To investigate the presence and functionality of oestrogen receptor alpha (ERalpha) in interleukin (IL)1beta-treated rabbit articular chondrocytes in culture, and to determine the mechanisms of 17beta oestradiol (E2) effects on IL1beta-induced inducible nitric oxide synthase (iNOS) expression., Methods: The presence and functionality of ERalpha were investigated by immunocytochemistry and transient expression of an E2-responsive reporter construct. iNOS expression and production were determined by transient expression of a chimeric iNOS promoter-luciferase construct and protein immunoblotting. Nitric oxide (NO) production was determined by the Griess reaction. DNA-binding activities of nuclear factor-kappaB (NF-kappaB) and activated protein 1 were determined by electrophoretic mobility shift assay (EMSA)-ELISA assays. Nuclear translocation of p65 was studied by immunocytochemistry., Results: ERalpha was identified in the nucleus of chondrocytes. ERalpha efficiently transactivated a transiently expressed E2-responsive construct. On IL1beta treatment, ERalpha partially diffused from its nuclear localisation into the cytoplasm and its transactivation ability was impaired. Nevertheless, E2, tamoxifen and raloxifene efficiently inhibited IL1beta-induced NO production (-34%, -31% and -36%, respectively). E2 decreased IL1beta-induced iNOS protein expression (-40%). Transient expression of an iNOS promoter construct strongly suggested that iNOS expression was inhibited at the transcriptional level, and EMSA-ELISA assays showed that E2 reduced (-60%) the IL1beta-induced p65 DNA-binding capacity. Finally, the p65 nuclear translocation induced by IL1beta was also strongly decreased by E2., Conclusions: Our data support a reciprocal antagonism between oestrogens and IL1beta, ultimately resulting in the decrease of cytokine-dependent NO production through transcriptional inhibition of iNOS expression. This effect was associated with selective inhibition of p65 DNA binding and nuclear translocation.

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 RARalpha 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 RARalpha 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.

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.

Study Design: Anulus fibrosus cells from rabbits were grown in primary culture 1) to study their ability to produce prostaglandin E2 and Type II phospholipase A2, and to express stromelysin-1 messenger ribonucleic acid; and 2) to study the effect of interleukin 1 beta on this production and on proteoglycan aggregation., Objectives: To investigate the potency of anulus fibrosus cells to respond to interleukin 1 beta by producing degradative and inflammatory agents as compared with the potency of articular chondrocytes in the same animal., Summary of Background Data: Interleukin 1 beta has been implicated in the degradation of intervertebral discs. The way anulus fibrosus cells differ from articular chondrocytes in their responses to interleukin 1 beta remains to be established., Methods: Anulus fibrosus cells and articular chondrocytes were obtained from young rabbits, grown in primary culture, and incubated with interleukin 1 beta. The newly synthesized proteoglycan was measured by labeling with [35S]-sulfate. Proteoglycan aggregation was analyzed by the elution profile on Sepharose 2B columns. The contents of collagen Type II and stromelysin-1 messenger ribonucleic acid were assessed by Northern blot analysis. The Type II phospholipase A2 activity was measured using a fluorometric substrate. Prostaglandin E2 production was evaluated by radioimmunoassay., Results: Anulus fibrosus cells had 2.5-fold less Type II collagen messenger ribonucleic acid than articular chondrocytes, and interleukin 1 beta had no significant effect on this. Anulus fibrosus cells synthesized and secreted four-fold less proteoglycan than articular chondrocytes. Interleukin 1 beta reduced the anulus fibrosus content of total [35S]-sulfated proteoglycan by 35% (P < 0.01), and that of articular cells by 41% and decreased proteoglycan aggregation. Interleukin 1 beta induced the production of stromelysin-1 messenger ribonucleic acid in both cell types. The stromelysin-1 messenger ribonucleic acid content of anulus fibrosus cells was one half that of articular cells. Interleukin 1 beta increased the production of prostaglandin E2 and caused a dose-dependent secretion of Type II phospholipase A2 activity in both cell types. Its effect was 2.5-fold lower in anulus fibrosus cells than in articular chondrocytes., Conclusion: Anulus fibrosus cells can be stimulated by interleukin 1 beta to produce factors implicated in local degradative and inflammatory processes. This production is associated with decreased proteoglycan aggregation. Anulus fibrosus cells respond slightly less well to interleukin 1 beta in vitro than do articular cells.

