tretinoin and pirinixic-acid

Topics: Acyl-CoA Oxidase; Animals; Base Sequence; Clofibrate; Cloning, Molecular; Diethylhexyl Phthalate; DNA; Fatty Acids; Humans; Liver; Liver Neoplasms, Experimental; Mice; Microbodies; Molecular Sequence Data; Oxidoreductases; Promoter Regions, Genetic; Pyrimidines; Rats; Receptors, Cytoplasmic and Nuclear; Receptors, Retinoic Acid; Retinoid X Receptors; Risk Assessment; Transcription Factors; Tretinoin

The fatty acid sensing nuclear receptor families retinoid X receptors (RXRs) and peroxisome proliferator-activated receptors (PPARs) hold therapeutic potential in neurodegeneration. Valuable pleiotropic activities of Wy14,643 in models of such conditions exceed its known PPAR agonistic profile. Here, we characterize the compound as an RXR agonist explaining the pleiotropic effects and report its systematic structure-activity relationship analysis with the discovery of specific molecular determinants driving activity on PPARs and RXRs. We have designed close analogues of the drug comprising selective and dual agonism on RXRs and PPARs that may serve as superior pharmacological tools to study the role and interplay of the nuclear receptors in various pathologies. A systematically optimized high potency RXR agonist revealed activity in vivo and active concentrations in brain. With its lack of RXR/liver X receptor-mediated side effects and superior profile compared to classical rexinoids, it establishes a new class of innovative RXR modulators to overcome key challenges in RXR targeting drug discovery.

Topics: Animals; HEK293 Cells; Hep G2 Cells; Humans; Male; Mice, Inbred C57BL; Microsomes, Liver; Molecular Structure; Peroxisome Proliferator-Activated Receptors; Pyrimidines; Rats; Retinoid X Receptors; Structure-Activity Relationship

The ability of mammals to resist body fat accumulation is linked to their ability to expand the number of "brown adipocytes" within white fat depots. All-trans retinoic acid (t-RA) and peroxi-some proliferator-activated receptor-α (PPARα) have been implicated in "browning-like" or "browning" programs, respectively. However, a PPARα-agonist (WY14643) failed to regulate the expression of the uncoupling protein 1(UCP1) gene unless combined with retinoic acid. This study investigated the effects of the PPARα-agonist WY14643 combined with t-RA, on the "browning" of white adipocytes in mice mediated by UCP1, and the molecular mechanisms involved in this process. We compared the effects of WY14643 alone and WY14643 combined with t-RA or the p38 MAPK-inhibitor, SB203580, on white adipocytes after 24 h using the expression of UCP1, detected with RT-PCR and western blot. We also determined the mechanism by which p38 MAPK and phospho-p38 MAPK influence the process of "brown-ing" using western blot. All concentrations of WY14643 failed to in-duce UCP1 mRNA expression, protein expression, or phosphorylation of p38 MAPK (P < 0.05). WY14643 combined with t-RA was observed to induce UCP1 mRNA expression, protein expression, and phosphory-lation of p38 MAPK (P < 0.05). SB203580 combined with WY14643 and t-RA suppressed UCP1 mRNA expression, protein expression, and p38 MAPK phosphorylation (P < 0.05). WY14643 combined with t-RA can induce the transformation of white adipocytes to brown adipocytes through activation of the p38 MAPK signaling pathway.

Topics: 3T3-L1 Cells; Adipocytes, Brown; Adipocytes, White; Animals; Cell Differentiation; Drug Synergism; Gene Expression Regulation; Imidazoles; Ion Channels; Male; Mice; Mitochondrial Proteins; p38 Mitogen-Activated Protein Kinases; Phosphorylation; PPAR alpha; Pyridines; Pyrimidines; RNA, Messenger; Signal Transduction; Tretinoin; Uncoupling Protein 1

Recent literature highlights the importance of pro-inflammatory cytokines in the biology of breast cancer stem cells (CSCs), unraveling differences with respect to their normal counterparts. Expansion of mammospheres (MS) is a valuable tool for the in vitro study of normal and cancer mammary gland stem cells. Here, we expanded MSs from human breast cancer and normal mammary gland tissues, as well from tumorigenic (MCF7) and non-tumorigenic (MCF10) breast cell lines. We observed that agonists for the retinoid X receptor (6-OH-11-O-hydroxyphenanthrene), retinoic acid receptor (all-trans retinoic acid (RA)) and peroxisome proliferator-activated receptor (PPAR)-γ (pioglitazone (PGZ)), reduce the survival of MS generated from breast cancer tissues and MCF7 cells, but not from normal mammary gland or MCF10 cells. This phenomenon is paralleled by the hampering of pro-inflammatory Nuclear Factor-κB (NF-κB)/Interleukin-6 (IL6) axis that is hyperactive in breast cancer-derived MS. The hindrance of such pathway associates with the downregulation of MS regulatory genes (SLUG, Notch3, Jagged1) and with the upregulation of the differentiation markers estrogen receptor-α and keratin18. At variance, the PPARα agonist Wy14643 promotes MS formation, upregulating NF-κB/IL6 axis and MS regulatory genes. These data reveal that nuclear receptors agonists (6-OH-11-O-hydroxyphenanthrene, RA, PGZ) reduce the inflammation dependent survival of breast CSCs and that PPARα agonist Wy14643 exerts opposite effects on this phenotype.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Survival; Female; Humans; Inflammation; Interleukin-6; Neoplastic Stem Cells; NF-kappa B; Phenanthrenes; Pioglitazone; PPAR gamma; Pyrimidines; Receptors, Cytoplasmic and Nuclear; Receptors, Retinoic Acid; Retinoid X Receptors; Thiazolidinediones; Tretinoin

