Karim Bordji

Interleukin-1β down-regulates the expression of glucuronosyltransferase I, a key enzyme priming glycosaminoglycan biosynthesis influence of glucosamine on interleukin-1β-mediated effects in rat chondrocytes

OBJECTIVEnTo assess the variations of galactose-beta-1,3-glucuronosyltransferase I (GlcAT-I) expression related to the decrease in proteoglycan synthesis mediated by interleukin-1beta (IL-1beta) in rat chondrocytes, and to evaluate the influence of glucosamine on the effects elicited by this proinflammatory cytokine.nnnMETHODSnRat articular chondrocytes in primary monolayer cultures or encapsulated into alginate beads were treated with recombinant IL-1beta in the absence or presence (1.0-4.5 gm/liter) of glucosamine. Variations of GlcAT-I and expression of stromelysin 1 (matrix metalloproteinase 3 [MMP-3]) messenger RNA (mRNA) were evaluated by quantitative multistandard reverse transcriptase-polymerase chain reaction. In vitro enzymatic activity of GlcAT-I was measured by thin-layer chromatography, with radiolabeled UDP-glucuronic acid and a digalactoside derivative as substrates. Proteoglycan synthesis was determined by ex vivo incorporation of Na2-35SO4. Nitric oxide synthase and cyclooxygenase activities were monitored by the evaluation of nitrite (NO2-) and prostaglandin E2 (PGE2) produced in the culture medium, respectively.nnnRESULTSnIL-1beta treatment resulted in a marked inhibition of GlcAT-I mRNA expression and in vitro catalytic activity, together with a decrease in proteoglycan synthesis. In addition, glucosamine was able to prevent, in a dose-dependent manner, the inhibitory effects of IL-1beta. In the same way, the amino sugar reduced NO2- and PGE2 production induced by IL-1beta. Finally, the up-regulation of stromelysin 1 (MMP-3) mRNA expression by IL-1beta was fully prevented by glucosamine.nnnCONCLUSIONnThe results of this study suggest that the deleterious effect of IL-1beta on the anabolism of proteoglycan could involve the repression of GlcAT-I, a key enzyme in the biosynthesis of glycosaminoglycan. Glucosamine was highly effective in preventing these IL-1beta-mediated suppressive effects. The amino sugar also prevented the production of inflammatory mediators induced by the cytokine. This action could account for a possible beneficial effect of glucosamine on osteoarthritic articular cartilage.

A plant steroid, diosgenin, induces apoptosis, cell cycle arrest and COX activity in osteosarcoma cells

Cyclooxygenases (COXs) are key enzymes in the conversion of arachidonic acid into prostanoids which are involved in apoptosis and inflammation. Two distinct COXs have been identified: COX‐1 which is constitutively expressed and COX‐2 which is induced by different products such as tumor promoters or growth factors. Previously, we demonstrated that a plant steroid, diosgenin, was a new megakaryocytic differentiation inducer of human erythroleukemia cells. In our study, we investigated the effect of diosgenin on the proliferation rate, cell cycle distribution and apoptosis in the human osteosarcoma 1547 cell line. The effects of this compound were also tested on COX expression and COX activities. Diosgenin treatment caused an inhibition of 1547 cell growth with a cycle arrest in G1 phase and apoptosis induction. Moreover, we found a correlation between p53, p21 mRNA expression and nuclear factor‐κB activation and we observed a time‐dependent increase in PGE2 synthesis after diosgenin treatment.

Glucosamine modulates IL-1-induced activation of rat chondrocytes at a receptor level, and by inhibiting the NF-κB pathway

We recently reported that glucosamine reversed the decrease in proteoglycan synthesis and in UDP‐glucuronosyltransferase I mRNA expression induced by interleukin‐1β (IL‐1β) [Arthritis Rheum. 44 (2001) 351–360]. In the present work, we show that glucosamine does not exert the same effects when chondrocytes were stimulated with reactive oxygen species (ROS). In order to better understand its mechanism of action, we determined if glucosamine could prevent the binding of IL‐1β to its cellular receptors or could interfere with its signaling pathway at a post‐receptor level. Addition of glucosamine to rat chondrocytes treated with IL‐1β or with ROS decreased the activation of the nuclear factor κB, but not the activator protein‐1. After treatment with IL‐1β, glucosamine increased the expression of mRNA encoding the type II IL‐1β receptor. These results emphasize the potential role of two regulating proteins of the IL‐1β signaling pathway that could account for the beneficial effect of glucosamine in osteoarthritis.

