Specific treatment of age-related aortic wall arteriosclerosis and stiffening is lacking. As anti-inflammatory ligands for peroxisome proliferator-activated receptor-gamma have beneficial effects on the arterial wall in atherosclerosis, we investigated whether chronic pioglitazone treatment protects against another form of vascular wall disease, arteriosclerosis. In a rat model of elastocalcinotic arteriosclerosis (hypervitaminosis D and nicotine, VDN), we evaluated whether pioglitazone attenuated arteriosclerosis and its consequences, aortic wall rigidity, increased aortic pulse pressure and left ventricular hypertrophy. In VDN rats, medial calcification was associated with monocyte/macrophage infiltration and induction of TNF-alpha and IL-1B. Pioglitazone decreased TNF-alpha and IL-1B mRNA expression, blunted aortic wall calcification and prevented fragmentation of elastic fibers. Pioglitazone reduced aortic wall stiffness, aortic pulse pressure and left ventricular hypertrophy. Our results may be clinically relevant in elderly patients suffering from aortic wall stiffening and isolated systolic hypertension.

Avec l’âge, les vaisseaux deviennent plus sensibles au stress oxydant et à l’inflammation, responsables d’altérations fonctionnelles et structurales comme l’artériosclérose. Cette dernière se caractérise par un remodelage et une rigidité de la paroi. La rigidité conduit à une hyperpulsatilité tensionnelle, une hypertension systolique isolée puis une hypertrophie ventriculaire gauche, facteurs de risque de morbi-mortalité cardiovasculaire. Depuis plus de 15 ans, nous avons validé un modèle murin d’artériosclérose liée à une élastocalcinose (calcification et dégradation du réseau élastique artériel) par hypervitaminose D3 et nicotine (VDN).

This study tests the hypothesis that the activators of peroxisome proliferator-activated receptors (PPARs) and 9-cis-retinoic acid receptor (RXR) regulate human semaphorin 6B (Sema6B) gene expression. The human MCF-7 breast adenocarcinoma cell line was chosen because it expresses Sema6B at a high level. The Sema6B mRNA level was analyzed by RT-PCR and the semaphorin 6B protein content was determined using a polyclonal antibody that we have produced and characterized. Treatments with fenofibrate (a PPARalpha activator) and troglitazone (a PPARgamma ligand) strongly decreased the Sema6B mRNA. The drop in Sema6B mRNA level and in protein content was more important when the treatment combined the action of fenofibrate or troglitazone and 9-cis-retinoic acid. On the other hand, no significant change was observed in the Sema6B mRNA and protein levels when the cells were exposed to the combined action of GW610742 (a PPARbeta activator) and 9-cis-retinoic acid. These data suggest that PPARalpha/RXR and PPARgamma/RXR heterodimers are involved in the regulation of Sema6B gene expression and open new perspectives concerning the participation of these nuclear receptors in cell recognition and migration.

Specific treatment of age-related aortic wall arteriosclerosis and stiffening is lacking. Because ligands for peroxisome proliferator-activated receptor γ have beneficial effects on the arterial wall in atherosclerosis, via an antiinflammatory mechanism, we investigated whether long-term pioglitazone (Pio) treatment protects against another form of vascular wall disease, arteriosclerosis. We evaluated, in a rat model of elastocalcinotic arteriosclerosis (hypervitaminosis D and nicotine [VDN]), whether Pio (3 mg · kg −1 per day for 1.5 month PO) attenuated arteriosclerosis and its consequences: aortic wall rigidity, increased aortic pulse pressure, and left ventricular hypertrophy. In VDN rats, medial calcification was associated with monocyte/macrophage infiltration and induction of tumor necrosis factor α and interleukin 1β. Pio increased nuclear peroxisome proliferator-activated receptor γ immunostaining in the aortic wall, decreased tumor necrosis factor α ( P <0.05 versus VDN Pio − ), tended to decrease interleukin 1β mRNA expression ( P =0.08 versus VDN Pio − ), blunted aortic wall calcification (271±69, P <0.05 versus VDN Pio − 562±87 μmol · g −1 dry weight) and prevented fragmentation of elastic fibers (segments per 10 000 μm 2 : 8.4±0.3; P <0.05 versus VDN Pio − 10.5±0.6). Pio reduced aortic wall stiffness (elastic modulus/wall stress: 4.8±0.6; P <0.05 versus VDN Pio − 10.0±1.6), aortic pulse pressure (30±2 mm Hg; P <0.05 versus VDN Pio − 39±4) and left ventricular hypertrophy (1.58±0.05 g · kg −1 ; P <0.05 versus VDN Pio − 1.76±0.06). In conclusion, long-term Pio treatment attenuates aortic wall elastocalcinosis and, thus, lowers aortic wall stiffness, aortic pulse pressure, and left ventricular hypertrophy.