  Retinoids have been used for the treatment of skin disorders such as acne, psoriasis, and photoaging. However, despite their beneficial effects, topical retinoids often cause severe local irritation called retinoid dermatitis. We previously developed a novel vitamin A derivative, retinyl retinoate, which induces less irritation and affords excellent tolerance. In this study, we examined whether co-treatment with topical peroxisome proliferator-activated receptor-α (PPARα) agonists (e.g. WY14643) reduce retinoid dermatitis in hairless mouse skin..   The effect of concomitant treatment with a PPARα agonist on retinoid dermatitis in hairless mouse epidermis was evaluated by measuring transepidermal water loss, epidermal histology, and cytokine expression..   Retinyl retinoate induced less severe retinoid dermatitis than retinoic acid. Topical application of a PPARα agonist improved the stratum corneum structure and function, reduced mRNA expression of interleukin (IL)-1α, tumor necrosis factor-α and IL-8, and inhibited ear edema induced by retinoic acid or retinyl retinoate..   Our results indicate that PPARα agonists can potentially be used to improve retinoid dermatitis. We suggest that co-treatment with retinyl retinoate and a PPARα agonist may reduce or prevent detrimental alterations in retinoid-treated skin.

Topics: Administration, Topical; Animals; Cell Differentiation; Cell Proliferation; Dermatitis; Enzyme Inhibitors; Epidermis; Female; Interleukin-1alpha; Interleukin-8; Keratolytic Agents; Liver X Receptors; Mice; Mice, Hairless; Orphan Nuclear Receptors; Palmitic Acid; PPAR alpha; Pyrimidines; Retinoids; Retinyl Esters; RNA, Messenger; Tretinoin; Tumor Necrosis Factor-alpha; Water Loss, Insensible

The pseudokinase tribbles homologue 3 (Drosophila) (TRIB3) negatively interferes with insulin-mediated phosphorylation and activation of v-akt murine thymoma viral oncogene homologue 1 (AKT1, also known as protein kinase B). Animal studies have shown that Trib3 expression was higher in the fasting state and in animal models of diabetes, promoting hyperglycaemia presumably by increasing glucose production in the liver. Less is known about the role of TRIB3 in insulin resistance in humans, although a gain-of-function mutation associated with abnormalities related to insulin resistance has been described in TRIB3.. We determined hepatic mRNA expression of TRIB3 and selected genes encoding enzymes, transcription factors and coactivators involved in glucose homeostasis. We also determined biochemical variables of intermediary metabolism in obese patients with varying degrees of insulin resistance.. In our study population hepatic TRIB3 mRNA expression was associated with surrogate markers of insulin resistance. TRIB3 expression was significantly increased in a subgroup with high HOMA of insulin resistance (HOMA-IR) compared with a low HOMA-IR group (p = 0.0033). TRIB3 transcript levels were correlated with PEPCK (also known as PCK2) mRNA expression (p = 0.0014) and mRNA expression of PPARGC1A (p = 0.0020), PPARGC1B (p < 0.0001), USF1 (p = 0.0017), FOXO1 (p = 0.0003) and SREBP-1c (also known as SREBF1; p = 0.0360). Furthermore ligands of peroxisome proliferator-activated receptor alpha/retinoid X receptor and overexpression of its coactivator PPARGC1A as well as overexpression of SREBP-1c and its coactivator PPARGC1B increased TRIB3 promoter activity in HepG2 cells.. We have found evidence for a role of aberrant hepatic TRIB3 transcript levels in insulin resistance in obese humans and identified potential transcriptional pathways involved in regulation of TRIB3 gene expression in the liver.