15-Deoxy-Δ12,14-PGJ2, but not troglitazone, modulates IL-1β effects in human chondrocytes by inhibiting NF-κB and AP-1 activation pathways

The activation of peroxisome proliferator‐activated receptor γ (PPARγ) has been shown to inhibit the production and the effects of proinflammatory cytokines. Since interleukin‐1β (IL‐1β) directly mediates cartilage degradation in osteoarthritis, we investigated the capability of PPARγ ligands to modulate IL‐1β effects on human chondrocytes. RT‐PCR and Western blot analysis revealed that PPARγ expression was decreased by IL‐1β. 15‐Deoxy‐Δ12,14‐prostaglandin J2 (15d‐PGJ2), in contrast to troglitazone, was highly potent to counteract IL‐1β‐induced cyclooxygenase‐2 and inductible nitric oxide synthase expression, NO production and the decrease in proteoglycan synthesis. Western blot and gel‐shift analyses demonstrated that 15d‐PGJ2 inhibited NF‐κB activation, while troglitazone was ineffective. Although 15d‐PGJ2 attenuated activator protein‐1 binding on the DNA, it potentiated c‐jun migration in the nucleus. The absence or the low effect of troglitazone suggests that 15d‐PGJ2 action in human chondrocytes is mainly PPARγ‐independent.

Relations between functional, inflammatory, and degenerative parameters during adjuvant arthritis in rats

We assessed the time-course of adjuvant arthritis (AA) in Lewis rats, using biotelemetry to monitor the rats spontaneous locomotor activity and body temperature, and studied the evolution of the arthritic index, circulating concentrations of inflammation-promoting cytokines, cartilage proteoglycan synthesis, and the effect of indomethacin as a cyclooxygenase inhibitor to evaluate prostaglandin (PG) contribution in AA. The injection of complete Freunds adjuvant on day 0( D0) induced a marked, transient loss of locomotor activity ( D1- D4; initial phase) and then a second phase of hypomobility peaking on D15 and thereafter irreversible ( D16- D20; arthritic phase). Fever peaked first on D1 and again between D13 and D17. The primary hyperthermia was associated with increases in plasma interleukin-6 and tumor necrosis factor-α concentrations and seemed to be partly PG dependent. Proteoglycan synthesis inhibition in the patellar cartilage increased gradually, spreading from the injected paw to the contralateral paw. It was corrected on D20 by preventive and curative indomethacin treatments. Indomethacin also greatly relieved hypomobility during the systemic phase of AA ( D10- D15). The combination of information about cartilage metabolism, body temperature, locomotor activity, and cytokine in this study permits analysis of analgesic, antipyretic, anti-inflammatory, and chondroprotective properties of drugs in the various phases of AA. Thus, using a new methodology, we have discriminated the different phases of the disease and confirmed the symptomatic and systemic inhibitory effect of indomethacin on fever, activity, and cartilage metabolism.

Modulation of IL‐1‐induced cartilage injury by NO synthase inhibitors: a comparative study with rat chondrocytes and cartilage entities

