[Pioglitazone protects against elastocalcinosis and improves aortic wall elasticity]. / Importance de l'activation des PPARgamma dans la pharmacologie de la rigidité artérielle.

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.

Up-regulation of fatty acid metabolizing-enzymes mRNA in rat spinal cord during persistent peripheral local inflammation.

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.

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.

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

We recently reported that glucosamine reversed the decrease in proteoglycan synthesis and in UDP-glucuronosyltransferase I mRNA expression induced by interleukin-1 beta (IL-1 beta) [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-1 beta 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-1 beta or with ROS decreased the activation of the nuclear factor kappa B, but not the activator protein-1. After treatment with IL-1 beta, glucosamine increased the expression of mRNA encoding the type II IL-1 beta receptor. These results emphasize the potential role of two regulating proteins of the IL-1 beta signaling pathway that could account for the beneficial effect of glucosamine in osteoarthritis.

Constitutive NF-kappaB activity influences basal apoptosis and radiosensitivity of head-and-neck carcinoma cell lines.

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 alpha 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 alpha 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.

Intracellular generation of free radicals and modifications of detoxifying enzymes in cultured neurons from the developing rat forebrain in response to transient hypoxia.

To address the influence of oxidative stress and defense capacities in the effects of transient hypoxia in the immature brain, the time course of reactive oxygen species generation was monitored by flow cytometry using dihydrorhodamine 123 and 2',7'-dichlorofluorescein-diacetate in cultured neurons issued from the fetal rat forebrain and subjected to hypoxia/reoxygenation (6 h/96 h). Parallel transcriptional and activity changes of superoxide dismutases, glutathione peroxidase and catalase were analyzed, in line with cell outcome. The study confirmed hypoxia-induced delayed apoptotic death, and depicted increased mitochondrial and cytosolic productions of free radicals (+30%) occurring over the 48-h period after the restoration of oxygen supply, with sequential stimulations of superoxide dismutases. Whereas catalase mRNA levels and activity were augmented by cell reoxygenation, glutathione peroxidase activity was transiently repressed (-24%), along with reduced glutathione reductase activity (-27%) and intracellular glutathione depletion (-19%). Coupled with the neuroprotective effects of the glutathione precursor N-acetyl-cysteine (50 microM), these data suggest that hypoxia/reoxygenation-induced production of reactive oxygen species can overwhelm glutathione-dependent antioxidant capacity, and thus may contribute to the resulting neuronal apoptosis.

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-kappaB activation and we observed a time-dependent increase in PGE2 synthesis after diosgenin treatment.

Activation of the activator protein-1 by the peroxisome proliferator clofibric acid in rat H4IIEC3 hepatoma cells.

Clofibric acid (CA), a potent peroxisome proliferator (PP), has been shown to cause tumor formation in rat liver. The precise mechanism of action of PPs remains largely unknown. However, it has been proposed that they act by increasing reactive oxygen species (ROS), leading to a cellular oxidative stress. In the present study, we have investigated the effect of CA on the activator protein-1 (AP-1) expression in PP-responsive H4IIEC3 rat hepatoma cells. Electrophoretic mobility shift assays demonstrated that AP-1 activation was induced in cells treated with CA for 24 h at all concentrations of the fibrate. This activation was prolonged up to 48 h. Using transfection experiments with H4IIEC3 cells, we found that CA induced the expression of a reporter gene driven by AP-1 and that of the glutathione S-transferase P target gene. By supershift experiments, jun and fos proteins were identified as components of the CA-activated AP-1 complexes. Western blot analyses revealed that the induction of the AP-1 activity was not dependent to an increase in the levels of jun and fos proteins. Cotreatment of H4IIEC3 cells with CA and the antioxidant N-acetylcysteine or calphostin C, a specific inhibitor of protein kinase C (PKC), blocked the AP-1 activation and the expression of the AP-1-driven luciferase reporter gene. These results demonstrate that CA activates AP-1 in H4IIEC3 cells and that this induction is mediated via ROS and PKC.

