L-FABP is exclusively expressed in alveolar macrophages within the myeloid lineage: evidence for a PPARalpha-independent expression

Peroxisome proliferator-activated receptors (PPARs) play a role in inflammation and, in particular, PPARgamma is involved in monocyte/macrophage differentiation. Members of the fatty acid-binding protein (FABP) family have been reported to function as transactivators for PPARs. Therefore, the expression of PPARs and FABPs in the myeloid lineage was investigated by real-time PCR and immunofluorescence analysis. We found adipocyte-, epidermal-, and heart-type FABP to be ubiquitously expressed within the myeloid lineage. In contrast, liver-type FABP was exclusively detected in murine alveolar macrophages (AM), confirmed on protein level by double fluorescence analysis. The PPAR subtypes also showed a temporally and spatially regulated expression pattern in myeloid cells: the beta-subtype was expressed in bone marrow, peritoneal, and alveolar macrophages, whereas it was not detected in dendritic cells (DCs). The gamma1-isoform was present in all cells, however, at different levels, whereas the gamma2-isoform was expressed in alveolar macrophages and dendritic cells. A low level PPARalpha mRNA could be detected in peritoneal macrophages and immature dendritic cells but not in mature dendritic cells and bone marrow macrophages. Interestingly, PPARalpha mRNA was also absent in the alveolar macrophages although liver-type FABP was expressed, indicating that gene expression of liver-type FABP was independent of PPARalpha. Since liver-type FABP is known as transactivator of PPARgamma the simultaneous expression of both proteins may have general implications for the activation of PPARgamma in alveolar macrophages.     

Peroxisome proliferator-activated receptors and skin development

PPARs are nuclear hormone receptors. PPAR subtypes (alpha, gamma, delta, the latter a xPPARbeta homologue) were initially investigated in skin because of their known role in regulating lipid metabolism. Studies adding specific PPAR ligand activators to cultured skin or skin cells are compatible with the concepts that PPARalpha activation mediates early lipogenic steps common to the function of both skin epidermal cells (keratinocytes) and sebaceous cells (sebocytes), PPARgamma activation plays a unique role in stimulating sebocyte lipogenesis, and PPARdelta activation may contribute to lipid biosynthesis in both sebocytes and keratinocytes under certain circumstances. Epidermal keratinocytes appear to express small amounts of PPARalpha and PPARdelta mRNA and a trace of PPARgamma mRNA which is up-regulated with differentiation. Sebocytes express all subtypes; PPARgamma gene expression excedes that in epidermis. The emerging data on PPAR protein expression suggests that epidermis normally expresses predominantly PPARalpha, while sebocytes express more PPARgamma than PPARalpha. These expression patterns may change during hyperplasia, differentiation and inflammation. Gene disruption studies in mice are compatible with a contribution of PPARalpha to skin barrier function, suggest that PPARgamma is necessary for sebocyte differentiation, and indicate that PPARdelta can ameliorate inflammatory responses in skin. PPARs appear to play a role in keratinocyte synthesis of the lipids that they export to the intercellular space to form the skin permeability barrier. They also appear to be important for sebocyte formation of the intracellular fused lipid droplets that constitute the holocrine secretion of the sebaceous gland. In addition, they may play roles in keratinocyte growth and differentiation and the inhibition of skin inflammation by diverse mechanisms not necessarily related to fat metabolism.     

