RESUMO
The peroxisome proliferator-activated receptor (PPAR) was discovered a quarter of a century ago. PPAR was soon recognized as a general transcriptional regulator of lipid homeostasis, and several hypolipidemic and antidiabetic agents were shown to be effective ligands for it. Since then, many attempts to develop more potent drugs have been made worldwide, but most were unsuccessful due to serious side effects. It appears that the PPAR boom has ended. This review summarizes the short history of PPAR studies, including our own results, and discusses the lessons learned from the rise and fall of studies in this field for next-generation basic studies and drug development research.
Assuntos
Descoberta de Drogas/tendências , Hipoglicemiantes , Hipolipemiantes , Receptores Ativados por Proliferador de Peroxissomo/fisiologia , Pesquisa/tendências , Animais , Humanos , Hipoglicemiantes/efeitos adversos , Hipolipemiantes/efeitos adversos , Ligantes , Metabolismo dos Lipídeos , PPAR alfa , Tiazolidinedionas/efeitos adversosRESUMO
We identified and characterized a new eukaryotic-type protein kinase (PkaE) from Streptomyces coelicolor A3 (2) M145. PkaE, consisting of 510 amino acid residues, is a cytoplasmic protein kinase and contains the catalytic domain of eukaryotic protein kinases in the N-terminal region. Recombinant PkaE was found to be autophosphorylated at threonine residues only. The disruption of chromosomal pkaE resulted in the overproduction of the actinorhodin-related blue pigment antibiotics. pkaE was expressed during the late growth phase in S. coelicolor A3 (2) M145, which corresponded to the production time of blue pigments. This result indicated that PkaE acts as a negative regulator for production of the secondary metabolites. In addition, PkaE was able to phosphorylate KbpA, a regulator involved in the AfsK-AfsR regulatory pathway.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Streptomyces coelicolor/metabolismo , Antibacterianos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Transporte/metabolismo , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Streptomyces coelicolor/genéticaRESUMO
Plants contain potentially toxic compounds for animals and animals have developed physiological strategies to detoxify the ingested toxins during evolution. Feeding mice with various plant seeds and grains showed unexpected result that only sesame killed PPARα-null mice but not wild-type mice at all. A detailed analysis of this observation revealed that PPARα is involved in the metabolism of toxic compounds from plants as well as endobiotic substrates by inducing phase I and phase II detoxification enzymes. PPARα plays a vital role in direct or indirect activation of the relevant genes via the complex network among other xenobiotic nuclear receptors. Thus, PPARα plays its wider and more extensive role in energy metabolism from natural food intake to fat storage than previously thought.
RESUMO
Recent research in neural development has highlighted the importance of markers to discriminate phenotypic alterations of neural cells at various developmental stages. We isolated a new monoclonal antibody, 4F2, which was shown to be specific for an oligodendrocyte lineage. In primary cultures of oligodendroglial and mixed neural cells, the 4F2 antibody labeled a large proportion of Sox2(+) , Sox10(+) , A2B5(+) , NG2(+) , Olig2(+) , O4(+) , and myelin basic protein (MBP)(+) cells but did not label any GFAP(+) or NeuN(+) cells. In immunohistochemisty of rat embryos, the 4F2 antibody labeled a portion of neuroepithelial cells of the neural tube at embryonic day 9. The 4F2-positive cells were located initially in the ventricular zone as Musashi1(+) Tuj1(-) populations and distributed throughout the striatum; thereafter, they populated the whole brain and spinal cord. These cells showed ramified processes during embryonal development. The 4F2 antigen was associated with all four isoforms of MBP in coimmunoprecipitation experiments using brain homogenates or cell lysates of cultured oligodendrocytes. Immunoscreening of a brain cDNA library identified the antigen as DEAD (Asp-Glu-Ala-Asp) box polypeptide 54 (Ddx54), a member of the DEAD box family of RNA helicases involved in RNA metabolism, transcription, and translation. Cotransfection of the Ddx54 gene with MBP isoform genes increased the nuclear localization of the 21.5-kDa MBP isoform, which has been reported to function as a nuclear signal transduction molecule. These data indicate that Ddx54 might be not only a useful marker for investigating the ontogeny of oligodendrocytes but also an important factor in oligodendrocyte differentiation and myelination.
