Expression of the peroxisome proliferator-activated receptor (PPAR) in the mouse colonic mucosa

The peroxisome proliferator-activated receptor (PPAR), a member of the steroid nuclear receptor superfamily, has been shown to be activated by various compounds such as fibrates, thiazolidinediones, prostaglandins, and fatty acids. Here we demonstrate expression of PPAR in mouse colonic and small intestinal mucosa by Western blot analysis and immunohistochemistry, indicating a higher expression level in the differentiated colonic epithelial cells facing the intestinal lumen. Quantification of PPAR mRNA by ribonuclease protection assay revealed relatively high expression of PPAR gamma and Nuc1 in the colon as compared to the small intestine. In contrast, PPAR alpha expression was higher in the small intestine as compared to the colon. These results demonstrate the presence of PPAR in the intestinal mucosa; however, the physiological roles of the various isoforms in the intestine remain to be established.     

Role of peroxisome proliferator-activated receptor alpha in disease of pancreatic beta cells

Expression of peroxisome proliferator-activated receptor alpha (PPARalpha) and enzymes of fatty acid (FA) oxidation is markedly reduced in the fat-laden, dysfunctional islets of obese, prediabetic Zucker diabetic fatty (fa/fa) rats with mutated leptin receptors (OB-R). Leptin, PPARalpha/retinoid x receptor ligands, and FA all up-regulate PPARalpha and enzymes of FA oxidation and stimulate [3H]-palmitate oxidation in normal islets but not in islets from fa/fa rats. Overexpression of normal OB-R in islets of fa/fa rats corrects all of the foregoing abnormalities and reverses the diabetic phenotype. PPARalpha is a OB-R-dependent factor required for normal fat homeostasis in islet cells.     

Expression of peroxisome proliferator-activated receptor gamma (PPARgamma) in rat aortic smooth muscle cells

Creation of isopenicillin N from delta-(L-alpha-aminodipyl)-L-cysteinyl-D-valine (ACV) in the penicillin and cephalosporin biosynthetic pathway is catalysed by isopenicillin N synthase (IPNS), a non-heme iron-containing dioxygenase. A tripeptide R-X-S motif which consists of arginine-281 and serine-283 (Cephalosporium acremonium IPNS numbering) was found to be conserved in IPNS and other related proteins. These two amino acids mentioned were proposed to have a role in ACV substrate binding by the recent Aspergillus nidulans IPNS crystal structure. Using site-directed mutagenesis arginine-281 in C. acremonium IPNS (cIPNS) was earlier found to be essential for catalysis by our group. Similarly, serine-283 in cIPNS was also altered by site-directed mutagenesis to determine its role in cIPNS. No measurable activity was detected from the resultant mutant using enzyme bioassays. It is most likely that the eliminatin of the mutant's substrate-binding capability similar to that of arginine-281 lead to the abolishment of the catalytic reaction. This highlights the importance of the R-X-S motif in the functionality of cIPNS.     

Analysis of catalytic action of transglutaminase induced in human promyelocytic leukemia (HL-60) and human hepatoblastoma (HepG2) cells

Transglutaminase is a calcium-dependent enzyme that catalyzes an amine incorporation and a cross-linking of proteins. Intracellular transglutaminase is induced when human promyelocytic leukemia HL-60 cells are treated with retinoic acid and human hepatoblastoma HepG2 cells, with interleukin-6. To find whether the intracellular reaction catalyzed by transglutaminase increased when the enzyme is induced in these cells, the transglutaminase-catalyzed incorporation of 14C-labeled methylamine into cellular proteins was measured. The incorporation level of the labeled methylamine into proteins of HL-60 and HepG2 cells did not increase after the transglutaminase had been induced. The presence of the calcium ionophore A23187 did not affect these results. These findings suggested that even after the enzyme induction the catalytic action of intracellular transglutaminase is maintained at a constant level in these cells by unknown regulatory mechanism(s).     

