RESUMEN
Type 2 diabetes is a metabolic disorder associated with abnormal glucose homeostasis and is characterized by intrinsic defects in ß-cell function and mass. Trimethylguanosine synthase 1 (TGS1) is an evolutionarily conserved enzyme that methylates small nuclear and nucleolar RNAs and that is involved in pre-mRNA splicing, transcription, and ribosome production. However, the role of TGS1 in ß-cells and glucose homeostasis had not been explored. Here, we show that TGS1 is upregulated by insulin and upregulated in islets of Langerhans from mice exposed to a high-fat diet and in human ß-cells from type 2 diabetes donors. Using mice with conditional (ßTGS1KO) and inducible (MIP-CreERT-TGS1KO) TGS1 deletion, we determined that TGS1 regulates ß-cell mass and function. Using unbiased approaches, we identified a link between TGS1 and endoplasmic reticulum stress and cell cycle arrest, as well as and how TGS1 regulates ß-cell apoptosis. We also found that deletion of TGS1 results in an increase in the unfolded protein response by increasing XBP-1, ATF-4, and the phosphorylation of eIF2α, in addition to promoting several changes in cell cycle inhibitors and activators such as p27 and Cyclin D2. This study establishes TGS1 as a key player regulating ß-cell mass and function. We propose that these observations can be used as a stepping-stone for the design of novel strategies focused on TGS1 as a therapeutic target for the treatment of diabetes.
Asunto(s)
Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Animales , Diabetes Mellitus Tipo 2/enzimología , Diabetes Mellitus Tipo 2/metabolismo , Glucosa/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/enzimología , Células Secretoras de Insulina/metabolismo , Metiltransferasas/metabolismo , Ratones , Ratones NoqueadosRESUMEN
Phosphodiesterase 1C (PDE1C) is expressed in mammalian heart and regulates cardiac functions by controlling levels of second messenger cyclic AMP and cyclic GMP (cAMP and cGMP, respectively). However, molecular mechanisms of cardiac Pde1c regulation are currently unknown. In this study, we demonstrate that treatment of wild type mice and H9c2 myoblasts with Wy-14,643, a potent ligand of nuclear receptor peroxisome-proliferator activated receptor alpha (PPARα), leads to elevated cardiac Pde1C mRNA and cardiac PDE1C protein, which correlate with reduced levels of cAMP. Furthermore, using mice lacking either Pparα or cardiomyocyte-specific Med1, the major subunit of Mediator complex, we show that Wy-14,643-mediated Pde1C induction fails to occur in the absence of Pparα and Med1 in the heart. Finally, using chromatin immunoprecipitation assays we demonstrate that PPARα binds to the upstream Pde1C promoter sequence on two sites, one of which is a palindrome sequence (agcTAGGttatcttaacctagc) that shows a robust binding. Based on these observations, we conclude that cardiac Pde1C is a direct transcriptional target of PPARα and that Med1 may be required for the PPARα mediated transcriptional activation of cardiac Pde1C.