Topics: Carrier Proteins; Cell Cycle Proteins; Gene Expression; Heat-Shock Proteins; Hep G2 Cells; Humans; Insulin Resistance; Liver; Obesity; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; PPAR alpha; Protein Serine-Threonine Kinases; Pyrimidines; Repressor Proteins; RNA-Binding Proteins; RNA, Messenger; Sterol Regulatory Element Binding Protein 1; Transcription Factors; Tretinoin

Insulin resistance is the failure of insulin to stimulate the transport of glucose into its target cells. A highly regulatable supply of glucose is important for cardiomyocytes to cope with situations of metabolic stress. We recently observed that isolated adult rat cardiomyocytes become insulin resistant in vitro. Insulin resistance is combated at the whole body level with agonists of the nuclear receptor complex peroxisome proliferator-activated receptor gamma (PPARgamma)/retinoid X receptor (RXR). We investigated the effects of PPARgamma/RXR agonists on the insulin-stimulated glucose transport and on insulin signaling in insulin-resistant adult rat cardiomyocytes. Treatment of cardiomyocytes with ciglitazone, a PPARgamma agonist, or 9-cis retinoic acid (RA), a RXR agonist, increased insulin- and metabolic stress-stimulated glucose transport, whereas agonists of PPARalpha or PPARbeta/delta had no effect. Stimulation of glucose transport in response to insulin requires the phosphorylation of the signaling intermediate Akt on the residues Thr308 and Ser473 and, downstream of Akt, AS160 on several Thr and Ser residues. Phosphorylation of Akt and AS160 in response to insulin was lower in insulin-resistant cardiomyocytes. However, treatment with 9-cis RA markedly increased phosphorylation of both proteins. Treatment with 9-cis RA also led to better preservation of microtubules in cultured cardiomyocytes. Disruption of microtubules in insulin-responsive cardiomyocytes abolished insulin-stimulated glucose transport and reduced phosphorylation of AS160 but not Akt. Metabolic stress-stimulated glucose transport also involved AS160 phosphorylation in a microtubule-dependent manner. Thus, the stimulation of glucose uptake in response to insulin or metabolic stress is dependent in cardiomyocytes on the presence of intact microtubules.

Topics: Alitretinoin; AMP-Activated Protein Kinase Kinases; Animals; Cells, Cultured; Cytoskeleton; Glucose; Glucose Transporter Type 4; Insulin; Insulin Resistance; Male; Microtubules; Myocytes, Cardiac; Phenoxyacetates; PPAR gamma; Protein Kinases; Pyrimidines; Rats; Rats, Sprague-Dawley; Retinoid X Receptors; Signal Transduction; Thiazolidinediones; Tretinoin

In rodents, treatment with peroxisome proliferator-activated receptor alpha (PPARalpha) agonists results in peroxisome proliferation, hepatocellular hypertrophy, and hepatomegaly. Drugs in the fibrate class of PPARalpha agonists have also been reported to produce rare skeletal muscle toxicity. Although target-driven hepatic effects of PPARalpha treatment have been extensively studied, a characterization of the transcriptional effects of this nuclear receptor/transcription factor on skeletal muscle responses has not been reported. In this study we investigated the effects of PPARalpha agonists on skeletal muscle gene transcription in rats. Further, since statins have been reported to preferentially effect type II muscle fibers, we compared PPARalpha signaling effects between type I and type II muscles. By comparing the transcriptional responses of agonists that signal through different nuclear receptors and using a selection/deselection analytical strategy based on ANOVA, we identified a PPARalpha activation signature that is evident in type I (soleus), but not type II (quadriceps femoris), skeletal muscle fibers. The fiber-type-selective nature of this response is consistent with increased fatty acid uptake and beta-oxidation, which represent the major clinical benefits of the hypolipidemic compounds used in this study, but does not reveal any obvious off-target pathways that may drive adverse effects.

Topics: Animals; Bezafibrate; Fatty Acids; Female; Fenofibrate; Gene Expression Profiling; Glucose; Hypolipidemic Agents; Liver; Muscle Fibers, Slow-Twitch; Muscle, Skeletal; PPAR alpha; Pyrimidines; Rats; Rats, Inbred Strains; Rosiglitazone; Thiazolidinediones; Tretinoin

Microsomal triglyceride transfer protein (MTP) is rate-limiting in the assembly and secretion of lipoproteins containing apolipoprotein (apo) B. Previously we demonstrated that Wy 14,643 (Wy), a peroxisome proliferator-activated receptor (PPAR) alpha agonist, increases apoB-100 secretion despite decreased triglyceride synthesis. In this study, we sought to determine whether PPARalpha activation increases MTP expression and activity. Treatment with Wy increased hepatic MTP expression and activity in rats and mice and increased MTP expression in primary cultures of rat and mouse hepatocytes. Addition of actinomycin D blocked this increase and the MTP promoter (-136 to +67) containing a conserved DR1 element was activated by Wy, showing that PPARalpha activates transcription of the gene. Wy did not affect MTP expression in the intestine or in cultured hepatocytes from PPARalpha-null mice. A retinoid X receptor agonist (9-cis-retinoic acid), but not a PPARgamma agonist (rosiglitazone), increased MTP mRNA expression in cultured hepatocytes from both wild type and PPARalpha-null mice. In rat hepatocytes incubated with Wy, MTP mRNA levels increased between 6 and 24 h, and MTP protein expression and apoB-100 secretion increased between 24 and 72 h. In conclusion, PPARalpha activation stimulates hepatic MTP expression via increased transcription of the Mtp gene. This effect is paralleled by a change in apoB-100 secretion, indicating that the effect of Wy on apoB-100 secretion is mediated by increased expression of MTP.

