Marie-Thérèse Corvol

Different mechanisms are involved in intracellular calcium increase by insulin‐like growth factors 1 and 2 in articular chondrocytes: Voltage‐gated calcium channels, and/or phospholipase C coupled to a pertussis‐sensitive G‐protein

This study describes the mechanisms involved in the IGF‐1 and IGF‐2‐induced increases in intracellular calcium concentration [Ca2+]i in cultured chondrocytes and the involvement of type 1 IGF receptors. It shows that IGF‐1, IGF‐2, and insulin increased the cytosolic free calcium concentration [Ca2+]i in a dose‐dependent manner, with a plateau from 25 to 100 ng/ml for both IGF‐1 and IGF‐2 and from 1 to 2 μg/ml for insulin. The effect of IGF‐1 was twice as great as the one of IGF‐2, and the effect of insulin was 40% lower than IGF‐1 effect. Two different mechanisms are involved in the intracellular [Ca2+]i increase. 1) IGF‐1 and insulin but not IGF‐2 involved a Ca2+ influx through voltage‐gated calcium channels: pretreatment of the cells by EGTA and verapamil diminished the IGF‐1 or insulin‐induced[Ca2+]i but did not block the effect of IGF‐2.2)IGF‐1, IGF‐2, and insulin also induced a Ca2+ mobilization from the endoplasmic reticulum: phospholipase C (PLC) inhihitors, neomycin, or U‐73122 partially blocked the intracellular [Ca2+]i increase induced by IGF‐1 and insulin and totally inhibited the effect of IGF‐2. This Ca2+ mobilization was pertussis toxin (PTX) dependent, suggesting an activation of a PLC coupled to a PTX‐sensitive G‐protein. Lastly, preincubation of the cells with IGF1 receptor antibodies diminished the IGF‐1‐induced Ca2+ spike and totally abolished the Ca2+ influx, but did not modify the effect of IGF‐2. These results suggest that IGF‐1 action on Ca2+ influx involves the IGF1 receptor, while part of IGF‐1 and all of IGF‐2 Ca2+ mobilization do not implicate this receptor. J. Cell. Biochem. 64:414–422.

Oestrogens inhibit interleukin 1β‐mediated nitric oxide synthase expression in articular chondrocytes through nuclear factor‐κB impairment

Objectives: To investigate the presence and functionality of oestrogen receptor α (ERα) in interleukin (IL)1β-treated rabbit articular chondrocytes in culture, and to determine the mechanisms of 17β oestradiol (E2) effects on IL1β-induced inducible nitric oxide synthase (iNOS) expression. Methods: The presence and functionality of ERα 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-κB (NF-κB) and activated protein 1 were determined by electrophoretic mobility shift assay (EMSA)–ELISA assays. Nuclear translocation of p65 was studied by immunocytochemistry. Results: ERα was identified in the nucleus of chondrocytes. ERα efficiently transactivated a transiently expressed E2-responsive construct. On IL1β treatment, ERα partially diffused from its nuclear localisation into the cytoplasm and its transactivation ability was impaired. Nevertheless, E2, tamoxifen and raloxifene efficiently inhibited IL1β-induced NO production (−34%, −31% and −36%, respectively). E2 decreased IL1β-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 IL1β-induced p65 DNA-binding capacity. Finally, the p65 nuclear translocation induced by IL1β was also strongly decreased by E2. Conclusions: Our data support a reciprocal antagonism between oestrogens and IL1β, 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.

Involvement of the notch pathway in the regulation of matrix metalloproteinase 13 and the dedifferentiation of articular chondrocytes in murine cartilage

OBJECTIVE To demonstrate the activation of the Notch signaling pathway during changes in the phenotype of chondrocytes in vitro, and to assess the influence of Notch on the production of chondrocyte markers. METHODS Serial monolayer primary cultures of murine articular chondrocytes (MACs), as a model of chondrocyte dedifferentiation, were prepared. MACs were cultured with or without a Notch inhibitor and transfected with different Notch-expressing vectors. The Notch pathway and chondrocyte marker profiles were assessed by quantitative reverse transcription-polymerase chain reaction, immunoblotting, and immunocytochemistry. RESULTS Successive passages of MACs resulted in a loss of type II collagen and aggrecan (chondrocyte differentiation markers), an increase in type I collagen (dedifferentiation marker), an increase in Notch ligands, and augmented target gene activity. The Notch inhibitor decreased the type II collagen protein content but had no effect on Col2a1 messenger RNA, while transfection with the constitutive active forms of the Notch1 receptor led to a decrease in type II collagen in transfected cells. In assays to investigate the mechanism of type II collagen breakdown, matrix metalloproteinase 13 (MMP-13) synthesis was regulated in a Notch-dependent manner, whereas MMP-2 synthesis was unchanged. CONCLUSION The Notch signaling pathway is associated with decreased type II collagen production during the dedifferentiation of MACs in vitro. This may be correlated with the increase in MMP-13 production linked to activation of Notch.

Natural chondroitin sulphates increase aggregation of proteoglycan complexes and decrease adamts-5 expression in interleukin 1β-treated chondrocytes

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.

