Arnaud Bianchi

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.

Differential expression of peroxisome proliferator-activated receptors (PPARs) in the developing human fetal digestive tract.

SUMMARY We investigated the spatiotemporal distributions of the different peroxisome proliferator-activated receptor (PPAR) isotypes (α, β, and α) during development (Week 7 to Week 22 of gestation) of the human fetal digestive tract by immunohistochemistry using specific polyclonal antibodies. The PPAR subtypes, including PPARα, are expressed as early as 7 weeks of development in cell types of endodermal and mesodermal origin. The presence of PPARα was also found by Western blotting and nuclease-S1 protection assay, confirming that this subtype is not adipocyte-specific. PPARα, PPARβ, and PPARα exhibit different patterns of expression during morphogenesis of the digestive tract. Whatever the stage and the gut region (except the stomach) examined, PPARα is expressed at a high level, suggesting some fundamental role for this receptor in development and/or physiology of the human digestive tract.

Free radical production and changes in superoxide dismutases associated with hypoxia/reoxygenation-induced apoptosis of embryonic rat forebrain neurons in culture

Following hypoxia/reoxygenation (6h/96h), cultured neurons from the embryonic rat forebrain undergo delayed apoptosis. To evaluate the participation of oxidative stress and defense mechanisms, temporal evolution of intraneuronal free radical generation was monitored by flow cytometry using dihydrorhodamine 123, in parallel with the study of transcriptional, translational, and activity changes of the detoxifying enzymes Cu/Zn-SOD and Mn-SOD. Two distinct peaks of radical generation were depicted, at the time of reoxygenation (+ 27%) and 48 h later (+ 25%), respectively. Radical production was unaffected by caspase inhibitors YVAD-CHO or DEVD-CHO, which prevented neuronal damage, suggesting that caspase activation is not an upstream initiator of radicals in this model. Cell treatment by vitamin E (100 microM) displayed significant neuroprotection, whereas the superoxide generating system xanthine/xanthine oxidase induced apoptosis. Transcript and protein levels of both SODs were reduced 1 h after the onset of hypoxia, but activities were transiently stimulated. Reoxygenation was associated with an increased expression (139%), but a decreased activity (21%) of the inducible Mn-SOD, whereas Cu/Zn-SOD protein and activity were low and progressively increased until 48 h post-hypoxia, when the second rise in radicals occurred. In spite of a temporal regulation of SODs, which parallels radical formation, oxidative stress might account for neurotoxicity induced by hypoxia.

Induction of MnSOD gene by arachidonic acid is mediated by reactive oxygen species and p38 MAPK signaling pathway in human HepG2 hepatoma cells

Metabolism of arachidonic acid (AA) is known to induce in different cell types an oxidative stress via the production of reactive oxygen species. As these latter may be scavenged by antioxidant enzymes as manganese and copper/zinc-dependent superoxide dismutase (MnSOD and Cu/ZnSOD, respectively), we investigated the effects of AA on their expression in human HepG2 hepatoma cells. RT-PCR and Western blot data revealed that AA induced an increase in the MnSOD, but not Cu/ZnSOD, expression at the mRNA and protein levels, respectively. This induction was also marked by an increase in MnSOD activity. The AA-induced MnSOD expression required de novo transcription as demonstrated by cotreatment of HepG2 cells with AA and actinomycin D. The fact that MnSOD expression was not induced when HepG2 cells were cultured with 5,8,11,14-eicosatetraynoic acid (ETYA), a nonmetabolizable analog of AA, or with different inhibitors of the AA metabolism pathways suggested that the metabolism of AA was required. Further investigations into the mechanisms by which AA induced MnSOD expression showed that superoxide anions released from AA metabolism act as second messengers via a signal-controlling pathway involving protein kinase C and p38 mitogen activated protein kinase (MAPK). These results define a novel role of p38 MAPK dependent-pathway in the regulation of MnSOD gene.

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.