Protein expression changes in the Sprague Dawley rat liver proteome following administration of peroxisome proliferator activated receptor alpha and gamma ligands

Peroxisome proliferator activated receptors (PPARs) are members of the nuclear receptor superfamily and are intimately involved in lipid metabolism and energy homeostasis. Activation of these receptors in rodents can lead to hepatomegaly and ultimately hepatic carcinogenesis although the mechanisms by which these processes occur are poorly understood. To further our understanding of these processes and to discriminate between different PPAR mediated signalling pathways, a proteomic approach has been undertaken to identify changes in protein expression patterns in Sprague Dawley rat liver following dosing with a PPARalpha agonist (Wyeth 14643), a PPARgamma agonist (Troglitazone) and a compound with mixed PPARalpha/gamma agonist activity (SB-219994). Using one-and-two-dimensional electrophoresis of tissue lysates a diverse range of protein abundance changes was observed in these tissues. Whilst a number of these proteins have PPAR response elements (PPREs) in their respective promoters, another group was detected whose expression has been documented to be sensitive to peroxisome proliferator administration. Most notably within these groups, proteins involved in lipid catabolism displayed increased expression following drug administration. A further subset of proteins, with less obvious biological implications, also showed altered expression patterns. Where available, sequences upstream of the coding regions of genes not previously known to have PPREs were searched with positional consensus matrices for the presence of PPREs in an attempt to validate these changes. Using such an approach putative PPARgamma and PPARdelta response elements were discovered upstream of the tubulin beta coding region. There was limited overlap in observed protein abundance changes between the three groups, and where this was the case (cytosolic epoxide hydrolase, peroxisomal bifunctional enzyme, hydroxymethyl glutaryl CoA, synthase, long chain acyl-CoA thioesterase), expression of these proteins had previously been shown to be under the control of PPAR activity.     

Peroxisome proliferator-activated receptor alpha-responsive genes induced in the newborn but not prenatal liver of peroxisomal fatty acyl-CoA oxidase null mice.

Mice deficient in fatty acyl-CoA oxidase (AOX(-/-)), the first enzyme of the peroxisomal beta-oxidation system, develop specific morphological and molecular changes in the liver characterized by microvesicular fatty change, increased mitosis, spontaneous peroxisome proliferation, increased mRNA and protein levels of genes regulated by peroxisome proliferator-activated receptor alpha (PPARalpha), and hepatocellular carcinoma. Based on these findings it is proposed that substrates for AOX function as ligands for PPARalpha. In this study we examined the sequential changes in morphology and gene expression in the liver of wild-type and AOX(-/-) mice at Embryonic Day 17.5, and during postnatal development up to 2 months of age. In AOX(-/-) mice high levels of expression of PPARalpha-responsive genes in the liver commenced on the day of birth and persisted throughout the postnatal period. We found no indication of PPARalpha activation in the livers of AOX(-/-) mice at embryonic age E17.5. In AOX(-/-) mice microvesicular fatty change in liver cells was evident at 7 days. At 2 months of age livers showed extensive steatosis and the presence in the periportal areas of clusters of hepatocytes with abundant granular eosinophilic cytoplasm rich in peroxisomes. These results suggest that the biological ligands for PPARalpha vis a vis substrates for AOX either are not functional in fetal liver or do not cross the placental barrier during the fetal development and that postnatally they are likely derived from milk and diet.     

Interactive effects of benzo(a)pyrene and cadmium and effects of di(2-ethylhexyl) phthalate on antioxidant and peroxisomal enzymes and peroxisomal volume density in the digestive gland of mussel Mytilus galloprovincialis Lmk