Resveratrol, Potential Therapeutic Interest in Joint Disorders: A Critical Narrative Review

Trans-resveratrol (t-Res) is a natural compound of a family of hydroxystilbenes found in a variety of spermatophyte plants. Because of its effects on lipids and arachidonic acid metabolisms, and its antioxidant activity, t-Res is considered as the major cardioprotective component of red wine, leading to the “French Paradox” health concept. In the past decade, research on the effects of resveratrol on human health has developed considerably in diverse fields such as cancer, neurodegenerative and cardiovascular diseases, and metabolic disorders. In the field of rheumatic disorders, in vitro evidence suggest anti-inflammatory, anti-catabolic, anti-apoptotic and anti-oxidative properties of t-Res in various articular cell types, including chondrocytes and synoviocytes, along with immunomodulation properties on T and B lymphocytes. In preclinical models of osteoarthritis and rheumatoid arthritis, resveratrol has shown joint protective effects, mainly mediated by decreased production of pro-inflammatory and pro-degradative soluble factors, and modulation of cellular and humoral responses. Herein, we comprehensively reviewed evidence supporting a potential therapeutic interest of t-Res in treating symptoms related to rheumatic disorders.

First Combined in Vivo X-Ray Tomography and High-Resolution Molecular Electron Paramagnetic Resonance (EPR) Imaging of the Mouse Knee Joint Taking into Account the Disappearance Kinetics of the EPR Probe:

Although laboratory data clearly suggest a role for oxidants (dioxygen and free radicals derived from dioxygen) in the pathogenesis of many age-related and degenerative diseases (such as arthrosis and arthritis), methods to image such species in vivo are still very limited. This methodological problem limits physiopathologic studies about the role of those species in vivo, the effects of their regulation using various drugs, and the evaluation of their levels for diagnosis of degenerative diseases. In vivo electron paramagnetic resonance (EPR) imaging and spectroscopy are unique, noninvasive methods used to specifically detect and quantify paramagnetic species. However, two problems limit their application: the anatomic location of the EPR image in the animal body and the relative instability of the EPR probes. Our aim is to use EPR imaging to obtain physiologic and pathologic information on the mouse knee joint. This article reports the first in vivo EPR image of a small tissue, the mouse knee joint, with good resolution (≈ 160 μm) after intra-articular injection of a triarylmethyl radical EPR probe. It was obtained by combining EPR and x-ray micro-computed tomography for the first time and by taking into account the disappearance kinetics of the EPR probe during image acquisition to reconstruct the image. This multidisciplinary approach opens the way to high-resolution EPR imaging and local metabolism studies of radical species in vivo in different physiologic and pathologic situations.

Contraceptive steroids from pharmaceutical waste perturbate junctional communication in Sertoli cells

The potential health impact of pharmaceutical waste is now a growing concern. Contraceptive steroids are prominent environmental contaminants and thus may act as endocrine disruptors. Numerous xenobiotics hamper Sertoli cells junctional communication which is known to participate in spermatogenesis control. This has been associated with male subfertility and testicular cancer. We investigated three contraceptive molecules found in the environment for their potential impact on Sertoli cells gap junction functionality: 17a-ethynylestradiol, medroxyprogesterone acetate and levonorgestrel. Four other non-steroid drugs also found in the environment were included in the study. Communication disruption was analyzed in vitro in murine seminiferous tubules and the 42GPA9 Sertoli cell line. Steroids modulated connexin43 trafficking and impaired junctional communication through rapid effects apparently acting on the cell membrane but not on Cx43 expression. The 4 non-steroid compounds showed no effect. Longer exposure to steroids increased gap junction impairment, which was associated in part with Na/K ATPase internalization. Estrogen receptors (ER) did not appear to be involved in gap junction disruption: Sertoli cells are devoid of ERalpha and only express the cytoplasmic beta isoform. ERbeta localization was not modified by either steroid. The threshold level was surprisingly low, around 10(-16) M. We conclude that steroidal pollutants disrupt Sertoli cells junctional communication in vitro at concentrations that can be found in the environment.

Hes1, a new target for interleukin 1β in chondrocytes

Objectives To investigate the effects of interleukin 1β (IL1β) treatment on the Notch1/Hes1 pathway in chondrocytes in vitro. Methods Mouse articular chondrocytes in primary culture were challenged with IL1β, alone or combined with Notch1 and IL1β pathway inhibitors. Notch1 and Hes1 expressions were investigated by immunocytochemistry, western blot and real-time quantitative (q)PCR. IL1β-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. IL1β 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. IL1β induced increase in Hes1 mRNA was transcriptional, occurred through nuclear factor (NF)κB activation and appeared to be associated with downregulation by its own protein. Hes1 induction was insensitive to the γ-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 IL1β-induced downregulation of Sox9, type II collagen and aggrecan transcription but mediated IL1β 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 IL1β. Conclusion Hes1 is a novel IL1β target gene in chondrocytes which influences a discrete subset of genes linked to cartilage matrix remodelling and/or degradation.

Lipid composition of two types of chondrocytes in primary culture.

SummaryChondrocytes from articular and growth plate cartilage were grown in primary culture. The lipid content, distribution into different types, as well as the fatty acid patterns of these lipids were compared when the cells had reached stationary phase and were synthesizing maximal amounts of proteoglycans. Numerous significant differences were observed, depending on the origin of the chondrocytes. In particular, growth plate chondrocytes showed increased dry weight, increased lipid content (phosphatides and triglycerides), and decreased cholesterol to phosphatide ratio when compared to articular chondrocytes; they also incorporated more of C18:1 and less C16:0 into their major lipid types. Whether these differences arise from specific metabolic regulation or are a consequence of chondrocyte organization in primary culture remains unclear.