This prospective longitudinal study was undertaken to examine the short-term effects (6 months) of estrogen withdrawal on the circulating IGF system. A series of 40 patients suffering from endometriosis was studied before and after a 6-month treatment period with gonadotrophin releasing hormone (GnRH) agonist and calcium, with or without nasal salmon calcitonin. The plasma concentrations of insulin-like growth factor I (IGF-I) and insulin-like growth factor II (IGF-II) were measured by radioimmunoassay and radioreceptor assay respectively. Plasma IGF binding proteins (IGFBPs) were quantified and characterized by ligand blot and immunoblot. In all patients, a secondary hypoestrogenism was observed, including a 4% decrease in lumbar bone mineral density (L-BMD). The plasma IGF-I and IGF-II concentrations increased after treatment (24%, p < 0.0005 and 40%, p < 0.004 respectively), with no significant difference between the treatment groups. There was a positive correlation between plasma IGF-I (but not IGF-II) changes and changes in urinary deoxypyridinoline (r = 0.32, p < 0.05), urinary C telopeptide of type 1 collagen (r = 0.33, p < 0.04) and total plasma alkaline phosphatases (r = 0.33, p < 0.04). No correlation was found between IGF-I and L-BMD changes, while there was a positive correlation between the changes in plasma IGF-II and L-BMD (r = 0.32, p < 0.05). Ligand blot analysis revealed a significant increase in IGF-II binding to a 29-31 kilodalton region where positive staining with specific antibodies to IGFBP-3 or IGFBP-1 was observed. In conclusion, IGF-I and IGF-II plasma concentrations are both increased following a short period of treatment with a GnRH agonist. The changes in individual IGF peptides are differently correlated with changes in markers of bone remodelling and L-BMD.

The IR-IGF1 production by rabbit epiphyseal chondrocytes cultured in serum-free medium was analyzed. Cell proliferation was induced by the addition of 10 ng/ml basic fibroblast growth factor (bFGF) without or with 100 ng/ml recombinant human growth hormone (hGH). GH alone induced no cell multiplication. Chondrocytes treated with bFGF alone secreted an IR-IGF1 activity proportional to the mitotic activity of the cells. A specific positive IGF1 immunostaining was localized in the Golgi of control and hGH-treated cells. The IR-IGF1 activity recovered into culture medium was mainly composed of three fractions of apparent MW 6-8 kDa, 9-14 kDa, and 16-18 kDa. [35S]Methionine pulse-chase experiments indicated that the radiolabeled 16-18 kDa IR-IGF1 fraction was partly converted into the 9-14 kDa and 6-8 kDa fractions. At equilibrium, 70% of the chondrocyte IR-IGF1 activity was recovered as 9- to 18-kDa forms which contained high IR-proIGF1A activity. The 6-8 kDa fraction had biochemical characteristics similar to those of the mature IGF1 peptide. Similar results were observed when 4% fetal calf serum was added to the culture. The addition of 100 ng/ml of hGH significantly and specifically increased IGF1 precursor material, which thus represented 90% of total IR-IGF1 activity. On Day 16 of the culture, when cells stopped dividing, the amount of chondrocyte IR-IGF1 was significantly lower than during cell proliferation, and hGH had no effect on this production. These data indicate that cultured chondrocytes produce more IGF1 precursors than mature IGF1 and that GH specifically stimulates biosynthesis of IGF1 precursors but not IGF1 per se. A GH-dependent biological function of IGF1 proforms in chondrocytes remains to be demonstrated.

This paper reports data on the in vitro effects of insulin-like growth factor I (IGF-I) and basic fibroblast growth factor (bFGF) on the phenotypic expression of epiphyseal chondrocytes grown in serum-free (SF) culture medium. bFGF mostly stimulates chondrocyte DNA and inhibits sulfated proteoglycan synthesis and type II collagen mRNA. On the contrary, IGF-I is poorly mitogenic but strongly stimulates protein synthesis and type II collagen mRNA. In addition, IGF-I prevents the expression of type I collagen gene. Lastly, chondrocytes cultured in SF medium are able to locally produce IGF-I peptides. In conclusion, IGF-I and bFGF have opposite effects on the phenotypic expression of chondrocytes in vitro: bFGF is mostly mitogenic and IGF-I appears to be a differentiating factor.

Chondrocytes from normal cartilage of a 2 month old child have been cultured. In vitro the cells maintained their morphological identity and synthesised proteoglycans with electrophoretic properties similar to those synthesised by cartilage in vivo. Sulphation was stimulated by vitamin D metabolites and by somatomedin. This technique has been used to study biopsies of abnormal cartilage.

Chondrocyte cultures were prepared from rabbit growth plate (GPC) and articular (ARC) chondrocytes. These two cell types have distinct morphological characteristics. The cells reached maximum numbers by days 10 and 21 for ARC and GPC, respectively. The proteoglycans (PG) contained in the cellular pool were extracted and purified by DEAE cellulose chromatography. The effect of a partially purified somatomedin peptide with insulin-like activity on [35S]sulphate incorporation into PG was evaluated. In both ARC and GPC a significant stimulation of [35S]sulphate uptake into PG subunits was obtained with 1 ng Eq./ml of somatomedin peptide. In order to obtain the same stimulatory effect with porcine insulin, a 1000-fold greater concentration was required. The electrophoretic patterns of the PG subunits on acrylamide-agarose electrophoresis were identical on control incubations and after stimulation with the somatomedin peptide. These data demonstrate in vitro biological activity of this peptide on well differentiated articular and epiphyseal growth plate chondrocytes in culture. These cultures appear to provide a sensitive biological assay for somatomedin peptides.