Peroxisome proliferator-activated receptor (PPAR): structure, mechanisms of activation and diverse functions

The structurally diverse xenobiotic peroxisome proliferators (PPs) increase the number of peroxisomes per cell and the levels of several enzymes, and cause hepatomegaly, often leading to hepatocarcinogenesis in a species- and tissue-specific manner. The deadlocked problems of the molecular mechanism of PP action and its physiological meanings have begun to be understood through cDNA cloning of a PP-activated receptor (PPAR). PPAR, a member of the steroid/thyroid/vitamin superfamily of nuclear receptors, has isoforms and differentially heterodimerizes with other nuclear receptors, providing potential mechanisms not only for species- and tissue-specific actions but also for diverse actions of PPs. Recent findings related to PPAR are summarized, and its possible role in lipid metabolism and involvement in PP-induced hepatocarcinogenesis are discussed.     

Polarity and specific sequence requirements of peroxisome proliferator-activated receptor (PPAR)/retinoid X receptor heterodimer binding to DNA. A functional analysis of the malic enzyme gene PPAR response element

The malic enzyme (ME) gene is a target for both thyroid hormone receptors and peroxisome proliferator-activated receptors (PPAR). Within the ME promoter, two direct repeat (DR)-1-like elements, MEp and MEd, have been identified as putative PPAR response elements (PPRE). We demonstrate that only MEp and not MEd is able to bind PPAR/retinoid X receptor (RXR) heterodimers and mediate peroxisome proliferator signaling. Taking advantage of the close sequence resemblance of MEp and MEd, we have identified crucial determinants of a PPRE. Using reciprocal mutation analyses of these two elements, we show the preference for adenine as the spacing nucleotide between the two half-sites of the PPRE and demonstrate the importance of the two first bases flanking the core DR1 in 5'. This latter feature of the PPRE lead us to consider the polarity of the PPAR/RXR heterodimer bound to its cognate element. We demonstrate that, in contrast to the polarity of RXR/TR and RXR/RAR bound to DR4 and DR5 elements respectively, PPAR binds to the 5' extended half-site of the response element, while RXR occupies the 3' half-site. Consistent with this polarity is our finding that formation and binding of the PPAR/RXR heterodimer requires an intact hinge T region in RXR while its integrity is not required for binding of the RXR/TR heterodimer to a DR4.     

Cross-talk between orphan nuclear hormone receptor RZRalpha and peroxisome proliferator-activated receptor alpha in regulation of the peroxisomal hydratase-dehydrogenase gene

The genes encoding the peroxisomal beta-oxidation enzymes enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (HD) and fatty acyl-CoA oxidase (AOx) are coordinately regulated by peroxisome proliferator-activated receptor alpha (PPARalpha)/9-cis-retinoic acid receptor (RXRalpha) heterodimers that transactivate these genes in a ligand-dependent manner via upstream peroxisome proliferator response elements (PPRE). Here we demonstrate that the monomeric orphan nuclear hormone receptor, RZRalpha, modulates PPARalpha/RXRalpha-dependent transactivation in a response-element dependent manner. Electrophoretic mobility shift analysis showed that RZRalpha bound specifically as a monomer to the HD-PPRE but not the AOx-PPRE. Determinants in the HD-PPRE for binding of RZRalpha were distinct from those required for interaction with PPARalpha/RXRalpha heterodimers. In transient transfections, RZRalpha stimulated ligand-mediated transactivation by PPARalpha from an HD-PPRE luciferase reporter in the absence of exogenously added RXRalpha, but did not affect PPARalpha-dependent transactivation of an AOx-PPRE reporter gene. These data illustrate cross-talk between the RZRalpha and PPARalpha signaling pathways at the level of the HD-PPRE in the regulation of the HD gene and characterize additional factors governing the regulation of peroxisomal beta-oxidation.     