Exposure of marine animals to certain organic and metal pollutants is thought to enhance reactive oxygen species (ROS) production with concomitant alterations of antioxidant defence mechanisms. Some of these organic pollutants cause peroxisome proliferation, a process resulting also in possible enhanced production of ROS. The aim of this study was to investigate the effects of two organic xenobiotics, benzo(a)pyrene (B(a)P) and di(2-ethylhexyl)phthalate (DEHP), as well as the effects of cadmium (Cd), on antioxidant and peroxisomal enzymes and on peroxisomal volume density in the digestive gland of mussel, Mytilus galloprovincialis Lmk., experimentally exposed for 21 days. Special attention was paid to the interactive effects of organic and metal compounds by exposing one group of mussels to a mixture of B(a)P and Cd. Exposure of mussels to Cd caused a decrease in superoxide dismutase (SOD) activity, in Mn-SOD protein levels and in volume density of peroxisomes. B(a)P exposure significantly increased catalase and glutathione peroxidase (GPX) and inhibited Mn-SOD after 21 days of exposure. B(a)P also caused a slight increase in acyl-CoA oxidase (AOX) activity and peroxisomal volume density after 21 days of exposure. Cd tended to inhibit changes provoked by B(a)P, indicating that responses to organic xenobiotics can be modulated by concomitant exposure to metal contaminants. Exposure to DEHP increased catalase and AOX and inhibited SOD activity and Mn-SOD protein levels. In conclusion, peroxisome proliferation, measured as an increase of the peroxisomal enzymes catalase and AOX (up to 1.53-fold for AOX), is a specific response to organic contaminants such as B(a)P and DEHP, whereas Cd does not cause peroxisome proliferation. Thus, peroxisome proliferation may be a specific biomarker of organic pollutants in mussels. Both organic and metal pollutants inhibited SOD activity and protein levels (up to 0.21-fold for Mn-SOD protein levels), the latter offering potential as general marker of pollution.     

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.     

Adipocyte-specific gene expression and adipogenic steatosis in the mouse liver due to peroxisome proliferator-activated receptor gamma1 (PPARgamma1) overexpression

Peroxisome proliferator activated-receptor (PPAR) isoforms, alpha and gamma, function as important coregulators of energy (lipid) homeostasis. PPARalpha regulates fatty acid oxidation primarily in liver and to a lesser extent in adipose tissue, whereas PPARgamma serves as a key regulator of adipocyte differentiation and lipid storage. Of the two PPARgamma isoforms, PPARgamma1 and PPARgamma2 generated by alternative splicing, PPARgamma1 isoform is expressed in liver and other tissues, whereas PPARgamma2 isoform is expressed exclusively in adipose tissue where it regulates adipogenesis and lipogenesis. Since the function of PPARgamma1 in liver is not clear, we have, in this study, investigated the biological impact of overexpression of PPARgamma1 in mouse liver. Adenovirus-PPARgamma1 injected into the tail vein induced hepatic steatosis in PPARalpha(-/-) mice. Northern blotting and gene expression profiling results showed that adipocyte-specific genes and lipogenesis-related genes are highly induced in PPARalpha(-/-) livers with PPARgamma1 overexpression. These include adipsin, adiponectin, aP2, caveolin-1, fasting-induced adipose factor, fat-specific gene 27 (FSP27), CD36, Delta(9) desaturase, and malic enzyme among others, implying adipogenic transformation of hepatocytes. Of interest is that hepatic steatosis per se, induced either by feeding a diet deficient in choline or developing in fasted PPARalpha(-/-) mice, failed to induce the expression of these PPARgamma-regulated adipogenesis-related genes in steatotic liver. These results suggest that a high level of PPARgamma in mouse liver is sufficient for the induction of adipogenic transformation of hepatocytes with adipose tissue-specific gene expression and lipid accumulation. We conclude that excess PPARgamma activity can lead to the development of a novel type of adipogenic hepatic steatosis.     

Presence and inducibility of peroxisomes in a human glioblastoma cell line

We investigated the effect of the peroxisomal proliferator (PP) perfluorodecanoic acid (PFDA), alone or in combination with 9-cis-retinoic acid (RX) on the human glioblastoma cell line Lipari (LI). Cell proliferation, apoptotic rate, peroxisome morphology and morphometry, peroxisomal enzyme activities and the presence of peroxisome proliferator-activated receptors (PPARs) were examined. We show that PFDA alone produces pleiotropic effects on LI cells and that RX enhances some of these effects. Peroxisomal number and relative volume, as well as palmitoyl-CoA oxidase activity and protein, are increased by PFDA treatment, with a synergistic effect by RX. The latter, alone or in association with PFDA, induces catalase activity and protein, increases apoptosis and decreases cell proliferation. PPAR isotypes alpha and gamma were detected in LI cells. While the former is apparently unaffected by either treatment, the latter increases in response to PFDA, independent of the presence of RX. The results of this study are discussed in terms of PPARalpha activation and PPARgamma induction by PFDA, by either a direct or an indirect mechanism.     