Topics: Alitretinoin; Animals; Apolipoproteins B; Carrier Proteins; Cell Line; Cells, Cultured; Dactinomycin; Gene Expression Regulation; Hepatocytes; Liver; Male; Mice; Mice, Inbred C57BL; PPAR alpha; Promoter Regions, Genetic; Pyrimidines; Rats; Rats, Sprague-Dawley; RNA, Messenger; Rosiglitazone; Thiazolidinediones; Transcription, Genetic; Tretinoin

Several contaminants detected in aquatic ecosystems are agonists of peroxisome proliferator-activated receptors (PPARs). Peroxisome proliferator-activated receptors interact with the retinoid X receptor (RXR) to activate the transcription of genes that control a variety of physiological functions. We cloned and sequenced partial cDNA fragments of rainbow trout (Oncorhynchus mykiss) PPARalpha and PPARbeta from rainbow trout (rt) gill-W1 cells, a cell line derived from rainbow trout gills; predicted amino acid identities are 77% and 82% compared with their respective human homologs and 83 to 88% and 91 to 98% identical to fish homologs. A reporter gene assay was developed by transfecting rt-gill-W1 cells with a reporter gene construct containing the peroxisome proliferator response element (PPRE) of the rat liver 3-ketoacyl-CoA thiolase B (TB) gene, which drives luciferase expression. Agonists of both PPARalpha (WY14,643 and gemfibrozil) and PPARbeta (bezafibrate) induced luciferase activity, while rosiglitazone, a PPARgamma agonist, was not effective. The fibrate drug, bezafibrate increased luciferase activity in a dose-dependent manner, but addition of 50 nM 9-cis-retinoic acid to the transfected rt-gill-W1 cell culture maximized the sensitivity of the assay so that bezafibrate could be detected at concentrations as low as 6 nM. Extracts from treated domestic wastewater containing fibrate drugs induced luciferase activity in the transfected gill cells. This in vitro reporter gene assay shows promise as a rapid and sensitive technique for screening environmental samples for PPAR-active substances.

Topics: Acetyl-CoA C-Acyltransferase; Alitretinoin; Angiogenesis Inhibitors; Animals; Bezafibrate; Cloning, Molecular; DNA; DNA, Complementary; Dose-Response Relationship, Drug; Environmental Monitoring; Gemfibrozil; Genes, Reporter; Humans; Hypolipidemic Agents; In Vitro Techniques; Liver; Luciferases; Oncorhynchus mykiss; Peroxisome Proliferator-Activated Receptors; Peroxisome Proliferators; Plasmids; PPAR alpha; PPAR-beta; Promoter Regions, Genetic; Pyrimidines; Rats; Retinoid X Receptors; Transcription, Genetic; Transfection; Tretinoin; Water Pollutants, Chemical

To determine the effects of peroxisome proliferator-activated receptor alpha (PPARalpha) and retinoid X receptor (RXR) agonists on insulin action, we investigated the effects of Wy-14643 and 9- cis-retinoic acid (9- cis-RA) on insulin signalling and glucose uptake in human myotubes.. Primary cultures of differentiated human skeletal muscle cells, established from healthy subjects and Type 2 diabetic patients, were used to study the effects of Wy-14643 and 9- cis-RA on the expression and activity of proteins involved in the insulin signalling cascade. Glucose transport was assessed by measuring the rate of [(3)H]2-deoxyglucose uptake.. Wy-14643 and 9- cis-RA increased IRS-2 and p85alpha phosphatidylinositol 3-kinase (PI 3-kinase) mRNA and protein expression in myotubes from non-diabetic and Type 2 diabetic subjects. This resulted in increased insulin stimulation of protein kinase B phosphorylation and increased glucose uptake in cells from control subjects. Myotubes from diabetic patients displayed marked alterations in the stimulation by insulin of the IRS-1/PI 3-kinase pathway. These alterations were associated with blunted stimulation of glucose transport. Treatment with Wy-14643 and 9- cis-RA did not restore these defects but increased the basal rate of glucose uptake.. These results demonstrate that PPARalpha and RXR agonists can directly affect insulin signalling in human muscle cells. They also indicate that an increase in the IRS-2/PI 3-kinase pathway does not overcome the impaired stimulation of the IRS-1-dependent pathway and does not restore insulin-stimulated glucose uptake in myotubes from Type 2 diabetic patients.