Assuntos
Anticorpos Monoclonais/metabolismo , RNA Helicases DEAD-box/imunologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteína Básica da Mielina/metabolismo , Oligodendroglia/metabolismo , Fatores Etários , Sequência de Aminoácidos , Animais , Antígenos de Diferenciação/metabolismo , Células Cultivadas , Córtex Cerebral/citologia , RNA Helicases DEAD-box/metabolismo , Embrião de Mamíferos , Feminino , Imunoprecipitação , Masculino , Neurônios/metabolismo , Gravidez , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratos , Ratos Wistar , TransfecçãoRESUMO
Statins are the most common type of medicine used to treat hypercholesterolemia; however, they are associated with a low incidence of myotoxicity such as myopathy and rhabdomyolysis. The mechanisms for the adverse effects remain to be fully elucidated for safer chronic use and drug development. The results of our earlier work suggested that hydrophobic statins induce autophagy in cultured human rhabdomyosarcoma A204 cells. In this study, we first confirmed the statin-induced autophagy by assessing other criteria, including induced expression of the autophagy-related genes, enhanced protein degradation of autophagy marker protein p62 and electron microscopic observation of induced formation of autophagosome. We next demonstrated that the extent of inhibition of HMG-CoA reductase in the cell is parallel with the ability of a statin to induce autophagy. Thus, the primary activity of statins causes autophagy in A204 cells. Considering the mechanism for the induction, we showed that statins induce autophagy by depleting cellular levels of geranylgeranyl diphosphate (GGPP) mostly through an unknown pathway that does not involve two major small G proteins, Rheb and Ras. Finally, we demonstrated that the ability of statins to induce autophagy parallels their toxicity to A204 cells and that both can be suppressed by GGPP.
Assuntos
Autofagia/efeitos dos fármacos , Interações Hidrofóbicas e Hidrofílicas , Inibidores de Hidroximetilglutaril-CoA Redutases/química , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Fosfatos de Poli-Isoprenil/metabolismo , Rabdomiossarcoma/patologia , Linhagem Celular Tumoral , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Monoméricas de Ligação ao GTP/antagonistas & inibidores , Neuropeptídeos/antagonistas & inibidores , Proteína Enriquecida em Homólogo de Ras do EncéfaloRESUMO
Peroxisome-proliferator activated receptor α (PPARα) is a ligand-activated transcription factor, playing a key role in several essential pathways including lipid metabolism. Although nuclear localization of PPARα is essential for its transactivation activity, mechanisms underlying intracellular traffics of PPARα remain undefined. We here identify and characterize a nuclear localization signal (NLS) residing in the junction between DNA-binding domain and hinge regions of PPARα. The NLS consists of two basic-amino acid clusters locating in the sequence encompassing amino acid residues at 144-187. We evidently show by mutational analysis that the basic residues in this NLS are essential for the nuclear import. Moreover, the PPARα NLS binds well-known nuclear transporters, importin α and importin ß, in a manner independent of DNA-binding activity.
Assuntos
Transporte Ativo do Núcleo Celular/fisiologia , Aminoácidos Básicos/metabolismo , PPAR alfa/metabolismo , Transporte Ativo do Núcleo Celular/genética , Aminoácidos Básicos/genética , Animais , Células COS , Chlorocebus aethiops , Camundongos , PPAR alfa/genética , Ligação Proteica , alfa Carioferinas/metabolismo , beta Carioferinas/metabolismoRESUMO
The liver is not a storage site of excess energy as triacylglycerides but a major site of carbohydrate storage, playing a vital role in glucose homeostasis, and the hepatic lipid droplets (LDs) should have a distinct physiologic role from those in lipid-storing tissues. Most studies so far have been limited to characterization of the LDs in cultured cells or of the liver of animals maintained on a normal laboratory diet, and little is known about the properties of the LDs in the liver responding to dietary excess, irregular fats, and potentially toxic compounds contained in a natural food diet. We started to characterize the hepatic LDs in wild-type and peroxisome proliferator-activated receptor alpha (PPARalpha)-null mice fed various natural diets by identifying the liver-enriched LD-associated proteins and the changes in lipid compositions. Based on the currently available data, we propose the hypothesis that hepatic LDs play vital protective roles against diet-derived excess fatty acids and potentially toxic hydrophobic compounds by temporarily storing them as neutral lipids or compounds until completion of the remodeling of fatty acids and detoxification of the compounds in a PPARalpha-dependent manner.