Mechanism of action of the nongenotoxic peroxisome proliferators: role of the peroxisome proliferator-activator receptor alpha

Peroxisome proliferators are a diverse group of chemicals that include several therapeutically used drugs (e.g., hypolipidemic agents), plasticizers and organic solvents used in the chemical industry, herbicides, and naturally occurring hormones. As the name implies, peroxisome proliferators cause an increase in the number and size of peroxisomes in the liver, kidney, and heart tissue of susceptible species, such as rats and mice. Long-term administration of peroxisome proliferators can cause liver cancer in these animals, a response that has been the central issue of research on peroxisome proliferators for many years. Peroxisome proliferators are representative of the class of nongenotoxic carcinogens that cause cancer through mechanisms that do not involve direct DNA damage. The fact that humans are frequently exposed to these agents makes them of particular concern to government regulatory agencies responsible for assuring human safety. Whether frequent exposure to peroxisome proliferators represents a hazard to humans is unknown; however, increased cancer risk has not been shown to be associated with long-term therapeutic administration of the hypolipidemic drugs gemfibrozil, fenofibrate, and clofibrate. To make sound judgments regarding the safety of peroxisome proliferators, the validity of extrapolating results from rodent bioassays to humans must be based on the agents' mechanism of action and species differences in biologic activity and carcinogenicity. The peroxisome proliferator-activated receptor alpha (PPARalpha), a member of the nuclear receptor superfamily, has been found to mediate the activity of peroxisome proliferators in mice. Gene-knockout mice lacking PPARalpha are refractory to peroxisome proliferation and peroxisome proliferator-induced changes in gene expression. Furthermore, PPARalpha-null mice are resistant to hepatocarcinogenesis when fed a diet containing a potent nongenotoxic carcinogen WY-14,643. Recent studies have revealed that humans have considerably lower levels of PPARalpha in liver than rodents, and this difference may, in part, explain the species differences in the carcinogenic response to peroxisome proliferators.     

Identification of an alpha2-macroglobulin receptor in human mammary epithelial cells

Several cases of zinc (Zn) deficiency in human infants caused by abnormally low concentrations of Zn in breast milk were recently reported, the underlying mechanism of which is not known. Alpha2-macroglobulin (alpha2-M), a major Zn-binding ligand in serum, presents a potential vehicle for mammary Zn uptake. This study was conducted to determine if an alpha2-M receptor is present in human mammary epithelial cells, where it may be involved in the endocytosis of alpha2-M into the mammary gland. Normal human mammary epithelial cells were grown to confluency in serum-free medium. For all binding and uptake studies, alpha2-M, preactivated with methylamine and labeled with 125I, was added to cells for varied lengths of time to determine saturation over time and at varied concentrations to determine saturation over increasing concentration of ligand. Nonspecific and competitive binding were measured by addition of a 100-fold molar excess of unlabeled alpha2-M and serum albumin or lactoferrin, respectively. Binding at 4 degreesC was specific for alpha2-M and approached saturation kinetics at 56 nmol/L. Scatchard plot analysis of the binding data demonstrated more than one binding site: a high affinity, saturable binding site and a low affinity, nonsaturable binding site. Uptake of alpha2-M at 37 degreesC was rapid and continuous over increasing concentrations of alpha2-M, and internalized alpha2-M was rapidly degraded. Results from this study present evidence for receptor-mediated uptake of alpha2-M in human mammary epithelial cells, which in turn, provides a potential mechanism for Zn acquisition by the cell.     