Chondrocytes from epihyseal growth plate cartilage of prepubertal rabbits were cultured. Cells from the proliferative zone showed a very different adhesiveness: they did not stick to the flasks until the 8th to 10th days. They need more oxygen tension to develop and grew in small separated colonies. Each colony was composed by several layers of round or polygonal cells, metachromatic with toluidine blue and they were surrounded by one layer of fibroblastic-like cells. At the end of primary culture the colonies looked like "craters". Cells from the resting zone multiplied as monolayer culture identical to articular chondrocytes. The stage of differentiation of the two different kinds of cells is discussed.

Proteoglycans synthesized by articular and epiphyseal chondrocytes in culture were compared. Proteoglycans extruded by the two types of cells into the culture medium are of identical Mr. On the other hand, the proteoglycans of cells or pericellular matrix synthesized by the articular chondrocytes are characterized by an heterogeneous fraction of low-Mr which is not present in the material derived from epiphyseal chondrocytes. There are at least two components in this fraction: the first seems to be a precursor of aggregated proteoglycans, the other may represent a component of cell coat. Stimulation of the cell cultures with vitamin D metabolites and somatomedin enhances proteoglycan biosynthesis but no modification is observed in the proteoglycan Mr.

Interactions between cartilage and vitamin D metabolism were studied in vitro. Chondrocytes were isolated from rabbit growth plate cartilage and cultured. These cells are able to transform 25-hydroxycholecalciferol in 24, 25-dihydroxycholecalciferol. Metabolic transformation is modulated by 1, 25-dihydroxycholecalciferol but not by parathyroid hormone neither by extracellular calcium concentrations. 24, 25-(OH) 2D3 and 1, 25-(OH) 2D3 are active on the cellular metabolism of cultured chondrocytes in a different way : 24, 25-(OH) 2D3 stimulates the proteoglycan synthesis in "mature" chondrocytes 1, 25-(OH) 2D3 increases DNA polymerase activities in chondrocytes during the logarithmic phase of division. Finally, specific sites of nuclear receptors of 24, 25-(OH) 2D3 are present in chondrocytes.

This study concerns the immunoreactive somatomedin C secretion by prepubertal rabbit epiphyseal chondrocytes cultured in a defined serum-free medium. In such culture conditions, chondrocytes mainly synthesized Type II collagen (80% of total collagen) during 10 days. A small amount of Type I collagen was also found with a significant (p less than 0.05) higher level during the period of cell multiplication (6.4 +/- 1.5%) than when cells reached confluency (0.9 +/- 0.2%). During the 10 days of culture without serum and without hormone added, a Sm-C/IGF1 activity was measured by RIA at a mean level of 30 +/- 5 mU/ml/10 micrograms DNA. This value was significantly higher (p less than 0.001) than in the medium not incubated with the cells (1.7 +/- 0.9 mU/ml). When hGH was added to the culture medium during the period of cell division, the level of Sm-C/IGF1 activity was significantly elevated at 39 +/- 4 mU/ml/10 micrograms DNA (p less than 0.05) and at 55 +/- 3 mU/ml/10 micrograms DNA (p less than 0.001) with 50 ng/ml and 100 ng/ml hGH concentrations respectively. On the contrary, no difference was observed at confluency in treated and non treated cells.

To determine whether growth hormone has a direct effect on skeletal tissues not mediated by somatomedins, and to better define the role of somatomedin-C/insulin-like growth factor I (Sm-C/IGF-I) in skeletal development, bovine growth plate, and rabbit articular and growth plate chondrocytes in primary culture were evaluated under a variety of experimental conditions designed to elicit growth hormone and Sm-C/IGF-I stimulation. Under none of these conditions did bovine growth plate chondrocytes respond to either homologous bovine growth hormone or heterologous hGH. Under the same conditions, these cells were highly responsive to human Sm-C/IGF-I with respect to both [3H]thymidine and [35S]sulfate incorporation, indices of mitotic and differentiated cell functions, respectively. Similarly, both rabbit articular and growth plate chondrocytes showed enhanced incorporation of [3H] thymidine and [35S]sulfate in the presence of Sm-C/IGF-I, but did not respond to either native or recombinant hGH. Cells at different stages of maturation within the bovine growth plate differed in their reaction to Sm-C/IGF-I with proliferative zone cells manifesting a greater response to the peptide than cells of the reserve zone. These results suggest that the action of Sm-C/IGF-I on growth plate and articular chondrocytes is direct and that the effect of GH on these cells is indirect. The data further suggest that within the growth plate, the transition from reserve to proliferative status is associated with an increased Sm-C/IGF-I responsiveness, a change which may contribute to the functional differences in these cells.