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 1/genética , Miocardio/metabolismo , PPAR alfa/metabolismo , Animales , Línea Celular , AMP Cíclico/metabolismo , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 1/metabolismo , Subunidad 1 del Complejo Mediador/genética , Subunidad 1 del Complejo Mediador/metabolismo , Ratones , Ratones Endogámicos C57BL , PPAR alfa/genética , Regiones Promotoras Genéticas , Unión Proteica , Activación TranscripcionalRESUMEN
PIMT/NCOA6IP, a transcriptional coactivator PRIP/NCOA6 binding protein, enhances nuclear receptor transcriptional activity. Germline disruption of PIMT results in early embryonic lethality due to impairment of development around blastocyst and uterine implantation stages. We now generated mice with Cre-mediated cardiac-specific deletion of PIMT (csPIMT-/-) in adult mice. These mice manifest enlargement of heart, with nearly 100% mortality by 7.5 months of age due to dilated cardiomyopathy. Significant reductions in the expression of genes (i) pertaining to mitochondrial respiratory chain complexes I to IV; (ii) calcium cycling cardiac muscle contraction (Atp2a1, Atp2a2, Ryr2); and (iii) nuclear receptor PPAR- regulated genes involved in glucose and fatty acid energy metabolism were found in csPIMT-/- mouse heart. Elevated levels of Nppa and Nppb mRNAs were noted in csPIMT-/- heart indicative of myocardial damage. These hearts revealed increased reparative fibrosis associated with enhanced expression of Tgfß2 and Ctgf. Furthermore, cardiac-specific deletion of PIMT in adult mice, using tamoxifen-inducible Cre-approach (TmcsPIMT-/-), results in the development of cardiomyopathy. Thus, cumulative evidence suggests that PIMT functions in cardiac energy metabolism by interacting with nuclear receptor coactivators and this property could be useful in the management of heart failure.
Asunto(s)
Cardiomiopatías/genética , Cardiomiopatías/metabolismo , Metabolismo Energético , Eliminación de Gen , Coactivadores de Receptor Nuclear/genética , Proteína D-Aspartato-L-Isoaspartato Metiltransferasa/genética , Animales , Cardiomiopatías/patología , Cardiomiopatía Dilatada/genética , Cardiomiopatía Dilatada/metabolismo , Cardiomiopatía Dilatada/patología , Modelos Animales de Enfermedad , Fibrosis , Expresión Génica , Glucosa/metabolismo , Glucógeno/metabolismo , Ratones , Ratones Noqueados , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Contracción Miocárdica/genética , Miocardio/metabolismo , Miocardio/patología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismoRESUMEN
OBJECTIVE: MED1 (mediator 1) interacts with transcription factors to regulate transcriptional machinery. The role of MED1 in macrophage biology and the relevant disease state remains to be investigated. APPROACH AND RESULTS: To study the molecular mechanism by which MED1 regulates the M1/M2 phenotype switch of macrophage and the effect on atherosclerosis, we generated MED1/apolipoprotein E (ApoE) double-deficient (MED1ΔMac/ApoE-/-) mice and found that atherosclerosis was greater in MED1ΔMac/ApoE-/- mice than in MED1fl/fl/ApoE-/- littermates. The gene expression of M1 markers was increased and that of M2 markers decreased in both aortic wall and peritoneal macrophages from MED1ΔMac/ApoE-/- mice, whereas MED1 overexpression rectified the changes in M1/M2 expression. Moreover, LDLR (low-density lipoprotein receptor)-deficient mice received bone marrow from MED1ΔMac mice showed greater atherosclerosis. Mechanistically, MED1 ablation decreased the binding of PPARγ (peroxisome proliferator-activated receptor γ) and enrichment of H3K4me1 and H3K27ac to upstream region of M2 marker genes. Furthermore, interleukin 4 induction of PPARγ and MED1 increased the binding of PPARγ or MED1 to the PPAR response elements of M2 marker genes. CONCLUSIONS: Our data suggest that MED1 is required for the PPARγ-mediated M2 phenotype switch, with M2 marker genes induced but M1 marker genes suppressed. MED1 in macrophages has an antiatherosclerotic role via PPARγ-regulated transactivation.