Hypolipidemic drugs (HP drugs) are xenobiotics belonging to the peroxisome proliferator family which are used as pharmaceuticals in the treatment of human hyperlipidemia and hypercholesterolemia. They cause hepatocarcinogenesis in rodents by increasing cell proliferation. One hypothesis is that this hepatocarcinogenic effect is caused by induced oxidative stress resulting from the overproduction of reactive oxygen species (ROS) and from a decreasing antioxidant defense. In addition, ROS play a role in hepatocellular proliferation by activation of NF-kappa B and AP-1, leading to an increase in mitogenic cytokines such as tumor necrosis factor-alpha. No liver cancer incidence has been noted in individuals treated with HP drugs for brief periods of time, However, the observation that old rats and mice are more susceptible than young individuals to the hepatocarcinogenic effect caused by long term exposure to HP drugs raises the question of a potential health risk for the human population. In vitro, HP drugs cause an apoptogenic effect in human hepatocytes. This effect is related to a moderate antioxidant response, dysfunction of mitochondria caused by an overproduction of ROS and release of apoptogenic factors, Finally, the apoptogenic effect of HP drugs is observed in human hepatomas, suggesting a clinical interest of these agents in antitumoral activity.

The peroxisome proliferator-activated receptors (PPARs) are ligand-inducible transcription factors and belong to the nuclear hormone receptor superfamily. They form heterodimers with the retinoid X receptor and bind to specific peroxisome proliferator-response elements. The latter are direct repeat elements of two hexanucleotides with the consensus sequence TG(A/T)CCT separated by a single nucleotide spacer. Such a sequence, or a similar one, has been found in numerous PPAR-inducible genes. We developed an affinity method to isolate human genomic fragments containing binding sites for PPARs and to identify novel PPAR target genes. For this, an antibody raised against all PPAR subtypes was used. Immunoselected fragments were amplified and sequenced and one of them, ISF5148, was found to bind specifically to PPARs in gel mobility shift, supershift, and competition assays and to exhibit a down transregulation potentiality in transfection experiments under clofibrate (a PPARα agonist) treatment. ISF5148 was mapped by BLAST analysis 8.5 kb upstream of the human semaphorin 6B [(HSA)SEMA6B] gene. The latter encodes a member of the semaphorin family of axon guidance molecules. Expression of this gene in human glioblastoma T98G cells was strongly down regulated after treatment with clofibrate or Wy-14,643, two PPARα agonists. Our study establishes for the first time that PPAR activators diminish the expression of the human (HSA)SEMA6B gene. These data are relevant to the fact that PPARs are implicated in brain development, neuronal differentiation, and lipid metabolism in the central nervous system. In addition, cross talk between the peroxisome proliferator and retinoic acid pathways is suggested.

We showed that the metabolism of arachidonic acid (AA) in HepG2 cells generates reactive oxygen species (ROS), which activate the p38 mitogen-activated protein kinase (MAPK) pathway and the redox-sensitive transcription factors AP-1 and NF-kappaB, leading to the induction of the antioxidant manganese superoxide dismutase gene. The present study reports that AA decreases the HepG2 cell growth by 40% and 55% after a treatment for 24 and 48 h, respectively. This effect was blocked by an inhibitor of lipoxygenase/cytochrome P450 monooxygenase pathways and by the antioxidants. In addition, AA induced an oxidative stress, as an accumulation of malondialdehyde (MDA)-modified proteins, resulting to a generation of MDA and H2O2 was observed after 24 h. This AA-induced oxidative stress was associated with the lack of an increase in the H2O2-degrading enzyme level. In contrast, 5,8,11,14-eicosatetraynoic acid, a nonmetabolizable analog of AA, had not effect. The peroxisome proliferator-activated receptor gamma (PPARgamma) with AA metabolites as ligands was upregulated by the fatty acid but was not involved in the AA effect because its transcriptional activity estimated by reporter gene assays was negatively controlled by p38 MAPK pathway. These findings suggest that the effect of AA on human hepatoma cell growth by inducing an oxidative stress may present a clinical interest in the treatment of the liver cancer. (C) 2004 Elsevier SAS. All rights reserved.