Assuntos
17-Hidroxiesteroide Desidrogenases/metabolismo , Metabolismo dos Lipídeos , Lipídeos , Fígado/metabolismo , PPAR alfa/metabolismo , Animais , Ácidos Graxos/metabolismo , Camundongos , Camundongos Knockout , Organelas/metabolismoRESUMO
Fatty aldehyde dehydrogenase (FALDH; also known as ALDH3A2 or ALDH10) oxidizes medium- or long-chain aliphatic aldehydes. FALDH deficiency in humans is known to be the cause of Sjögren-Larsson syndrome, in which individuals display neurological symptoms and cutaneous abnormality. FALDH-V, a splice isoform of FALDH, is localized in the peroxisome and contributes to the oxidization of pristanal, an intermediate of the alpha-oxidation pathway. FALDH-N, another splice isoform of FALDH, is induced by peroxisomal proliferator-activated receptor alpha ligands, although its activation mechanism has not been clarified. In the present study, we show that transcriptional activation of FALDH is directly regulated by peroxisomal proliferator-activated receptor alpha through a direct repeat-1 site located in the FALDH promoter. In addition, FALDH is efficiently induced by linoleic acid in rat hepatoma Fao cells through transcriptional activation by peroxisomal proliferator-activated receptor alpha. Furthermore, ectopic expression of endoplasmic reticulum-localizing FALDH-N, but not peroxisome-localizing FALDH-V, suppresses endoplasmic reticulum stress caused by linoleic acid in HEK293 cells. These results suggest the autocatalytic nature of the FALDH-N system against endoplasmic reticulum stress that is induced by polyunsaturated fatty acid; polyunsaturated fatty acid binds to peroxisomal proliferator-activated receptor alpha to activate the expression of FALDH-N, which then detoxifies polyunsaturated fatty acid-derived fatty aldehydes and protects cells from endoplasmic reticulum stress.
Assuntos
Aldeído Oxirredutases/genética , Retículo Endoplasmático/metabolismo , Ácidos Graxos Insaturados/farmacologia , PPAR alfa/metabolismo , Regulação para Cima , Aldeído Oxirredutases/metabolismo , Animais , Células Cultivadas , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Estresse Oxidativo , PPAR alfa/antagonistas & inibidores , Regiões Promotoras Genéticas , Ratos , TransfecçãoRESUMO
Primary human hepatocytes are extensively used to study the potential of drugs to induce cytochrome P450 (CYP). However, the activities of these enzymes decrease rapidly during culture. Previously we reported that in a layered co-culture system with HepG2 and bovine endothelial cells, the expression levels of various CYP genes were significantly increased compared with the monolayer cultured HepG2 cells. Here, we examined the induction of CYP gene expression by an inducer by examining the effect of phenobarbital treatment on CYP gene expression in the co-culture system. In the layered co-cultured HepG2, expression of the CYP2C and CYP3A family genes was induced by phenobarbital treatment. We also detected CYP3A4 enzyme induction using this co-culture system. Moreover, the induction of hepatic drug transporters by phenobarbital was detected. These results suggest that functional regulation of the CYP and transporter gene pathway is retained in these layered co-cultured cells. Thus, this system may serve as a useful model for in vitro pharmacological studies on the coordinated regulation of transport and metabolism.
Assuntos
Sistema Enzimático do Citocromo P-450/biossíntese , Células Endoteliais/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Fenobarbital/metabolismo , Linhagem Celular Tumoral , Técnicas de Cocultura , Sistema Enzimático do Citocromo P-450/genética , Relação Dose-Resposta a Droga , Células Endoteliais/citologia , Células Endoteliais/enzimologia , Indução Enzimática , Expressão Gênica/efeitos dos fármacos , Hepatócitos/citologia , Hepatócitos/enzimologia , Humanos , Fígado/enzimologia , Fígado/patologia , Fenobarbital/farmacologiaRESUMO
We previously reported that using thermo-responsive culture surfaces, a layered co-culture was achieved by placing an endothelial cell sheet onto a layer of human hepatoma cell line HepG2 in order to develop a culture model that mimics hepatic lobules. In the layered co-culture cells, the expression levels of liver-specific genes gradually increased. A cross-sectional view of the layered co-culture cells showed that the thickness of the layer slowly increased after layering, as did extracellular matrix (ECM) deposition around HepG2 cells. In this study, we report that the molecular compositions of ECM and cell-adhesion molecules changed in the layered co-culture cells. Gene expression of integrin alpha4 and decorin gradually increased after layering, and the time-course pattern of these genes was correlated with that of liver-specific genes. Moreover, the layered co-culture system has the ability to assemble a branching network of fibronectin fibrils. These results suggest that a vastly different extracellular environment in layered co-culture cells may induce an increase in liver-specific functions.