Metabolism of hydroperoxy-phospholipids in human hepatoma HepG2 cells

Two enzymatic mechanisms have been proposed for the metabolism of hydroperoxy-phospholipids: i) the combined action of phospholipase A2 and glutathione peroxidase, and/or ii) direct enzymatic reduction. The latter reaction may be catalyzed by selenium-dependent phospholipid hydroperoxide glutathione peroxidase and/or by glutathione S-transferase alpha. To study the pathway of this reaction, we used human hepatoma HepG2 cells into which was incorporated labeled, hydroperoxy-phospholipids. The major product of incorporated l-palmitoyl-2-(13-hydroperoxy-cis-9, trans-11-octadecadienoyl)-L-3-phosphatidylcholine was the corresponding hydroxy-phospholipid with no hydroxy- or hydroperoxy-fatty acids. The contributions to reduction of hydroperoxy-phospholipids in HepG2 cells from glutathione S-transferase Al and phospholipid hydroperoxide glutathione peroxidase were calculated to be 0.5% and 99.5%, respectively. Increasing selenium in the cell culture medium led to increases in selenium-dependent phospholipid hydroperoxide glutathione peroxidase activity but not in glutathione S-transferase alpha. This increase in the selenium-dependent enzyme was paralleled by a concomitant increase in the extent of reduction of the incorporated hydroperoxy-phospholipid. We conclude that the main metabolic fate of hydroperoxy-phospholipids in HepG2 cells is by direct reduction to hydroxy-phospholipids by phospholipid hydroperoxide glutathione peroxidase but also by glutathione S-transferase alpha, and that phospholipase A2/selenium-dependent glutathione peroxidase does not play a significant role in the reduction.     

Corticosteroid-binding globulin synthesis regulation by cytokines and glucocorticoids in human hepatoblastoma-derived (HepG2) cells

Plasma corticosteroid-binding globulin (CBG) concentrations decrease dramatically in patients with septic shock or burn injury. This decrease suggests that mediators of the acute phase response, such as cytokines and glucocorticoid hormones, might influence clearance as well as liver synthesis of CBG in humans. The present study investigated the effects of interleukin-6 (IL-6), IL-1 beta, and dexamethasone on CBG synthesis by a clone of human hepatoblastoma-derived (HepG2) cell line. In culture medium from HepG2 cells, the immunoconcentration of CBG and the levels of CBG messenger ribonucleic acid (mRNA) were dose dependently decreased in the presence of IL-6 concentrations ranging from 0.1-10 ng/mL. The percent decrease in CBG immunoconcentration was quantitatively similar to the percent decrease in CBG mRNA levels (29 +/- 6% and 39 +/- 15%, respectively, of control values). In contrast, and as expected, IL-6 dose dependently increased the mRNA levels (164 +/- 22% of control values) of alpha 1-antitrypsin, a positive acute phase protein, but did not affect the immunoconcentration of sex hormone-binding globulin, another liver protein. Dexamethasone alone did not significantly affect CBG secretion or mRNA levels, but did dose-dependently increase tyrosine amino-transferase mRNA levels, which increased to 252 +/- 16% of the control values. However, in combination with IL-6, dexamethasone had a significant additive effect on IL-6 inhibition of CBG secretion and mRNAs in HepG2 cells. IL-1 beta dose-dependently stimulated CBG secretion (156 +/- 10% of control values) with no significant effect on CBG mRNA levels. In addition, IL-1 beta significantly decreased the inhibitory effect of IL-6 on CBG secretion, but had no effect on the inhibitory effect of IL-6 on CBG mRNA levels. These results suggest that IL-1 beta acts on the posttranslation processing and/or secretion mechanisms of CBG in HepG2 cells. Together, the present results strongly support the hypothesis that the decrease in plasma CBG concentrations is associated with the increase in IL-6 and glucocorticoid levels reported in patients with septic shock and burn injury.     

Ligands for peroxisome proliferator-activated receptorgamma and retinoic acid receptor inhibit growth and induce apoptosis of human breast cancer cells in vitro and in BNX mice

Induction of differentiation and apoptosis in cancer cells through ligands of nuclear hormone receptors (NHRs) is a novel and promising approach to cancer therapy. All-trans-retinoic acid (ATRA), an RA receptor-specific NHR ligand, is now used for selective cancers. The NHR, peroxisome proliferator-activated receptor gamma (PPARgamma) is expressed in breast cancer cells. Activation of PPARgamma through a synthetic ligand, troglitazone (TGZ), and other PPARgamma-activators cause inhibition of proliferation and lipid accumulation in cultured breast cancer cells. TGZ (10(-5) M, 4 days) reversibly inhibits clonal growth of MCF7 breast cancer cells and the combination of TGZ (10(-5) M) and ATRA (10(-6) M, 4 days) synergistically and irreversibly inhibits growth and induces apoptosis of MCF7 cells, associated with a dramatic decrease of their bcl-2 protein levels. Similar effects are noted with in vitro cultured breast cancer tissues from patients, but not with normal breast epithelial cells. The observed apoptosis mediated by TGZ and ATRA may be related to the striking down-regulation of bcl-2, because forced over-expression of bcl-2 in MCF7 cells cultured with TGZ and ATRA blocks their cell death. TGZ significantly inhibits MCF7 tumor growth in triple immunodeficient mice. Combined administration of TGZ and ATRA causes prominent apoptosis and fibrosis of these tumors without toxic effects on the mice. Taken together, this combination may provide a novel, nontoxic and selective therapy for human breast cancers.     