Topics: Adult; Diabetes Mellitus, Type 2; Glucose; Humans; Insulin; Insulin Receptor Substrate Proteins; Intracellular Signaling Peptides and Proteins; Muscle Fibers, Skeletal; Muscle, Skeletal; Phosphatidylinositol 3-Kinases; Phosphoproteins; Pyrimidines; Reference Values; Signal Transduction; Tretinoin

We have reported that human TR2 orphan nuclear receptor (TR2) can modulate the transcriptional activity of the reporter gene containing an AGGTCA direct repeat-hormone response element.. The aim of this study is to investigate the potential role and regulation of TR2 in human HaCaT keratinocytes.. We performed mainly chloramphenicol acetyltransferase reporter gene assays (CAT assays), and Western blot analysis.. From CAT assays, TR2 can suppress retinoic acid (RA)-induced transactivation by 44.7% in HaCaT keratinocytes. This suppression is similar to our previous report showing TR4 orphan nuclear receptor (TR4) can suppress RA-induced transactivation. However, TR4 but not TR2 can significantly repress Wy-14643-mediated peroxisome proliferator-activated receptor alpha (PPAR alpha) transactivation by 95%. Western blot analysis suggested that Wy-14643 can differentially regulate the expression of TR2 and TR4 (by increasing the expression of TR4 protein and decreasing that of TR2) in HaCaT keratinocytes.. Our data not only provides the first evidence to demonstrate that close members of orphan nuclear receptors group, such as TR2 and TR4, can have distinct functions, but also suggests the existence of differential and bi-directional regulation between PPAR alpha and TR2/TR4, that may play some important roles in the PPAR alpha signaling pathway in human keratinocytes.

Topics: Binding, Competitive; Cell Line; Down-Regulation; Humans; Keratinocytes; Ligands; Nuclear Receptor Subfamily 2, Group C, Member 1; Peroxisome Proliferators; Pyrimidines; Receptors, Cytoplasmic and Nuclear; Receptors, Steroid; Receptors, Thyroid Hormone; Response Elements; Transcription Factors; Transcriptional Activation; Tretinoin; Up-Regulation

We investigated whether and how could various modulators of arachidonic acid metabolism affect apoptosis induced by tumour necrosis factor-alpha (TNF-alpha) in human myeloid leukaemia HL-60 cells. These included arachinonyltrifluoromethyl ketone (AACOCF3; cytosolic phospholipase A2 inhibitor), indomethacin (cyclooxygenase inhibitor), MK-886 (3-[1-(4-chlorobenzyl)-3-t-butyl-thio-5-isopropylindol-2-yl]-2,2-dimethyl propanoic acid; 5-lipoxygenase-activating protein inhibitor), nordihydroguaiaretic acid (general lipoxygenase inhibitor), and arachidonic acid itself. Incubation of HL-60 cells with nordihydroguaiaretic acid resulted in apoptosis and it was characterised by mitochondria membrane depolarisation, release of cytochrome c from mitochondria into cytosol and activation of caspase-3. Indomethacin and nordihydroguaiaretic acid synergistically potentiated TNF-alpha-induced apoptosis, while arachidonic acid, AACOCF3 and MK-886 did not modulate its effects. Furthermore, indomethacin potentiated apoptosis in cells treated with a differentiating agent, all-trans retinoic acid, which induces resistance to TNF-alpha. However, the observed effects were probably not associated either with the cyclooxygenase- or lipoxygenase-dependent activities of indomethacin and nordihydroguaiaretic acid, respectively. Since indomethacin may reportedly activate peroxisome proliferator-activated receptors (PPARs), the effects of specific ligands of PPARs on apoptosis were studied as well. It was found that selective PPARs ligands had no effects on TNF-alpha-induced apoptosis. The findings suggest that arachidonic acid metabolism does not play a key role in regulation of apoptosis induced by TNF-alpha in the present model. Nevertheless, our data raise the possibility that indomethacin could potentially be used to improve the treatment of human myeloid leukaemia.

Topics: Apoptosis; Arachidonic Acid; Caspase 3; Caspases; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Cyclooxygenase Inhibitors; Cytochrome c Group; Cytosol; Dose-Response Relationship, Drug; Drug Synergism; HL-60 Cells; Humans; Indomethacin; Isoenzymes; Masoprocol; Membrane Proteins; Peroxisome Proliferators; Phospholipases A; Phospholipases A2; Prostaglandin-Endoperoxide Synthases; Pyrimidines; Receptors, Cytoplasmic and Nuclear; Thiazoles; Thiazolidinediones; Transcription Factors; Tretinoin; Tumor Necrosis Factor-alpha

Regulation of p85a phosphatidylinositol-3-kinase (p85alphaPI-3K) expression by peroxisome proliferator-activated receptor (PPAR) activators was studied in human skeletal muscle cells. Activation of PPARgamma or PPARbeta did not modify the expression of p85alphaPI-3K. In contrast, activation of PPARalpha increased p85alphaPI-3K mRNA. This effect was potentiated by 9-cis-retinoic acid, an activator of RXR. Up-regulation of p85alphaPI-3K gene expression resulted in a rise in p85alphaPI-3K protein level and in an increase in insulin-induced PI3-kinase activity. According to the role of p85alphaPI-3K in insulin action, these results suggest that drugs with dual action on both PPARgamma and PPARalpha can be of interest for the treatment of insulin resistance.