Persistent peripheral inflammation is associated with repetitive painful inputs into the spinal cord, leading to a chronic pain state. Related dramatic changes occur in the central nervous system (CNS) including central sensitization, which results in hyperalgesia. This neural plasticity involves in part fatty acids as functional and structural compounds. We hypothesized that central modification of fatty acids metabolism might occur after prolonged peripheral noxious stimulation. In the present study, the regulation of genes involved in fatty acids metabolism in the rat CNS was investigated during a chronic pain state. Using semiquantitative RT-PCR, we explored in the neuraxis the mRNA expression of brain acyl-CoA synthetases (ACS) and acyl-CoA oxidase (ACO), which are major fatty acid-metabolizing enzymes, following complete Freund's adjuvant (CFA) injection into a hind paw. Similar spinal up-regulation of the isoforms ACS2, ACS3, ACS4, and of ACO was detected early after 30 min, reaching a maximal after 6 h post-injection. Other peaks were also observed after 4 and 21 days post-inoculation, corresponding to the acute and chronic inflammation, respectively. Induction occurred only in the lumbar spinal cord ipsilaterally to the inflamed paw and was completely inhibited by a local anaesthesia of the sciatic nerve, suggesting a neural transmission of the inducing signal. Moreover, intrathecal injection of MK801, a noncompetitive NMDA antagonist, partially prevented these inductions, highlighting the involvement of the neurotransmitter glutamate in the central ACS and ACO up-regulation. These findings suggest that the fatty metabolism is stimulated in the CNS during a chronic pain state.

Peroxisome proliferator-activated receptor alpha (PPARalpha) is a member of the nuclear hormone receptor superfamily that can be activated by natural fatty acids and various xenobiotics, including clofibrate. This transcription factor primarily regulates genes involved in lipid metabolism and homeostasis. We present the expression pattern of the PPARalpha subtype in the adult jerboa Jaculus orientalis, determined by RT-PCR and Western blotting using specific probes and a polyclonal antibody for PPARalpha, respectively. PPARalpha is highly expressed in liver and kidney, and to a lesser extent in duodenum and colon. PPARalpha expression is increased at the mRNA and protein levels in liver and duodenum of jerboa treated for 2 weeks with the peroxisome proliferator (PP) clofibrate. The induction is tissue-specific as no significant changes are observed in kidney and colon. The present data indicate that the PP-induced PPARalpha gene expression is not dependent on the PPARalpha content in target cells.

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors belonging to the nuclear receptor superfamily. Different subtypes of PPARs (α, β, and γ) 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 PPARα and PPARβ, but not PPARγ 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 PPARα was homogenous whereas PPARβ 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, PPARα was localized exclusively in astrocytes while PPARβ was present in oligodendrocytes. The possible functions of PPARα and PPARβ expressed in both white and gray matters of the spinal cord will be discussed but need further studies.

Regulation of growth arrest and apoptosis are, in part, controlled by the tumor suppressor p53 after its phosphorylation which causes a determinant role in its functional activation. Moreover, PPAR regulate many functions such as proliferation and apoptosis. We compared the biological activity of diosgenin with hecogenin and tigogenin, plant steroids structurally close to diosgenin, on proliferation rate, cell cycle distribution and apoptosis in human 1547 osteosarcoma cells. We found that all three molecules have an antiproliferative effect but gel shift analysis demonstrated that none of the plant steroids transactivated PPAR in human 1547 osteosarcoma cells whereas these molecules induced NF-kappaB binding to DNA. Although these plant steroids have a very close structure, only diosgenin caused a cell cycle arrest associated with strong apoptosis. This biological action seems correlated with a large increase of p53 protein expression. This fact was showed by immunofluorescence analysis which confirmed that diosgenin strongly enhanced the activation of p53 in contrast to hecogenin and tigogenin actions.

The expression of peroxisome proliferator-activated receptors alpha (PPARalpha) and gamma (PPARgamma) was studied in the human adenocarcinoma Caco-2 cells induced to differentiate by long term culture (15 days). The differentiation of Caco-2 cells was attested by increases in the activities of sucrase-isomaltase and alkaline phosphatase (two brush border enzymes), fatty acyl-CoA oxidase (AOX) and catalase (two peroxisomal enzymes), by an elevation in the protein levels of villin (a brush border molecular marker), AOX, peroxisomal bifunctional enzyme (PBE), catalase and peroxisomal membrane protein of 70 kDa (PMP70). and by the appearance of peroxisomes. The expression of PPARalpha and PPARgamma was investigated by Western blotting, immunocytochemistry, Northern blotting and S1 nuclease protection assay during the differentiation of Caco-2 cells. The protein levels of PPARalpha, PPARgamma, and PPARgamma2 increased gradually during the time-course of Caco-2 cell differentiation. Immunocytochemistry revealed that PPARalpha and gamma were localized in cell nuclei. The PPARgamma1 protein was encoded by PPARgamma3 mRNA because no signal was obtained for PPARgamma1 mRNA using a specific probe in S1 nuclease protection assay. The amount of PPARgamma3 mRNA increased concomitantly to the resulting PPARgamma1 protein. On the other hand, the mRNA of PPARalpha and PPARgamma2 were not significantly changed, suggesting that the increase in their respective protein was due to an elevation of the translational rate. The role played by the PPAR subtypes in Caco-2 cell differentiation is discussed.