Assuntos
Moléculas de Adesão Celular/metabolismo , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Matriz Extracelular/metabolismo , Animais , Bovinos , Moléculas de Adesão Celular/genética , Linhagem Celular Tumoral , Técnicas de Cocultura , Decorina , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Integrina alfa4/genética , Integrina alfa4/metabolismo , Transporte Proteico , Proteoglicanas/genética , Proteoglicanas/metabolismoRESUMO
Primary human hepatocytes are used extensively to study drug-metabolizing enzymes such as the cytochrome P450 (CYP) enzymes. However, the activities of these enzymes decrease rapidly during culture. In the present study, using a thermo-responsive culture dish, layered co-culture was achieved by placing a bovine pulmonary artery endothelial cell (BPAEC) sheet onto the human hepatoma cell line HepG2. In the BPAEC/HepG2 layered co-culture system, real-time polymerase chain reaction analysis showed that the expression levels of various CYP enzymes were more than 10 times greater 21 days after layering than with a HepG2 monolayer. The expression levels of CYP1B1, CYP2C9, CYP2E1, and CYP3A4 were up-regulated in a time-dependent manner, gradually increasing from day 10 after layering, and continuing to increase until at least day 21. The gene expression levels of the various CYP enzymes were almost identical to that of human liver. These results suggest that our layered co-culture system enhances the function of HepG2 cells and that our BPAEC/HepG2 layered co-culture system can serve as a useful model for the in vitro evaluation of CYP regulation.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Células Endoteliais/metabolismo , Hepatócitos/metabolismo , Animais , Bovinos , Linhagem Celular , Linhagem Celular Tumoral , Técnicas de Cocultura/métodos , Células Endoteliais/citologia , Regulação Enzimológica da Expressão Gênica , Hepatócitos/citologia , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para Cima/genéticaRESUMO
17beta-Hydroxysteroid dehydrogenase type 11 (17betaHSD11) is mostly localized on the endoplasmic reticulum (ER) membrane under normal conditions and redistributes to lipid droplets (LDs) when the formation of LDs is induced. In this study, confocal microscopy analyses of the subcellular localization of the mutated 17betaHSD11 proteins in cells with or without LDs revealed that both an N-terminal hydrophobic sequence and an adjacent sequence that has a weak homology with the PAT motif are independently necessary and both parts together (28 amino acid residues in total) are sufficient for the dual localization of 17betaHSD11. Mutation analyses suggest that the PAT-like motif in 17betaHSD11 will not be functionally similar to the canonical PAT motif. Hsp60 was identified as a possibly interacting protein with the PAT-like motif, and biochemical and microscopic analyses suggest that Hsp60 may be partly, but not necessarily involved in recognition of the PAT-like part of the targeting sequence of 17betaHSD11.
Assuntos
17-Hidroxiesteroide Desidrogenases/metabolismo , Retículo Endoplasmático/enzimologia , Sinais Direcionadores de Proteínas/fisiologia , 17-Hidroxiesteroide Desidrogenases/genética , Motivos de Aminoácidos/fisiologia , Animais , Células CHO , Chaperonina 60/genética , Chaperonina 60/metabolismo , Cricetinae , Cricetulus , Retículo Endoplasmático/genética , Humanos , Interações Hidrofóbicas e Hidrofílicas , Microscopia Confocal , Mutação , Transporte Proteico/fisiologiaRESUMO
We previously demonstrated that Nrf2 regulates oxidized LDL-mediated CD36 expression in macrophages. The current study aimed to determine the mechanism of Nrf2-mediated macrophage CD36 induction. Treatment with the Nrf2 activator diethylmaleate, but not PPARgamma specific ligands, caused marked upregulation of CD36 in mouse macrophage RAW264.7 cells. Similarly, Nrf2 activators induced CD36 expression in bone marrow-derived macrophages in a Nrf2-dependent manner. Induced expression of the three alternative first exons of mouse CD36, deemed 1A, 1B, and 1C, occurred upon Nrf2 activation with exon1A mainly contributing to the CD36 expression. Four antioxidant response elements (AREs) lie within close proximity to these three exons, and chromatin immunoprecipitation assays demonstrated that two AREs upstream of exon1A, the distal 1A-ARE1, and the proximal 1A-ARE2, were Nrf2-responsive. Luciferase reporter assays conclusively demonstrated that 1A-ARE2 is the critical regulatory element for the Nrf2-mediated gene expression. Thus Nrf2 directly regulates CD36 gene expression by binding to 1A-ARE2.