Topics: Alitretinoin; Blotting, Western; Cells, Cultured; Female; Gene Expression Regulation, Enzymologic; Humans; Male; Middle Aged; Muscle, Skeletal; Phosphatidylinositol 3-Kinases; Pyrimidines; Receptors, Cytoplasmic and Nuclear; RNA, Messenger; Transcription Factors; Tretinoin; Up-Regulation

Peroxisome proliferator-activated receptor-alpha (PPARalpha) is a member of the steroid hormone receptor superfamily. In rodents, PPARalpha alters genes involved in cell cycle regulation in hepatocytes. Some of these genes are implicated in neuronal cell death. Therefore, in this study, we examined the toxicological consequence of PPARalpha activation in rat primary cultures of cerebellar granule neurons. Our studies demonstrated the presence of PPARalpha mRNA in cultures by reverse transcriptase-polymerase chain reaction. After 10 days in vitro, cerebellar granule neuron cultures were incubated with the selective PPARalpha activator 4-chloro-6-(2,3-xylidino)2-pyrimidinylthioacetic acid (Wy-14,643). The inherent toxicity of Wy-14,643 and the effect of PPARalpha activation following toxic stimuli were assessed. In these studies, neurotoxicity was induced through reduction of extracellular [KCl] from 25 mM to 5.36 mM. We observed no inherent toxicity of Wy-14,643 (24 hr) in cultured cerebellar granule cells. However, after reduction of [KCl], cerebellar granule cell cultures incubated with Wy-14,643 showed significantly greater toxicity than controls. These results suggest a possible role for PPARalpha in augmentation of cerebellar granule neuronal death after toxic stimuli.

Topics: Alitretinoin; Animals; Apoptosis; Cells, Cultured; Cerebellar Cortex; L-Lactate Dehydrogenase; Nerve Tissue Proteins; Neurons; Potassium Chloride; Pyrimidines; Rats; Rats, Sprague-Dawley; Receptors, Cytoplasmic and Nuclear; Receptors, Retinoic Acid; Retinoid X Receptors; Reverse Transcriptase Polymerase Chain Reaction; Transcription Factors; Tretinoin; Up-Regulation

The peroxisome proliferator-activated receptor-gamma (PPARgamma) is activated by 15-deoxy-delta(12,14) prostaglandin J2 (15d-PGJ2), anti-diabetic thiazolidinediones and several non-steroidal anti-inflammatory drugs (NSAIDs). In rat glial cells, lipopolysaccharide and interferon-gamma (LPS/IFN-gamma) induced expression of both inducible nitric oxide synthase (iNOS) and heme oxygenase-1 (HO-1). PPARgamma activators inhibited iNOS expression by LPS and IFN-gamma. However, PPARgamma activator alone induced HO-1 expression and further enhanced LPS/IFN-gamma-induced HO-1 expression. These results suggest that activation of PPARgamma negatively regulate iNOS expression and positively regulates HO-1 expression in glial cells.

Topics: Alitretinoin; Animals; Anti-Inflammatory Agents, Non-Steroidal; Brain; Cells, Cultured; Dinoprostone; Enzyme Induction; Gene Expression Regulation, Enzymologic; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; Interferon-gamma; Lipopolysaccharides; Neuroglia; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitrites; Prostaglandin D2; Pyrimidines; Rats; Rats, Wistar; Receptors, Cytoplasmic and Nuclear; Thiazoles; Thiazolidinediones; Transcription Factors; Tretinoin

The peroxisome proliferator-activated receptors (PPARs) are lipid-activated transcription factors involved in the regulation of lipid metabolism and adipocyte differentiation. Little is known, however, about the control of the expression of the genes encoding each of all three receptor subtypes: alpha, delta, and gamma. We have addressed this question in the brown adipocyte, the only cell type that co-expresses high levels of the three PPAR subtypes. Differentiation of brown adipocytes is associated with enhanced expression of PPAR genes. However, whereas PPARgamma and PPARdelta genes are already expressed in preadipocytes, the mRNA for PPARalpha appears suddenly in association with the acquisition of the terminally differentiated phenotype. Both retinoic acid isomers and PPAR agonists, specific for either PPARalpha or PPARgamma, regulate expression of each PPAR subtype gene in the opposite way: they up-regulate PPARalpha and down-regulate PPARgamma. The effects on PPARalpha mRNA are independent of protein synthesis, whereas inhibition of PPARgamma mRNA expression depends on protein synthesis, except when its specific ligand prostaglandin J2 is used. Our results indicate a strictly opposite autoregulation of PPAR subtypes, which supports specific physiological roles for them in controlling brown fat differentiation and thermogenic activity.