Oxidized low density lipoprotein activates peroxisome proliferator-activated receptor-alpha (PPARalpha) and PPARgamma through MAPK-dependent COX-2 expression in macrophages

It has been reported that oxidized low density lipoprotein (Ox-LDL) can activate both peroxisome proliferator-activated receptor-alpha (PPARalpha) and PPARgamma. However, the detailed mechanisms of Ox-LDL-induced PPARalpha and PPARgamma activation are not fully understood. In the present study, we investigated the effect of Ox-LDL on PPARalpha and PPARgamma activation in macrophages. Ox-LDL, but not LDL, induced PPARalpha and PPARgamma activation in a dose-dependent manner. Ox-LDL transiently induced cyclooxygenase-2 (COX-2) mRNA and protein expression, and COX-2 specific inhibition by NS-398 or meloxicam or small interference RNA of COX-2 suppressed Ox-LDL-induced PPARalpha and PPARgamma activation. Ox-LDL induced phosphorylation of ERK1/2 and p38 MAPK, and ERK1/2 specific inhibition abrogated Ox-LDL-induced COX-2 expression and PPARalpha and PPARgamma activation, whereas p38 MAPK-specific inhibition had no effect. Ox-LDL decreased the amounts of intracellular long chain fatty acids, such as arachidonic, linoleic, oleic, and docosahexaenoic acids. On the other hand, Ox-LDL increased intracellular 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) level through ERK1/2-dependent overexpression of COX-2. Moreover, 15d-PGJ(2) induced both PPARalpha and PPARgamma activation. Furthermore, COX-2 and 15d-PGJ(2) expression and PPAR activity were increased in atherosclerotic lesions of apoE-deficient mice. Finally, we investigated the involvement of PPARalpha and PPARgamma on Ox-LDL-induced mRNA expression of ATP-binding cassette transporter A1 and monocyte chemoattractant protein-1. Interestingly, specific inhibition of PPARalpha and PPARgamma suppressed Ox-LDL-induced ATP-binding cassette transporter A1 mRNA expression and enhanced Ox-LDL-induced monocyte chemoattractant protein-1 mRNA expression. In conclusion, Ox-LDL-induced increase in 15d-PGJ(2) level through ERK1/2-dependent COX-2 expression is one of the mechanisms of PPARalpha and PPARgamma activation in macrophages. These effects of Ox-LDL may control excess atherosclerotic progression.     

Peroxisome proliferator-activated receptor gamma agonists inhibit adipocyte expression of alpha1-acid glycoprotein

alpha1-Acid glycoprotein (alpha1-AGP) is an acute phase protein that can potentiate cytokine secretion by mononuclear cells and may induce thrombosis by stabilizing the inhibitory activity of plasminogen activator inhibitor-1. Thus, alpha1-AGP may promote pathobiologies associated with type 2 diabetes mellitus (T2DM) including insulin resistance and cardiovascular disease. Here, we demonstrate that antidiabetic peroxisome proliferator-activated receptor gamma (PPARgamma) agonists inhibited expression of 3T3-L1 adipocyte alpha1-AGP in a concentration- and time-dependent manner via an apparent PPARgamma-mediated mechanism. As a result, synthesis and secretion of the glycoprotein was reduced. While PPARgamma agonist regulation of genes with functional peroxisome proliferator response elements in their promoter such as phosphoenolpyruvate carboxykinase were unaffected when cellular protein synthesis was inhibited, downregulation of alpha1-AGP mRNA was ablated thereby supporting the proposition that PPARgamma activation inhibits alpha1-AGP expression indirectly. These results suggest a potential novel adipocytic mechanism by which PPARgamma agonists may ameliorate T2DM-associated insulin resistance and cardiovascular disease.