15-Deoxy-delta12,14-PGJ2, but not troglitazone, modulates IL-1beta effects in human chondrocytes by inhibiting NF-kappaB and AP-1 activation pathways.

The activation of peroxisome proliferator-activated receptor gamma (PPARgamma) has been shown to inhibit the production and the effects of proinflammatory cytokines. Since interleukin-1beta (IL-1beta) directly mediates cartilage degradation in osteoarthritis, we investigated the capability of PPARgamma ligands to modulate IL-1beta effects on human chondrocytes. RT-PCR and Western blot analysis revealed that PPARgamma expression was decreased by IL-1beta. 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2), in contrast to troglitazone, was highly potent to counteract IL-1beta-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-kappaB 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 PPARgamma-independent.

Down-regulation of peroxisome proliferator-activated receptor-gamma gene expression by sphingomyelins.

We recently demonstrated that the sphingomyelin (SM) content of adipocyte membranes was negatively correlated with the expression of peroxisome proliferator-activated receptor-gamma (PPARgamma) in the subcutaneous adipose tissue of obese women with variable degrees of insulin resistance. We have now investigated whether SM really does have an impact on the expression of PPARgamma in 3T3-F442A adipocytes. Adding SM to the culture medium for 24 h caused a significant increase in SM content of adipocyte membranes and an acyl chain length-dependent decrease in the levels of PPARgamma mRNA and protein. The longer the acyl chain of the fatty acid of SM, the greater was the decrease in PPARgamma. These data suggest that the nature of the fatty acid is important in the regulation of PPARgamma by the SM pathway.

Control of cell proliferation via transduction of sPLA(2)-I activity and possible PPAR activation at the nuclear level.

Pancreatic phospholipase A2 (PLA(2)-I) stimulates U(III) cells proliferation, a rat uterine cell line, after binding to membrane receptors, internalization and translocation. Here, we demonstrate that during these steps of internalization, PLA(2)-I retains its hydrolytic activity and thus could exert its proliferative effect via nuclear phospholipids hydrolysis. Since fatty acids and eicosanoids released by such activity are known to be ligands of PPAR, we study the expression of these nuclear receptors and demonstrate that, in the experimental conditions where PLA(2)-I stimulates U(III) cells proliferation, PLA(2)-I also regulates PPAR expression indicating a possible mechanism of its proliferative effect.

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.

Implications of peroxisome proliferator-activated receptors (PPARS) in development, cell life status and disease.

The past several years have seen an increasing interest in the peroxisome proliferator-activated receptors (PPARs). These transcriptional factors belong to the superfamily of the steroid/thyroid/retinoid receptors. They are activated by fatty acids or their metabolites as well as by different xenobiotic peroxisome proliferators. These receptors are expressed in both the embryo and the adult organism. They have been implicated in cell proliferation, differentiation and apoptosis. In this review, we will attempt to point out some of the more salient features of this expression pattern during development and the different steps of cell life. The current understanding of how PPARs are involved in some human diseases will also be described.

New molecular aspects of regulation of mitochondrial activity by fenofibrate and fasting.

Fenofibrate and fasting are known to regulate several genes involved in lipid metabolism in a similar way. In this study measuring several mitochondrial enzyme activities, we demonstrate that, in contrast to citrate synthase and complex II, cytochrome c oxidase (COX) is a specific target of these two treatments. In mouse liver organelles, Western blot experiments indicated that mitochondrial levels of p43, a mitochondrial T3 receptor, and mitochondrial peroxisome proliferator activated receptor (mt-PPAR), previously described as a dimeric partner of p43 in the organelle, are increased by both fenofibrate and fasting. In addition, in PPAR alpha-deficient mice, this influence was abolished for mt-PPAR but not for p43, whereas the increase in COX activity was not altered. These data indicate that: (1) PPAR alpha is involved in specific regulation of mt-PPAR expression by both treatments; (2) fenofibrate and fasting regulate the mitochondrial levels of p43 and thus affect the efficiency of the direct T3 mitochondrial pathway.

Regional distribution of PPARbeta in the cerebellum of the rat.