Asunto(s)
Aorta/metabolismo , Enfermedades de la Aorta/prevención & control , Aterosclerosis/prevención & control , Plasticidad de la Célula , Macrófagos Peritoneales/metabolismo , Subunidad 1 del Complejo Mediador/metabolismo , Acetilación , Animales , Aorta/inmunología , Aorta/patología , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/metabolismo , Enfermedades de la Aorta/patología , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Aterosclerosis/genética , Aterosclerosis/metabolismo , Aterosclerosis/patología , Sitios de Unión , Trasplante de Médula Ósea , Modelos Animales de Enfermedad , Epigénesis Genética , Regulación de la Expresión Génica , Predisposición Genética a la Enfermedad , Histonas/metabolismo , Inmunidad Innata , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/patología , Macrófagos Peritoneales/trasplante , Masculino , Subunidad 1 del Complejo Mediador/deficiencia , Subunidad 1 del Complejo Mediador/genética , Metilación , Ratones , Ratones Noqueados , PPAR gamma/metabolismo , Fenotipo , Placa Aterosclerótica , Células RAW 264.7 , Interferencia de ARN , Receptores de LDL/deficiencia , Receptores de LDL/genética , Elementos de Respuesta , Transducción de Señal , Transcripción Genética , Activación Transcripcional , TransfecciónRESUMEN
[This corrects the article DOI: 10.1371/journal.pone.0160755.].
RESUMEN
Mediator, an evolutionarily conserved multi-protein complex consisting of about 30 subunits, is a key component of the polymerase II mediated gene transcription. Germline deletion of the Mediator subunit 1 (Med1) of the Mediator in mice results in mid-gestational embryonic lethality with developmental impairment of multiple organs including heart. Here we show that cardiomyocyte-specific deletion of Med1 in mice (csMed1-/-) during late gestational and early postnatal development by intercrossing Med1fl/fl mice to α-MyHC-Cre transgenic mice results in lethality within 10 days after weaning due to dilated cardiomyopathy-related ventricular dilation and heart failure. The csMed1-/- mouse heart manifests mitochondrial damage, increased apoptosis and interstitial fibrosis. Global gene expression analysis revealed that loss of Med1 in heart down-regulates more than 200 genes including Acadm, Cacna1s, Atp2a2, Ryr2, Pde1c, Pln, PGC1α, and PGC1ß that are critical for calcium signaling, cardiac muscle contraction, arrhythmogenic right ventricular cardiomyopathy, dilated cardiomyopathy and peroxisome proliferator-activated receptor regulated energy metabolism. Many genes essential for oxidative phosphorylation and proper mitochondrial function such as genes coding for the succinate dehydrogenase subunits of the mitochondrial complex II are also down-regulated in csMed1-/- heart contributing to myocardial injury. Data also showed up-regulation of about 180 genes including Tgfb2, Ace, Atf3, Ctgf, Angpt14, Col9a2, Wisp2, Nppa, Nppb, and Actn1 that are linked to cardiac muscle contraction, cardiac hypertrophy, cardiac fibrosis and myocardial injury. Furthermore, we demonstrate that cardiac specific deletion of Med1 in adult mice using tamoxifen-inducible Cre approach (TmcsMed1-/-), results in rapid development of cardiomyopathy and death within 4 weeks. We found that the key findings of the csMed1-/- studies described above are highly reproducible in TmcsMed1-/- mouse heart. Collectively, these observations suggest that Med1 plays a critical role in the maintenance of heart function impacting on multiple metabolic, compensatory and reparative pathways with a likely therapeutic potential in the management of heart failure.