Nitric oxide (NO) is produced in diseased joints and may be a key mediator of IL‐1 effects on cartilage. Therefore, we compared the potency of new [aminoguanidine (AG), S‐methylisothiourea (SMT), S‐aminoethylisothiourea (AETU)] and classical [Nω‐monomethyl‐L‐arginine (L‐NMMA), Nω‐nitro‐L‐arginine methyl ester (L‐NAME)] NO synthase (NOS) inhibitors on the inhibitory effect of recombinant human interleukin‐1β (rhIL‐1β) on rat cartilage anabolism. Three different culture systems were used: (1) isolated chondrocytes encapsulated in alginate beads; (2) patellae and (3) femoral head caps. Chondrocyte beads and cartilage entities were incubated in vitro for 48u2003h in the presence of rhIL‐1β with a daily change of incubation medium to obtain optimal responses on proteoglycan synthesis and NO production. Proteoglycan synthesis was assessed by incorporation of radiolabelled sodium sulphate [Na235SO4] and NO production by cumulated nitrite release during the period of study. Chondrocytes and patellae, as well as femoral head caps, responded concentration‐dependently to IL‐1β challenge (0 to 250u2003Uu2003ml−1 and 0 to 15u2003Uu2003ml−1 respectively) by a large increase in nitrite level and a marked suppression of proteoglycan synthesis. Above these concentrations of IL‐1β (2500u2003Uu2003ml−1 and 30u2003Uu2003ml−1 respectively), proteoglycan synthesis plateaued whereas nitrite release still increased thus suggesting different concentration‐response curves. When studying the effect of NOS inhibitors (1 to 1000u2003μM) on NO production by cartilage cells stimulated with IL‐1β (25u2003Uu2003ml−1 or 5u2003Uu2003ml−1), we observed that: (i) their ability to reduce nitrite level decreased from chondrocytes to cartilage samples, except for L‐NMMA and AETU; (ii) they could be roughly classified in the following rank order of potency: AETU>L‐NMMASMT>AGL‐NAME and (iii) AETU was cytotoxic when used in the millimolar range. When studying the effect of NOS inhibitors on proteoglycan synthesis by cartilage cells treated with IL‐1β, we observed that: (i) they had more marked effects on proteoglycan synthesis in chondrocytes than in cartilage samples; (ii) they could be roughly classified in the following rank order of potency: L‐NAMEL‐NMMA>>AG>SMT>>AETU and (iii) potentiation of the IL‐1 effect by AETU was consistent with cytotoxicity in the millimolar range. D‐isomers of L‐arginine analog inhibitors (1000u2003μM) were unable to correct nitrite levels or proteoglycan synthesis in IL‐1β treated cells. L‐arginine (5000u2003μM) tended to reverse the correcting effect of L‐NMMA (1000u2003μM) on proteoglycan synthesis, thus suggesting a NO‐related chondroprotective effect. However, data with L‐NAME and SMT argued against a general inverse relationship between nitrite level and proteoglycan synthesis. Dexamethasone (0.1 to 100u2003μM) (i) failed to inhibit NO production in femoral head caps and chondrocytes beads whilst reducing it in patellae (50%) and (ii) did not affect or worsened the inhibitory effect of IL‐1β on proteoglycan synthesis. Such results suggested a corticosteroid‐resistance of rat chondrocyte iNOS. Data from patellae supported a possible contribution of subchondral bone in NO production. In conclusion, our results suggest that (i) NO may account only partially for the suppressive effects of IL‐1β on proteoglycan synthesis, particularly in cartilage samples; (ii) the chondroprotective potency of NOS inhibitors can not be extrapolated from their effects on NO production by joint‐derived cells and (iii) L‐arginine analog inhibitors are more promising than S‐substituted isothioureas for putative therapeutical uses.

15‐Deoxy‐Δ12,14‐prostaglandin J2 inhibits IL‐1β‐induced IKK enzymatic activity and IκBα degradation in rat chondrocytes through a PPARγ‐independent pathway

Peroxisome proliferator‐activated receptor γ (PPARγ) ligands have been shown to inhibit the effects of proinflammatory cytokines such as interleukin‐1β (IL‐1β). This cytokine plays a key role in articular pathophysiologies by inducing the production of inflammatory mediators such as nitric oxide (NO) and prostaglandin E2 (PGE2). We previously demonstrated that 15d‐PGJ2 was more potent than troglitazone to counteract IL‐1β effects on chondrocytes. Here, we studied the action of 15d‐PGJ2 on intracellular targets in nuclear factor‐κB (NF‐κB) signalling pathway in IL‐1β treated rat chondrocytes. We found that 15d‐PGJ2 decreased inhibitor κBα (IκBα) degradation but not its phosphorylation by specifically inhibiting IκB kinase β (IKKβ), but not IKKα, enzymatic activity. We further evaluated the involvement of PPARγ in the anti‐inflammatory action of its ligands. In chondrocytes overexpressing functional PPARγ protein, 15d‐PGJ2 pre‐treatment inhibited inducible NO synthase and COX‐2 mRNA expression, nitrite and PGE2 production, p65 translocation and NF‐κB activation. Troglitazone or rosiglitazone pre‐treatment had no effect. 15d‐PGJ2 exhibited the same effect in chondrocytes overexpressing mutated PPARγ protein. These results suggest that 15d‐PGJ2 exerts its anti‐inflammatory effect in rat chondrocytes by a PPARγ‐independent mechanism, which can be conferred to a partial inhibition of IκBα degradation.