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors belonging to the superfamily of steroid hormone receptors. Different subtypes of PPARs (alpha, beta and gamma) have been described, PPARalpha and PPARgamma presenting a more tissue specific distribution than PPARbeta. Specific polyclonal antibodies directed against each subtype of PPARs were produced and characterized. The general expression of PPAR proteins was investigated in rat brain and cerebellar extracts by Western blotting. In order to localize the PPAR proteins and transcripts in the cerebellum, immunocytochemical and in situ hybridization assays were performed. Our Western blot analysis revealed a 52 kDa band with the anti-PPARbeta antibody in brain and cerebellar homogenates, but no band with the anti-PPARalpha, gamma1/gamma2 and gamma2. By immunocytochemistry, a high expression of PPARbeta appeared in the nucleus of Purkinje cells. The in situ hybridization assays showed that PPARbeta transcripts were localized in the cytoplasm of the Purkinje cells. No labeling was observed for the other PPAR isoforms in the cerebellum. Purkinje cells represent the only efferent way from the cerebellar cortex and modulate spinal cord activity. The regional distribution of PPARbeta in these cells suggests some fundamental role for this subtype in this pathway.

A 45 kDa protein related to PPARgamma2, induced by peroxisome proliferators, is located in the mitochondrial matrix.

Besides their involvement in the control of nuclear gene expression by activating several peroxisome proliferator-activated receptors (PPARs), peroxisome proliferators influence mitochondrial activity. By analogy with the previous characterization of a mitochondrial T3 receptor (p43), we searched for the presence of a peroxisome proliferator target in the organelle. Using several antisera raised against different domains of PPARs, we demonstrated by Western blotting, immunoprecipitation and electron microscopy experiments, that a 45 kDa protein related to PPARgamma2 (mt-PPAR) is located in the matrix of rat liver mitochondria. In addition, we found that the amounts of mt-PPAR are increased by clofibrate treatment. Moreover, in EMSA experiments mt-PPAR bound to a DR2 sequence located in the mitochondrial D-loop, by forming a complex with p43. Last, studies of tissue-specific expression indicated that mt-PPAR is detected in mitochondria of all tissues tested except the brain in amounts positively related to p43 abundance. Besides their involvement in the control of nuclear gene expression by activating several peroxisome proliferator-activated receptors (PPARs), peroxisome proliferators influence mitochondrial activity. By analogy with the previous characterization of a mitochondrial T3 receptor (p43), we searched for the presence of a peroxisome proliferator target in the organelle. Using several antisera raised against different domains of PPARs, we demonstrated by Western blotting, immunoprecipitation and electron microscopy experiments, that a 45 kDa protein related to PPARgamma2 (mt-PPAR) is located in the matrix of rat liver mitochondria. In addition, we found that the amounts of mt-PPAR are increased by clofibrate treatment. Moreover, in EMSA experiments mt-PPAR bound to a DR2 sequence located in the mitochondrial D-loop, by forming a complex with p43. Last, studies of tissue-specific expression indicated that mt-PPAR is detected in mitochondria of all tissues tested except the brain in amounts positively related to p43 abundance.

Evidence for the presence of peroxisome proliferator-activated receptor (PPAR) alpha and gamma and retinoid Z receptor in cartilage. PPARgamma activation modulates the effects of interleukin-1beta on rat chondrocytes.