Assuntos
Antioxidantes/metabolismo , Antígenos CD36/genética , Éxons , Macrófagos/metabolismo , Fator 2 Relacionado a NF-E2/fisiologia , Elementos de Resposta/fisiologia , Animais , Antioxidantes/farmacologia , Western Blotting , Linhagem Celular , Imunoprecipitação da Cromatina , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Knockout , Fator 2 Relacionado a NF-E2/genética , Elementos de Resposta/efeitos dos fármacos , Elementos de Resposta/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The tissue distribution and disposition of carnitine, which plays an important role in the transport of long-chain fatty acids across the mitochondrial inner membrane for beta-oxidation, are well controlled by carnitine transporter organic cation/carnitine transporter 2 (OCTN2). Since little information is available on regulation of the expression of the OCTN2 gene, we examined the factors that affect the expression level of rat Octn2 (rOctn2), focusing on nuclear receptor peroxisome proliferator activated receptor alpha (PPARalpha), which regulates expression of genes associated with beta-oxidation of fatty acids. mRNA of rOctn2 was induced by the PPARalpha ligand fenofibrate in primary-cultured rat hepatocytes. Further, the PPARalpha ligand Wy14643 increased the expression of Octn2 in wild-type mice, but not in PPARalpha knockout mice. Analysis of the rOctn2 promoter region suggested the presence of putative cis elements of PPARalpha. Wistar rats treated with intraperitoneal fenofibrate administration showed increased expression of rOctn2 mRNA in liver, and uptake of [3H]carnitine by freshly isolated hepatocytes derived from those rats was also increased. In conclusion, our results indicate that the nuclear receptor PPARalpha directly up-regulates the expression of rOctn2 and increases the hepatic uptake of carnitine via rOctn2.
Assuntos
Proteínas de Transporte de Cátions Orgânicos/genética , PPAR alfa/fisiologia , Animais , Northern Blotting , Carnitina/metabolismo , Linhagem Celular Tumoral , Separação Celular , Células Cultivadas , Ensaio de Desvio de Mobilidade Eletroforética , Fenofibrato/farmacologia , Genes Reporter , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hipolipemiantes/farmacologia , Luciferases/metabolismo , Masculino , Proteínas de Transporte de Cátions Orgânicos/biossíntese , Proteínas de Transporte de Cátions Orgânicos/fisiologia , PPAR alfa/antagonistas & inibidores , Plasmídeos , Regiões Promotoras Genéticas/genética , Ratos , Ratos Wistar , Receptores Citoplasmáticos e Nucleares/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Membro 5 da Família 22 de Carreadores de Soluto , TransfecçãoRESUMO
17beta-Hydroxysteroid dehydrogenase (17betaHSD) type 13 is identified as a new lipid droplet-associated protein. 17betaHSD type 13 has an N-terminal sequence similar to that of 17betaHSD type 11, and both sequences function as an endoplasmic reticulum and lipid droplet-targeting signal. Localization of native 17betaHSD type 13 on the lipid droplets was confirmed by subcellular fractionation and Western blotting. In contrast to 17betaHSD type 11, however, expression of 17betaHSD type 13 is largely restricted to the liver and is not enhanced by peroxisome proliferator-activated receptor alpha and its ligand. Instead the expression level of 17betaHSD type 13 in the receptor-null mice was increased several-fold. 17betaHSD type 13 may have a distinct physiological role as a lipid droplet-associated protein in the liver.