Asunto(s)
Cardiomiopatía Dilatada/genética , Genes Letales , Insuficiencia Cardíaca/genética , Subunidad 1 del Complejo Mediador/genética , Mitocondrias/metabolismo , Miocitos Cardíacos/metabolismo , Animales , Apoptosis , Cadherinas/genética , Cadherinas/metabolismo , Canales de Calcio Tipo L/genética , Canales de Calcio Tipo L/metabolismo , Señalización del Calcio , Cardiomiopatía Dilatada/metabolismo , Cardiomiopatía Dilatada/patología , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 1/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 1/metabolismo , Embrión de Mamíferos , Metabolismo Energético , Femenino , Eliminación de Gen , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Edad Gestacional , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/patología , Subunidad 1 del Complejo Mediador/deficiencia , Ratones , Ratones Noqueados , Mitocondrias/patología , Contracción Miocárdica , Miocitos Cardíacos/patología , Receptores Activados del Proliferador del Peroxisoma/genética , Receptores Activados del Proliferador del Peroxisoma/metabolismo , Embarazo , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismoRESUMEN
Immunoglobulin (Ig) class switch recombination (CSR) is initiated by the transcription-coupled recruitment of activation-induced cytidine deaminase (AID) to Ig switch regions (S regions). During CSR, the IgH locus undergoes dynamic three-dimensional structural changes in which promoters, enhancers, and S regions are brought to close proximity. Nevertheless, little is known about the underlying mechanisms. In this study, we show that Med1 and Med12, two subunits of the mediator complex implicated in transcription initiation and long-range enhancer/promoter loop formation, are dynamically recruited to the IgH locus enhancers and the acceptor regions during CSR and that their knockdown in CH12 cells results in impaired CSR. Furthermore, we show that conditional inactivation of Med1 in B cells results in defective CSR and reduced acceptor S region transcription. Finally, we show that in B cells undergoing CSR, the dynamic long-range contacts between the IgH enhancers and the acceptor regions correlate with Med1 and Med12 binding and that they happen at a reduced frequency in Med1-deficient B cells. Our results implicate the mediator complex in the mechanism of CSR and are consistent with a model in which mediator facilitates the long-range contacts between S regions and the IgH locus enhancers during CSR and their transcriptional activation.
Asunto(s)
Cambio de Clase de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/genética , Subunidad 1 del Complejo Mediador/metabolismo , Complejo Mediador/metabolismo , Recombinación Genética/genética , Activación Transcripcional/genética , Animales , Linfocitos B/metabolismo , Células Cultivadas , Técnicas de Silenciamiento del Gen , Sitios Genéticos , Ratones , Unión Proteica , Transcripción GenéticaRESUMEN
The kidney is one of the target organs for various metabolic diseases, including diabetes, metabolic syndrome, and obesity. Most of the metabolic studies underscore glomerular pathobiology, although the tubulo-interstitial compartment has been underemphasized. This study highlights mechanisms concerning the pathobiology of tubular injury in the context of myo-inositol oxygenase (Miox), a tubular enzyme. The kidneys of mice fed a high fat diet (HFD) had increased Miox expression and activity, and the latter was related to phosphorylation of serine/threonine residues. Also, expression of sterol regulatory element-binding protein1 (Srebp1) and markers of cellular/nuclear damage was increased along with accentuated apoptosis and loss of tubular brush border. Similar results were observed in cells treated with palmitate/BSA. Multiple sterol-response elements and E-box motifs were found in the miox promoter, and its activity was modulated by palmitate/BSA. Electrophoretic mobility and ChIP assays confirmed binding of Srebp to consensus sequences of the miox promoter. Exposure of palmitate/BSA-treated cells to rapamycin normalized Miox expression and prevented Srebp1 nuclear translocation. In addition, rapamycin treatment reduced p53 expression and apoptosis. Like rapamycin, srebp siRNA reduced Miox expression. Increased expression of Miox was associated with the generation of reactive oxygen species (ROS) in kidney tubules of mice fed an HFD and cell exposed to palmitate/BSA. Both miox and srebp1 siRNAs reduced generation of ROS. Collectively, these findings suggest that HFD or fatty acids modulate transcriptional, translational, and post-translational regulation of Miox expression/activity and underscore Miox being a novel target of the transcription factor Srebp1. Conceivably, activation of the mTORC1/Srebp1/Miox pathway leads to the generation of ROS culminating into tubulo-interstitial injury in states of obesity.