Arachidonic acid activates a functional AP-1 and an inactive NF-κB complex in human HepG2 hepatoma cells

Exogenous arachidonic acid (AA) has been shown to induce the antioxidant manganese superoxide dismutase gene by reactive oxygen species (ROS) derived from AA metabolism and the participation of the p38 mitogen-activated protein kinase (MAPK) pathway in human HepG2 hepatoma cells. The goal of this study was to investigate the effect of AA on the activation of the two redox-sensitive transcription factors AP-1 and NF-kappaB in HepG2 cells. Using electrophoretic mobility shift assays, DNA-binding activities of AP-1 and NF-kappaB were markedly increased in AA-treated HepG2 cells. The c-Jun and c-Fos proteins were identified as components of the AA-induced AP-1 complex and their levels were increased. AA-activated NF-kappaB complex was constituted as a p50 homodimer resulting in a nuclear translocation for this protein only. Moreover, no degradation of IkappaBalpha was observed. These results were contrasted to the interleukin-1beta-activated p50/p65 complex used as a positive control. Using 5,8,11,14-eicosatetraynoic acid and inhibitors of AA metabolism, AP-1 and NF-kappaB activation required the lipoxygenase/cytochrome P450 monooxygenase pathways. In addition, antioxidants inhibited the AA-induced AP-1 and NF-kappaB activation, suggesting a role of ROS released from the AA metabolism. In reporter gene assays, AA induced the transcriptional activity of AP-1 but not that of NF-kappaB. Further investigations showed that the AA-induced transcriptional activity of AP-1 was regulated by protein kinase C and p38 MAPK pathways. These results suggest that the functional AP-1 activated by AA and coupled to that of p38 MAPK pathway may play an important role in response to ROS induced by AA metabolism in HepG2 cells without the involvement of the NF-kappaB pathway.

The inorganic pyrophosphate transporter ANK preserves the differentiated phenotype of articular chondrocyte.

The differentiated phenotype of chondrocyte is lost in pathological situations and after interleukin (IL)-1β challenge. Wnt proteins and the inorganic pyrophosphate (PPi) transporter Ank regulate the differentiation process in many cell types. We investigated the possible contribution of Ank and/or PPi to the maintenance of the differentiated chondrocyte phenotype with special care to Wnt signaling. Primary articular chondrocytes lost their phenotype upon IL-1β challenge, with cessation of type II collagen and Sox-9 expression. Ank expression and PPi transport were strongly reduced by IL-1β, whereas Wnt-5a was the only Wnt protein increased. Transient overexpression of Ank counteracted most of IL-1β effects on Type II collagen, Sox-9, and Wnt-5a expression. When resting chondrocytes were transfected with a siRNA against Ank, this reproduced the phenotype induced by IL-1β. In both cases, no markers for hypertrophic chondrocytes were detected. The conditioned supernatant from chondrocytes knocked-down for Ank contained Wnt-5a, which activated Tcf/Lef reporter plasmids and promoted translocation of β-catenin into the nucleus without activating the c-Jun N-terminal kinase (JNK) pathway. Supplementation with PPi compensated for most effects of Ank deficiency on Type II collagen, Sox-9, and Wnt-5 expression, both in IL-1β and Ank knock-down conditions. Phenotype changes induced by IL-1β were also supported by activation of the JNK pathway, but this latter was not sensitive to PPi supplementation. Altogether our data demonstrate that the transport of PPi by ANK contributed to the maintenance of the differentiated phenotype of chondrocyte by controlling the canonical Wnt pathway in a Wnt-5a-dependent manner.

Evidence for the presence of both peroxisome proliferator-activated receptors alpha and beta in the rat spinal cord.

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors belonging to the nuclear receptor superfamily. Different subtypes of PPARs (alpha, beta, and gamma) have been described. Their distinct physiological functions depend on their differential ligand activation profiles but also on their specific tissue expression. Previous studies have described their presence in the central nervous system. However, their expression in the adult rat spinal cord in normal physiological conditions has never been investigated. We demonstrated by using reverse-transcription-polymerase chain reaction, and Western blotting, the mRNA and protein expression of PPARalpha and PPARbeta, but not PPARgamma in cervical, thoracic, and lumbar segments of the spinal cord. Using immunohistochemistry, we also showed for the first time the specific cellular distribution of these transcription factors in the different segments of the spinal cord. In the gray matter, the distribution of PPARalpha was homogenous whereas PPARbeta was specifically localized in motoneurons and in medial part of laminae IV, V, VI, VII, VIII, and X. These latter areas are known as nociceptive afferent pathways to supra-spinal structures such as the medulla reticular nucleus and the thalamus. In the white matter, PPARalpha was localized exclusively in astrocytes while PPARbeta was present in oligodendrocytes. The possible functions of PPARalpha and PPARbeta expressed in both white and gray matters of the spinal cord will be discussed but need further studies.