Topics: Adipocytes; Adipose Tissue, Brown; Animals; Cell Differentiation; Cells, Cultured; DNA-Binding Proteins; Gene Expression Regulation; Ligands; Male; Mice; Prostaglandin D2; Protein Isoforms; Pyrimidines; Receptors, Cytoplasmic and Nuclear; RNA, Messenger; Transcription Factors; Tretinoin

Rodent brown adipocytes express peroxisome proliferator activated receptor-alpha (PPARalpha) and PPARgamma and while the rodent uncoupling protein-1 (UCP-1) gene contains a putative peroxisome proliferator response element (PPRE), only PPARgamma activation by thiazolidinediones increase UCP-1 mRNA levels. We have investigated this phenomenon in foetal rat brown adipocytes (FBA) and show that although transactivation occurs in FBA containing a plasmid encoding 4.5kb of the 5'-flanking region of the rat UCP-1 promoter ((-4551)-UCP-1-CAT) treated with either the selective PPARgamma agonist rosiglitazone (1.0 microM) or the selective PPARalpha agonist Wy 14643 (10.0 microM), only rosiglitazone induced transcription of UCP-1 mRNA. Furthermore, Wy 14643 (10 and 100.0 microM) abolished rosiglitazone-induced UCP-1 mRNA induction in spite of a transactivation event occurring with the combination treatment. Thus in FBA PPARalpha-activation with Wy 14643 elicits a "blind" transactivation of the UCP-1 promoter which can prevent PPARgamma-mediated UCP-1 mRNA transcription either by competition for the PPRE or by an unidentified post-transcriptional event.

Topics: Adipose Tissue, Brown; Alitretinoin; Animals; Carrier Proteins; Cells, Cultured; Gene Expression Regulation; Ion Channels; Membrane Proteins; Mitochondrial Proteins; Peroxisome Proliferators; Promoter Regions, Genetic; Pyrimidines; Rats; Rats, Sprague-Dawley; Receptors, Cytoplasmic and Nuclear; RNA, Messenger; Rosiglitazone; Thiazoles; Thiazolidinediones; Transcription Factors; Transcription, Genetic; Transcriptional Activation; Transfection; Tretinoin; Uncoupling Protein 1

Transcriptional regulation of gene expression by nuclear receptors requires negatively and positively acting cofactors. Recent models for receptor activation propose that certain receptors in the absence of ligands can recruit corepressors while ligand binding results in conformational changes leading to the recruitment of coactivators. Previous work has established a coactivator role for the SRC-1 family members as well as an involvement of the coactivators CBP/p300 in nuclear receptor signaling. However, in addition to coactivators, ligand-activated nuclear receptors bind a number of different proteins that possibly serve other functions. Using peroxisome proliferator-activated receptor-alpha (PPAR alpha) as bait in a yeast two-hybrid screening, we have isolated nuclear factor RIP140 whose function in receptor activation is unclear. We now report a detailed characterization of RIP140 action with a focus on the retinoid X receptor (RXR) heterodimeric receptors PPAR and thyroid hormone receptor (TR). We show that putative PPAR ligands enhance the interaction of RIP140 with the rat PPAR subtypes alpha and gamma in solution but not with PPAR/RXR heterodimers on DNA. However, RIP140 forms ternary complexes in the presence of RXR ligands. Similar experiments with TR support the high affinity of RIP140 to the RXR subunit and also suggest that either partner in the TR/RXR heterodimer can independently respond to ligand. Coactivation experiments in yeast and mammalian cells confirm the coactivator role for SRC-1, but not for RIP140. We provide important evidence that the in vitro binding of RIP140 and SRC-1 to nuclear receptors is competitive. Since RIP140 generally down-regulates receptor activity in mammalian cells and specifically down-regulates coactivation mediated by SRC-1, we propose a model in which RIP140 indirectly regulates nuclear receptor AF-2 activity by competition for coactivators such as SRC-1.

Topics: Adaptor Proteins, Signal Transducing; Alitretinoin; Amino Acid Sequence; Animals; Binding, Competitive; Cell Nucleus; Cloning, Molecular; Dimerization; DNA-Binding Proteins; Fatty Acids; Gene Expression Regulation; Gene Library; Histone Acetyltransferases; Humans; Macromolecular Substances; Molecular Sequence Data; Nuclear Proteins; Nuclear Receptor Co-Repressor 1; Nuclear Receptor Coactivator 1; Nuclear Receptor Coactivators; Nuclear Receptor Interacting Protein 1; Oncogene Proteins; Pyrimidines; Rabbits; Rats; Receptors, Cytoplasmic and Nuclear; Receptors, Retinoic Acid; Receptors, Thyroid Hormone; Recombinant Fusion Proteins; Repressor Proteins; Retinoid X Receptors; Rosiglitazone; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Thiazoles; Thiazolidinediones; Trans-Activators; Transcription Factors; Transcription, Genetic; Transfection; Tretinoin; Zinc Fingers