Peroxisome proliferator-activated receptor (PPAR) alpha, PPARgamma, and retinoid acid receptor-related orphan receptor (ROR) alpha are members of the nuclear receptor superfamily of ligand-activated transcription factors. Although they play a key role in adipocyte differentiation, lipid metabolism, or glucose homeostasis regulation, recent studies suggested that they might be involved in the inflammation control and especially in the modulation of the cytokine production. This strongly suggests that these transcriptional factors could modulate the deleterious effects of interleukin-1 (IL-1) on cartilage. However, to date, their presence in cartilage has never been investigated. By quantitative reverse transcription-polymerase chain reaction, Western blot, and immunocytochemistry analysis, we demonstrated, for the first time, the presence of PPARalpha, PPARgamma, and RORalpha in rat cartilage, at both mRNA and protein levels. Comparatively, the PPARalpha mRNA content in cartilage was much lower than in the liver but not significantly different to that of the adipose tissue. PPARgamma mRNA expression in cartilage was weak, when compared with adipose tissue, but similar to that found in the liver. RORalpha mRNA levels were similar in the three tissues. mRNA expression of the three nuclear receptors was very differently modulated by IL-1 or mono-iodoacetate treatments. This indicates that they should be unequally involved in the effects of IL-1 on chondrocyte, which is in accordance with results obtained in other cell types. Indeed, we showed that 15d-PGJ2 mainly, but also the drug troglitazone, that are ligands of PPARgamma could significantly counteract the decrease in proteoglycan synthesis and NO production induced by IL-1. By contrast, PPARalpha ligands such as Wy-14,643 or clofibrate had no effect on this process. Therefore, the presence of PPARgamma in chondrocytes opens up new perspectives to modulate the effects of cytokines on cartilage by the use of specific ligands. The function of the two other transcription factors, PPARalpha and RORalpha identified in chondrocytes remains to be explored.

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

We investigated the spatiotemporal distributions of the different peroxisome proliferator-activated receptor (PPAR) isotypes (alpha, beta, and gamma) 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 PPARgamma, are expressed as early as 7 weeks of development in cell types of endodermal and mesodermal origin. The presence of PPARgamma was also found by Western blotting and nuclease-S1 protection assay, confirming that this subtype is not adipocyte-specific. PPARalpha, PPARbeta, and PPARgamma exhibit different patterns of expression during morphogenesis of the digestive tract. Whatever the stage and the gut region (except the stomach) examined, PPARgamma is expressed at a high level, suggesting some fundamental role for this receptor in development and/or physiology of the human digestive tract.

Relationship between signal transduction and PPAR alpha-regulated genes of lipid metabolism in rat hepatic-derived Fao cells.

The goal of this study was to characterize phosphorylated proteins and to evaluate the changes in their phosphorylation level under the influence of a peroxisome proliferator (PP) with hypolipidemic activity of the fibrate family. The incubation of rat hepatic derived Fao cells with ciprofibrate leads to an overphosphorylation of proteins, especially one of 85 kDa, indicating that kinase (or phosphatase) activities are modified. Moreover, immunoprecipitation of 32P-labeled cell lysates shows that the nuclear receptor, PP-activated receptor, alpha isoform, can exist in a phosphorylated form, and its phosphorylation is increased by ciprofibrate. This study shows that PP acts at different steps of cell signaling. These steps can modulate gene expression of enzymes involved in fatty acid metabolism and lipid homeostasis, as well as in detoxication processes.

Phosphorylation of peroxisome proliferator-activated receptor alpha in rat Fao cells and stimulation by ciprofibrate.

The basic mechanism(s) by which peroxisome proliferators activate peroxisome proliferator-activated receptors (PPARs) is (are) not yet fully understood. Given the diversity of peroxisome proliferators, several hypotheses of activation have been proposed. Among them is the notion that peroxisome proliferators could activate PPARs by changing their phosphorylation status. In fact, it is well known that several members of the nuclear hormone receptor superfamily are regulated by phosphorylation. In this report, we show that the rat Fao hepatic-derived cell line, known to respond to peroxisome proliferators, exhibited a high content of PPARalpha. Alkaline phosphatase treatment of Fao cell lysate as well as immunoprecipitation of PPARalpha from cells prelabeled with [32P] orthophosphate clearly showed that PPARalpha is indeed a phosphoprotein in vivo. Moreover, treatment of rat Fao cells with ciprofibrate, a peroxisome proliferator, increased the phosphorylation level of the PPARalpha. In addition, treatment of Fao cells with phosphatase inhibitors (okadaic acid and sodium orthovanadate) decreased the activity of ciprofibrate-induced peroxisomal acyl-coenzyme A oxidase, an enzyme encoded by a PPARalpha target gene. Our results suggest that the gene expression controlled by peroxisome proliferators could be mediated in part by a modulation of the PPARalpha effect via a modification of the phosphorylation level of this receptor.