Assuntos
17-Hidroxiesteroide Desidrogenases/metabolismo , Metabolismo dos Lipídeos , Fígado/enzimologia , 17-Hidroxiesteroide Desidrogenases/análise , 17-Hidroxiesteroide Desidrogenases/genética , Sequência de Aminoácidos , Animais , Western Blotting , Células CHO , Cricetinae , Cricetulus , Camundongos , Camundongos Mutantes , Dados de Sequência Molecular , PPAR alfa/agonistas , PPAR alfa/genética , PPAR alfa/metabolismo , Sinais Direcionadores de Proteínas/genética , RNA Mensageiro/análise , RNA Mensageiro/metabolismoRESUMO
We identified and characterized the gene encoding a new eukaryotic-type protein kinase from Streptomyces coelicolor A3(2) M145. PkaD, consisting of 598 amino acid residues, contained the catalytic domain of eukaryotic protein kinases in the N-terminal region. A hydrophobicity plot indicated the presence of a putative transmembrane spanning sequence downstream of the catalytic domain, suggesting that PkaD is a transmembrane protein kinase. The recombinant PkaD was found to be phosphorylated at the threonine and tyrosine residues. In S. coelicolor A3(2), pkaD was transcribed as a monocistronic mRNA, and it was expressed constitutively throughout the life cycle. Disruption of chromosomal pkaD resulted in a significant loss of actinorhodin production. This result implies the involvement of pkaD in the regulation of secondary metabolism.
Assuntos
Proteínas de Bactérias/genética , Proteínas Serina-Treonina Quinases/genética , Fatores de Transcrição/genética , Antraquinonas/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/fisiologia , Sequência de Bases , Domínio Catalítico , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Membrana , Metabolismo , Dados de Sequência Molecular , Fosforilação , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/fisiologia , Streptomyces coelicolor , Fatores de Transcrição/química , Fatores de Transcrição/fisiologiaRESUMO
Statins are widely used to treat hypercholesterolemia, but they are associated with muscle-related adverse events, by as yet, inadequately resolved mechanisms. In this study, we report that statins induced autophagy in cultured human rhabdomyosarcoma A204 cells. Potency differed widely among the statins: cerivastatin induced autophagy at 0.1muM, simvastatin at 10muM but none was induced by pravastatin. Addition of mevalonate, but not cholesterol, blocked induction of autophagy by cerivastatin, suggesting that this induction is dependent on modulation of isoprenoid metabolic pathways. The statin-induced autophagy was not observed in other types of cells, such as human hepatoma HepG2 or embryonic kidney HEK293 cells. Muscle-specific abortive induction of autophagy by hydrophobic statins is a possible mechanism for statin-induced muscle-related side effects.
Assuntos
Autofagia/efeitos dos fármacos , Inibidores de Hidroximetilglutaril-CoA Redutases/administração & dosagem , Inibidores de Hidroximetilglutaril-CoA Redutases/química , Rabdomiossarcoma/patologia , Rabdomiossarcoma/fisiopatologia , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Humanos , Interações Hidrofóbicas e HidrofílicasRESUMO
A systematic analysis to examine the effects of peroxisome proliferator-activated receptor (PPAR)alpha agonists on the expression levels of all the nutrient/drug plasma-membrane transporters in the mouse small intestine was performed. Transporter mRNAs that were induced or repressed by two independent PPARalpha-specific agonists were identified by a genome-wide microarray method, and the changes were confirmed by real-time PCR using RNA isolated from the intestines and livers of wild-type and PPARalpha-null mice. Expression levels of seven nutrient/drug transporters (Abcd3, Octn2/Slc22a5, FATP2/Slc27a2, Slc22a21, Mct13/Slc16a13, Slc23a1 and Bcrp/Abcg2) in the intestine were up-regulated and the expression level of one (Mrp1/Abcc1) was down-regulated by PPARalpha; although the previously report that the H(+)/peptide co-transporter 1 (Pept1) is up-regulated by PPARalpha was not replicated in our study. We propose that the transport processes can be coordinately regulated with intracellular metabolism by nutrient nuclear receptors.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Coenzima A Ligases/genética , Regulação da Expressão Gênica , Intestino Delgado/metabolismo , Proteínas de Membrana/genética , Transportadores de Ácidos Monocarboxílicos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Transportadores de Ânions Orgânicos Dependentes de Sódio/genética , Proteínas de Transporte de Cátions Orgânicos/genética , PPAR alfa/agonistas , Simportadores/genética , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Transportadores de Sódio Acoplados à Vitamina C , Membro 5 da Família 22 de Carreadores de SolutoRESUMO
