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.

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.

Regulation of CYP4A1 and peroxisome proliferator-activated receptor alpha expression by interleukin-1beta, interleukin-6, and dexamethasone in cultured fetal rat hepatocytes.

The CYP4A1 isoenzyme induced in rodents by peroxisome proliferators is known to be repressed at a pretranslational level by interferon. Interleukin-1beta (IL-1beta) also reduces CYP4A1-related 12-laurate hydroxylase activity in cultured fetal rat hepatocytes after induction by clofibric acid. In this fetal hepatocyte model, IL-1beta and interleukin-6 (IL-6) were tested for their ability to reduce 12-laurate hydroxylase activity, CYP4A1 apoprotein content, and the CYP4A1 mRNA level. IL-1beta and IL-6 strongly diminished CYP4A1 activity and apoprotein and mRNA levels in a dose- and time-dependent manner. CYP4A1 expression is thus down-regulated at a pretranslational level by these cytokines. As it has been shown that the peroxisome proliferator-activated receptor alpha (PPAR alpha) mediates the induction of the CYP4A1 gene by a peroxisome proliferator, the capacity of IL-1beta or IL-6 to modulate the PPAR alpha mRNA level was tested. It was found that IL-1beta and IL-6 both repress the induction of PPAR alpha expression exerted by the combined action of clofibric acid and dexamethasone. However, even at the highest concentration (10 ng/mL) tested for both cytokines, IL-1beta as well as IL-6 failed to abolish the induction of CYP4A1 by dexamethasone. The mechanism of the protective effect of the synthetic glucocorticoid on CYP4A1 repression by interleukins is discussed.

Comparative effects of clofibrate on peroxisomal enzymes of human (Hep EBNA2) and rat (FaO) hepatoma cell lines.

We compared the responses of the human Hep EBNA2 and rat FaO hepatoma lines to the peroxisome proliferator, clofibrate. Using spectrophotometrical assays performed with peroxisome-enriched fractions, the dose- and time-dependent increase of catalase and acyl-CoA oxidase activities were determined. For catalase activity a maximum stimulation of 1.2-fold for Hep EBNA2 and 1.7-fold for FaO lines was obtained. This increase was neither dose- nor time-dependent. The activity of the initial enzyme of the peroxisomal beta-oxidation system, acyl-CoA oxidase, was tested using two different biochemical assays. The maximum stimulation of acyl-CoA oxidase was 2.4 to 3-fold for human Hep EBNA2 and 6 to 11-fold for rat FaO lines. The specific activity of acyl-CoA oxidase increased with the concentration of clofibrate and with the length of the treatment. Dot blot analyses carried out using mRNAs from FaO and Hep EBNA2 cells treated with 0.5 mM clofibrate for 5 days and from control cells, confirmed the increase in the level of acyl-CoA oxidase mRNAs from the clofibrate-treated cells. In the human cell line, the level of mRNA encoding for the peroxisomal bifunctional enzyme which is involved in the second and the third step of the beta-oxidation system, was also increased by clofibrate treatment.

Abnormalities in histamine pharmacodynamics in chronic urticaria.

Histamine plays a key role in the pathogenesis of chronic urticaria (CU). The authors of this paper have studied the effects of ingested histamine in 25 patients with CU. A 120 mg dose of histamine, well-tolerated in the healthy subject, was instillated into the duodenum. Concomitantly, plasma histamine (H) levels and plasma and urinary methylhistamine (MH) levels were measured. Intraduodenal administration of histamine was responsible for the development of an attack of urticaria in 64% of patients, while control subjects were asymptomatic. Plasma histamine levels were significantly higher after digestive histamine challenge (DHC) in patients with CU compared with controls. An abnormal increase in plasma histamine was observed in 72% of them. Plasma MH exhibited the same kinetic behaviour with a usually delayed time-pattern. Urinary MH concentration was higher in patients presenting with early-onset urticaria during the first hour than in those with the late-onset type between 1 and 12 hr after DHC. The coefficient of methylation (plasma MH/MH+H) was not significantly different in patients presenting with an attack of urticaria following DHC and in other subjects. Urinary excretion of MH and urinary flow increased significantly in patients presenting with an attack of urticaria following DHC which corresponds to increased absorption of histamine during the 5-hr period following DHC and its role on excretion by the kidney via vasodilation which it induces. This study demonstrates the abnormal frequency of disturbances in the metabolism of exogenous histamine in patients with CU. Increased plasma H accounts for the abnormal passage of H across the intestinal barrier which can result either from intestinal hyperpermeability and/or a deficit in the enzymatic catabolism of histamine. The systems of methylation and urinary clearance of MH appear to be effective. It is thus postulated that there is a deficit in diamine oxidase (DAO) in the enterocyte. The lack of correlation between the kinetic behaviour of plasma H and the onset of urticaria draws attention to the extent of individual variability in skin reactivity to histamine.

Intrinsic factor receptor during fetal development of the human intestine.

Intrinsic factor receptor activity was observed in mucosal homogenates from whole small intestine and colon of 10-19-week fetuses, whereas it was only detected in the distal part of the small intestine of a 25-week fetus. The receptor-intrinsic factor-cobalamin complex was eluted into the void-volume position when ileum mucosal extract was assayed for receptor activity by gel filtration after incubation with either fetal gastric extract or human gastric juice. The intrinsic-factor-binding capacity of intestinal mucosal extracts ranged from 2.6 to 30.5 fmol/mg and was correlated with the gestational age of six fetuses. The dissociation constant of the receptor for the intrinsic factor-cobalamin complex was estimated at 0.24-0.36 nM at pH 7.4. In conclusion, intrinsic-factor-receptor activity was detected in the whole intestine in 10-19-week fetuses, whereas it was only present in the distal ileum at the end of fetal development.