This work demonstrated the constitutive expression of peroxisome proliferator-activated receptor (PPAR)-gamma and PPAR-alpha in rat synovial fibroblasts at both mRNA and protein levels. A decrease in PPAR-gamma expression induced by 10 microg/ml lipopolysaccharide (LPS) was observed, whereas PPAR-alpha mRNA expression was not modified. 15-Deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) dose-dependently decreased LPS-induced cyclooxygenase (COX)-2 (-80%) and inducible nitric oxide synthase (iNOS) mRNA expression (-80%), whereas troglitazone (10 microM) only inhibited iNOS mRNA expression (-50%). 15d-PGJ(2) decreased LPS-induced interleukin (IL)-1 beta (-25%) and tumor necrosis factor (TNF)-alpha (-40%) expression. Interestingly, troglitazone strongly decreased TNF-alpha expression (-50%) but had no significant effect on IL-1 beta expression. 15d-PGJ(2) was able to inhibit DNA-binding activity of both nuclear factor (NF)-kappa B and AP-1. Troglitazone had no effect on NF-kappa B activation and was shown to increase LPS-induced AP-1 activation. 15d-PGJ(2) and troglitazone modulated the expression of LPS-induced iNOS, COX-2, and proinflammatory cytokines differently. Indeed, troglitazone seems to specifically target TNF-alpha and iNOS pathways. These results offer new insights in regard to the anti-inflammatory potential of the PPAR-gamma ligands and underline different mechanisms of action of 15d-PGJ(2) and troglitazone in synovial fibroblasts.

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.

Among the different hypotheses advanced to explain the peroxisome proliferator (PP)-induced hepatocarcinogenicity in rodents, one is based on the development of an oxidative stress due to an imbalance in the production of reactive oxygen species that leads to DNA damages and lipid peroxidation. On the other hand, human cells appear to be nonresponsive to PPs. As metallothionein proteins play an important antioxidant role, the aim of the present study was to investigate the expression of metallothionein IA (MTIA) and IIA (MTIIA) in HepG2 human hepatoma cells exposed to clofibric acid. When HepG2 cells were treated for 24 hr with 0.50 or 0.75 mM CA, a significant decrease was observed in MT protein-level determined by Western blotting and in the MTIIA mRNA content analyzed by RT-PCR and Northern blotting. No significant change was observed in the MTIA mRNA amount whatever the CA concentration and the duration of treatment. The decrease in MTIIA mRNA-level was not mediated via peroxisome proliferator-activated receptor alpha as attested by our data from gel mobility shift DNA binding assays, Dot blotting and cotransfection experiments with MTIIA promoter-driven luciferase reporter gene and PPARalpha expression vector. These results provide new insights about the pleiotropic effects of PPs on human cells.

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.

Purpose: Nuclear factor-kappaB (NF-kappaB) has been implicated in anti-apoptotic gene transactivation, according to its transcriptional activity. The present study was designed to investigate whether constitutive NF-kappaB activity could modulate basal apoptosis and intrinsic radiosensitivity of KB, head-and-neck carcinoma cell line and KB3 subline. The KB3 subline was more radiosensitive (SF2 = 0.48, alpha = 0.064) than the radioresistant KB parental cell line (SF2 = 0.80, alpha = 0.114).Methods and Materials: Constitutive NF-kappaB DNA-binding activity was determined using electrophoretic mobility shift assay. Modulation of NF-kappaB activity was performed by exposing both cell lines to tumor necrosis factor a or dexamethasone. Apoptotic cell population was analyzed using flow cytometry (annexin V/propidium iodide). Radiosensitivity was assessed from determination of the surviving fraction at 2 Gy (SF2), and alpha and beta parameters were determined using the linear-quadratic model.Results: Constitutive NF-kappaB activity was found to be significantly lower in KB3 than in KB. KB cell line exposure to dexamethasone significantly decreased NF-kappaB DNA-binding activity and, consequently, enhanced baseline apoptosis and radiosensitivity (alpha values: 0.114 vs. 0.052). Conversely, exposure of KB3 cells to tumor necrosis factor a increased NF-kappaB DNA-binding activity and resulted in a significant decrease (50%) in rate of apoptosis and in radiosensitivity (SF2 values: 0.48 vs. 0.63).Conclusions: Modulation of NF-kappaB DNA-binding activity influences baseline apoptosis and intrinsic radiosensitivity. (C) 2001 Elsevier Science Inc.