17beta-Hydroxysteroid dehydrogenase type 11 (17beta-HSD11) is a member of the short-chain dehydrogenase/reductase family involved in the activation and inactivation of sex steroid hormones. We recently identified 17beta-HSD11 as a gene that is efficiently regulated by peroxisome proliferator-activated receptor-alpha PPARalpha in the intestine and the liver [Motojima K (2004) Eur J Biochem271, 4141-4146]. In this study, we characterized 17beta-HSD11 at the protein level to obtain information about its physiologic role in the intestine and liver. For this purpose, specific antibodies against 17beta-HSD11 were obtained. Western blotting analysis showed that administration of a peroxisome proliferator-activated receptor-alpha agonist induced 17beta-HSD11 protein in the jejunum but not in the colon, and to a much higher extent than in the liver of mice. A subcellular localization study using Chinese hamster ovary cells and green fluorescent protein-tagged 17beta-HSD11 showed that it was mostly localized in the endoplasmic reticulum under normal conditions, whereas it was concentrated on lipid droplets when they were induced. A pulse-chase experiment suggested that 17beta-HSD11 was redistributed to the lipid droplets via the endoplasmic reticulum. Immunohistochemical analysis using tissue sections showed that 17beta-HSD11 was induced mostly in intestinal epithelia and hepatocytes, with heterogeneous localization both in the cytoplasm and in vesicular structures. A subcellular fractionation study of liver homogenates confirmed that 17beta-HSD11 was localized mostly in the endoplasmic reticulum when mice were fed a normal diet, but was distributed in both the endoplasmic reticulum and the lipid droplets of which formation was induced by feeding a diet containing a proliferator-activated receptor-alpha agonist. Taken together, these data indicate that 17beta-HSD11 localizes both in the endoplasmic reticulum and in lipid droplets, depending on physiologic conditions, and that lipid droplet 17beta-HSD11 is not merely an endoplasmic reticulum contaminant or a nonphysiologically associated protein in the cultured cells, but a bona fide protein component of the membranes of both intracellular compartments.
Assuntos
17-Hidroxiesteroide Desidrogenases/metabolismo , Regulação da Expressão Gênica , Intestinos/enzimologia , Fígado/enzimologia , PPAR alfa/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Citoplasma/metabolismo , Duodeno/enzimologia , Retículo Endoplasmático/metabolismo , Mucosa Intestinal/metabolismo , Jejuno/enzimologia , Lipídeos , Fígado/metabolismo , Camundongos , Especificidade de Órgãos , PPAR alfa/agonistasRESUMO
Fatty aldehyde dehydrogenase (FALDH, ALDH3A2) is thought to be involved in the degradation of phytanic acid, a saturated branched chain fatty acid derived from chlorophyll. However, the identity, subcellular distribution, and physiological roles of FALDH are unclear because several variants produced by alternative splicing are present in varying amounts at different subcellular locations. Subcellular fractionation experiments do not provide a clear-cut conclusion because of the incomplete separation of organelles. We established human cell lines heterologously expressing mouse FALDH from each cDNA without tagging under the control of an inducible promoter and detected the variant FALDH proteins using a mouse FALDH-specific antibody. One variant, FALDH-V, was exclusively detected in peroxisomal membranes. Human FALDH-V with an amino-terminal Myc sequence also localized to peroxisomes. The most dominant form, FALDH-N, and other variants examined, however, were distributed in the endoplasmic reticulum. A gas chromatography-mass spectrometry-based analysis of metabolites in FALDH-expressing cells incubated with phytol or phytanic acid showed that FALDH-V, not FALDH-N, is the key aldehyde dehydrogenase in the degradation pathway and that it protects peroxisomes from oxidative stress. In contrast, both FALDHs had a protective effect against oxidative stress induced by a model aldehyde for lipid peroxidation, dodecanal. These results suggest that FALDH variants are produced by alternative splicing and share an important role in protecting against oxidative stress in an organelle-specific manner.