Receptor-mediated endocytosis of the intrinsic factor-cobalamin complex in HT 29, a human colon carcinoma cell line.

A HT 29 cell line derived from human colonic carcinoma was shown to express the intrinsic factor receptor, with about 5000 binding sites per cell and an association constant of 20 x 10(9) 1/mol at pH 7.4 and 4 degrees C. The number of binding sites increased dramatically between 7 and 10 days of culture time. Endocytosis of the intrinsic factor-cobalamin-receptor complex was inhibited by two ways: at 4 degrees C and at 37 degrees C by incubating the cells with vinblastine, monensin and chloroquine. The plasma membrane receptor was cross-linked to [57Co]cobalamin-intrinsic factor and solubilized with Triton X-100. The cross-linked complex had a relative molecular mass of 330 kDa in native PAGE.

Synthesis and secretion of a cobalamin-binding protein by HT 29 cell line.

An HT 29 cell line derived from human colonic carcinoma was shown to synthesize and release a cobalamin-binding protein. The cobalamin-binding protein was classified as transcobalamin (TC). By gel filtration on Sephacryl S200 HR, we observed that the secreted protein bound to cobalamin had the same size as plasma transcobalamin. Like transcobalamin, the cobalamin-binding protein bound cobalamin but not cobinamide. Purification of the cobalamin-binding protein was performed by heparin-Sepharose affinity chromatography and by Sephacryl S200 gel filtration. The molecular mass of the purified protein was estimated at 44 kDa by SDS/PAGE. The isoelectric point was determined to be 6.4. The purified cobalamin-binding protein reacted with an antiserum produced against human transcobalamin. A 44 kDa band was also identified by SDS/PAGE of an immunoprecipitated homogenate from HT 29 cells labelled with [35S]methionine and in a Western blot of cell homogenates. The secretion of the cobalamin-binding protein was maximal between 10 and 12 days of cell culture and was inhibited by cycloheximide.

Binding assay and physicochemical characteristics of solubilized intrinsic factor receptor in ileal mucosal homogenates using phenyl-Sepharose to separate the saturated receptor from free intrinsic factor.

A radioisotopic assay was set to determine the physicochemical properties of the solubilized intrinsic factor receptor in pig mucosal extracts. In this assay, phenyl-Sepharose was used to separate the receptor-intrinsic factor-labelled cobalamin complex from the free saturated intrinsic factor. The association constant (at pH 7.4) of the receptor-intrinsic factor complex was estimated at 3.4 +/- 0.3 nM-1. Adsorption of the apo-receptor to phenyl-Sepharose allowed its binding site to be made accessible to intrinsic factor with an association constant in order of 6 nM-1. The receptor binding activity obtained with five mucosal extracts was closely correlated with that obtained by gel filtration of the intrinsic factor-receptor complex (r = 0.99). The radioisotope assay was used to detect the unsaturated receptor (apo-receptor) in sucrose density ultracentrifugation and in superose 6 gel filtration. The sedimentation coefficient was 9.5 s. The apo-receptor was eluted in three peaks in gel filtration, corresponding to the formation of oligomers. The peak of the monomer was increased in presence of EDTA. Its molecular mass was estimated at 270 kDa and its Stokes radius at 5.9 nm. It was concluded that calcium is involved in the oligomerisation of the apo-receptor.

Purification of intrinsic factor receptor from pig ileum using as affinity medium human intrinsic factor covalently bound to Sepharose.

Intrinsic factor receptor was purified from hog ileum using human intrinsic factor covalently bound to Sepharose. A yield of 49.6% and a specific activity of about 2500 pmol/mg protein were achieved. The purified receptor was very unstable: 24 h of storage or addition of sodium phosphate precipitated it. The association constant of the receptor for the cyano[57Co]cobalamin-intrinsic factor complex was estimated to be 2.1 nM-1. In native polyacrylamide gel electrophoresis it resolved in two 256 and 320 kDa bands; beta-mercaptoethanol treatment cleared it into four bands corresponding to molecular masses of 107, 81.8, 63.5 and 53.2 kDa. An additional 39.3 kDa band was considered to be an artefact due to the presence of Triton X-114. Isoelectric focusing polyacrylamide gel electrophoresis resolved the receptor into two isoproteins isoelectric at pH 4.7 and 5.1. A similar result was obtained in column electrofocusing with the 125I-iodinated receptor. The 125I-labelled receptor did not crossreact with rabbit anti-human intrinsic factor antiserum. The electrophoretic properties of the receptor purified with intrinsic factor covalently bound to Sepharose were compared to those of the receptor purified by the use of the classical cobalamin-affinity medium. It was concluded that a disassembled receptor was produced using the classical method.

Lectin binding to the porcine and human ileal receptor of intrinsic factor-cobalamin.

The purified porcine receptor for the intrinsic factor-cobalamin complex bound to concanavalin A, lentil lectin and wheat germ lectin covalently coupled to Sepharose and was eluted with the corresponding soluble sugars. In contrast, human intrinsic factor bound efficiently to concanavalin A, to some extent to lentil lectin, but only slightly to wheat germ agglutinin. The binding of IF-Cbl to the receptor was inhibited when the receptor was pre-incubated with soluble wheat germ agglutinin, with an inhibition constant estimated to be 1.9 mumol/l. After transfer of the purified receptor from SDS-PAGE to Immobilon, ligand blotting of the purified receptor with iodinated lectin showed that concanavalin A and lentil lectin bound to three (75, 56 and 43 kDa) components but that wheat germ agglutinin bound only to the 75 kDa component. These results showed that the alpha subunit of the receptor could bind to wheat germ agglutinin, resulting in an inhibition of its binding with intrinsic factor. Both binding sites of intrinsic factor and of wheat germ agglutinin could be located near to each other.