Fibrates are used to lower plasma triglycerides and cholesterol levels in hyperlipidemic patients. In addition, fibrates have been found to alter the plasma concentrations of fibrinogen, plasminogen activator inhibitor-1 (PAI-1) and apolipoprotein A-I (apo A-I). We have investigated the in vitro effects of fibrates on fibrinogen, PAI-1 and apo A-I synthesis and the underlying regulatory mechanisms in primary monkey hepatocytes. We show that fibrates time- and dose-dependently increase fibrinogen and apo A-I expression and decrease PAI-1 expression in cultured cynomolgus monkey hepatocytes, the effects demonstrating different potency for different fibrates. After three consecutive periods of 24 h the most effective fibrate. ciprofibrate (at 1 mmol/l), increased fibrinogen and apo A-I synthesis to 356% and 322% of control levels, respectively. Maximum inhibition of PAI-1 synthesis was about 50% of control levels and was reached by 1 mmol/l gemfibrozil or ciprofibrate after 48 h. A ligand for the retinoid-X-receptor (RXR), 9-cis retinoic acid, and specific activators of the peroxisome proliferator-activated receptor-alpha (PPARalpha), Wy14,643 and ETYA, influenced fibrinogen, PAI-1 and apo A-I expression in a similar fashion, suggesting a role for the PPARalpha/RXRalpha heterodimer in the regulation of these genes. When comparing the effects of the various compounds on PPARalpha transactivation activity as determined in a PPARalpha-sensitive reporter gene system and the ability of the compounds to affect fibrinogen, PAI-1 and apo A-I antigen production, a good correlation (r=0.80; p <0.01) between PPARalpha transactivation and fibrinogen expression was found. Apo A-I expression correlated only weakly with PPARalpha transactivation activity (r=0.47; p=0.24), whereas such a correlation was absent for PAI-1 (r=0.03; p=0.95). These results strongly suggest an involvement of PPARalpha in the regulation of fibrinogen gene expression.

Topics: 5,8,11,14-Eicosatetraynoic Acid; Alitretinoin; Animals; Apolipoprotein A-I; Cells, Cultured; Clofibrate; Clofibric Acid; Dimerization; Female; Fenofibrate; Fibric Acids; Fibrinogen; Gemfibrozil; Gene Expression Regulation; Genes, Reporter; Hypolipidemic Agents; Liver; Macaca fascicularis; Male; Peroxisome Proliferators; Plasminogen Activator Inhibitor 1; Pyrimidines; Receptors, Cytoplasmic and Nuclear; Receptors, Retinoic Acid; Retinoid X Receptors; Transcription Factors; Transcriptional Activation; Tretinoin

The human UGT1 gene is a single copy gene consisting of four common exons and more than 13 variable exons which span more than 200 kb of the human genome. A single variable exon is spliced to the four common exons to form the mRNA for synthesis of a single UDP-glucuronosyltransferase (UGT) isoenzyme. Treatment of humans or hepatoma cell lines with drugs such as phenobarbital causes the induction of hepatic bilirubin UGT by increased transcription from the UGT1 gene. The upstream region of UGT1*1 (bilirubin UGT) was sequenced and found to contain consensus sequences for several transcriptional regulatory elements including a 'BARBIE box'. An unusual 'TATA' promoter sequence A(TA)6TAA was also observed. The 5' region flanking the UGT1*1 exon when cloned into reporter constructs and transfected into four cells lines was capable of promoting reporter gene expression, but not when transfected into monkey kidney cell fibroblasts (COS-7 cells) indicating a cell specific expression. Sequential deletion of the 5' flanking region in the plasmid constructs did not cause any significant reduction in reporter expression. Treatment of cells transfected with these plasmid constructs with drugs did not cause a significant increase in reporter expression except with retinoic acid plus WY 14643. Introduction of an additional two base pairs (TA) into the 'TATA' box of the 5' gene sequence (as observed in Gilbert's patients) did not significantly change reporter expression levels. The regulation of the biliruibin UGT gene by drugs is not yet understood and it will be important to identify additional genetic elements possibly further than -2kb upstream of the UGT1*1 coding region, which regulate the expression of this gene.

Topics: Base Sequence; Binding Sites; Cells, Cultured; Consensus Sequence; Gene Expression Regulation; Genes, Reporter; Glucuronosyltransferase; Humans; Microsomes, Liver; Molecular Sequence Data; Phenobarbital; Pyrimidines; TATA Box; Transcription Factors; Tretinoin; Xenobiotics

Culture of mouse 3T3-L1 preadipocytes in medium containing delipidated bovine calf serum caused the cells to elongate and divide after reaching confluency. The continued proliferation correlated with sustained expression of the c-myc gene, which was repressed in control cells. Exposure of the cells to activators of peroxisome proliferator-activated receptor (PPAR), including clofibrate, WY-14,643, and 5,8,11,14-eicosatetraynoic acid, reversed and prevented the effects of culturing preadipocytes in delipidated serum. Continued exposure to PPAR activators led to adipose conversion, during which PPAR and its heterodimerization partner (retinoid X receptor) were induced. Retinoic acid (RA) had no effect on the growth or survival of preadipocytes grown in the presence of normal bovine serum. However, treatment of cells cultured in delipidated serum with RA caused death of the cells by apoptosis. Thus, preadipocyte phenotype and survival are regulated by activators of nuclear hormone receptors.

Topics: 3T3 Cells; 5,8,11,14-Eicosatetraynoic Acid; Adipose Tissue; Animals; Apoptosis; Cell Survival; Clofibrate; Culture Media; Mice; Microbodies; Phenotype; Pyrimidines; Receptors, Cytoplasmic and Nuclear; Receptors, Retinoic Acid; Retinoid X Receptors; Signal Transduction; Stem Cells; Transcription Factors; Tretinoin