Cyclic AMP-dependent protein kinase (cAK) in human B cells: co-localization of type I cAK (RI alpha 2 C2) with the antigen receptor during anti-immunoglobulin-induced B cell activation

Cyclic AMP (cAMP) inhibits antigen-stimulated B cell proliferation through activation of cAMP-dependent protein kinases (cAK). We have examined the molecular composition and cellular localization of cAK in human B cells. We find that human B cells contain substantial amounts of mRNA for RI alpha, RII alpha, C alpha and C beta, barely detectable levels of RI beta mRNA, and no detectable RII beta or C gamma mRNA. At the protein level, using Western blotting and subunit-specific antibodies against the different R subunits, we find RI alpha and RII alpha, but no RI beta or RII beta. The presence of catalytic subunits was demonstrated using a nonselective anti-C antiserum. By photoaffinity labeling of R subunits with 8-azido-[32P]cAMP, followed by immunoprecipitation with subunit-specific antibodies, we were also able to demonstrate low levels of RI beta. Immunofluorescence staining of RI alpha and RII alpha demonstrates a rather homogeneous intracellular (but extranuclear) distribution of RI alpha, whereas the RII alpha subunits of cAK are localized to distinct perinuclear structures, previously identified as centrosomes in other cell types. Upon anti-Ig-mediated capping of B cells, RI alpha subunits redistribute to the cap, co-localizing with the antigen-receptors, whereas the intracellular localization of RII alpha subunits remains unchanged.     

Expression of putative fatty acid transporter genes are regulated by peroxisome proliferator-activated receptor alpha and gamma activators in a tissue- and inducer-specific manner

Regulation of gene expression of three putative long-chain fatty acid transport proteins, fatty acid translocase (FAT), mitochondrial aspartate aminotransferase (mAspAT), and fatty acid transport protein (FATP), by drugs that activate peroxisome proliferator-activated receptor (PPAR) alpha and gamma were studied using normal and obese mice and rat hepatoma cells. FAT mRNA was induced in liver and intestine of normal mice and in hepatoma cells to various extents only by PPARalpha-activating drugs. FATP mRNA was similarly induced in liver, but to a lesser extent in intestine. The induction time course in the liver was slower for FAT and FATP mRNA than that of an mRNA encoding a peroxisomal enzyme. An obligatory role of PPARalpha in hepatic FAT and FATP induction was demonstrated, since an increase in these mRNAs was not observed in PPARalpha-null mice. Levels of mAspAT mRNA were higher in liver and intestine of mice treated with peroxisome proliferators, while levels in hepatoma cells were similar regardless of treatment. In white adipose tissue of KKAy obese mice, thiazolidinedione PPARgamma activators (pioglitazone and troglitazone) induced FAT and FATP more efficiently than the PPARalpha activator, clofibrate. This effect was absent in brown adipose tissue. Under the same conditions, levels of mAspAT mRNA did not change significantly in these tissues. In conclusion, tissue-specific expression of FAT and FATP genes involves both PPARalpha and -gamma. Our data suggest that among the three putative long-chain fatty acid transporters, FAT and FATP appear to have physiological roles. Thus, peroxisome proliferators not only influence the metabolism of intracellular fatty acids but also cellular uptake, which is likely to be an important regulatory step in lipid homeostasis.