Asunto(s)
Nefropatías Diabéticas/metabolismo , Inositol-Oxigenasa/metabolismo , Túbulos Renales/enzimología , Obesidad/metabolismo , Estrés Oxidativo , Procesamiento Proteico-Postraduccional , Regulación hacia Arriba , Animales , Apoptosis , Línea Celular , Nefropatías Diabéticas/etiología , Nefropatías Diabéticas/patología , Dieta Alta en Grasa/efectos adversos , Humanos , Inositol-Oxigenasa/antagonistas & inhibidores , Inositol-Oxigenasa/genética , Túbulos Renales/metabolismo , Túbulos Renales/patología , Masculino , Ratones , Obesidad/etiología , Obesidad/patología , Oxigenasas/antagonistas & inhibidores , Oxigenasas/genética , Oxigenasas/metabolismo , Fosforilación , Regiones Promotoras Genéticas , Proteínas/antagonistas & inhibidores , Proteínas/genética , Proteínas/metabolismo , Interferencia de ARN , Ratas , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/antagonistas & inhibidores , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genética , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Sus scrofaRESUMEN
The mechanisms underlying inflammation induced insulin resistance are poorly understood. Here, we report that the expression of PIMT, a transcriptional co-activator binding protein, was up-regulated in the soleus muscle of high sucrose diet (HSD) induced insulin resistant rats and TNF-α exposed cultured myoblasts. Moreover, TNF-α induced phosphorylation of PIMT at the ERK1/2 target site Ser(298). Wild type (WT) PIMT or phospho-mimic Ser298Asp mutant but not phospho-deficient Ser298Ala PIMT mutant abrogated insulin stimulated glucose uptake by L6 myotubes and neonatal rat skeletal myoblasts. Whereas, PIMT knock down relieved TNF-α inhibited insulin signaling. Mechanistic analysis revealed that PIMT differentially regulated the expression of GLUT4, MEF2A, PGC-1α and HDAC5 in cultured cells and skeletal muscle of Wistar rats. Further characterization showed that PIMT was recruited to GLUT4, MEF2A and HDAC5 promoters and overexpression of PIMT abolished the activity of WT but not MEF2A binding defective mutant GLUT4 promoter. Collectively, we conclude that PIMT mediates TNF-α induced insulin resistance at the skeletal muscle via the transcriptional modulation of GLUT4, MEF2A, PGC-1α and HDAC5 genes.
Asunto(s)
Transportador de Glucosa de Tipo 4/metabolismo , Proteína D-Aspartato-L-Isoaspartato Metiltransferasa/metabolismo , Transcripción Genética/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Animales , Glucemia/análisis , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Transportador de Glucosa de Tipo 4/genética , Células HEK293 , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Humanos , Resistencia a la Insulina , Factores de Transcripción MEF2/genética , Factores de Transcripción MEF2/metabolismo , Masculino , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Mioblastos Esqueléticos/citología , Mioblastos Esqueléticos/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Fosforilación/efectos de los fármacos , Proteína D-Aspartato-L-Isoaspartato Metiltransferasa/genética , Ratas , Ratas Wistar , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factor de Necrosis Tumoral alfa/sangreRESUMEN
Obesity poses an increased risk of developing metabolic syndrome and closely associated nonalcoholic fatty liver disease, including liver cancer. Satiety hormone leptin-deficient (ob/ob) mice, considered paradigmatic of nutritional obesity, develop hepatic steatosis but are less prone to developing liver tumors. Sustained activation of peroxisome proliferator-activated receptor α (PPARα) in ob/ob mouse liver increases fatty acid oxidation (FAO), which contributes to attenuation of obesity but enhances liver cancer risk. To further evaluate the role of PPARα-regulated hepatic FAO and energy burning in the progression of fatty liver disease, we generated PPARα-deficient ob/ob (PPARα(Δ)ob/ob) mice. These mice become strikingly more obese compared to ob/ob littermates, with increased white and brown adipose tissue content and severe hepatic steatosis. Hepatic steatosis becomes more severe in fasted PPARα(Δ)ob/ob mice as they fail to up-regulate FAO systems. PPARα(Δ)ob/ob mice also do not respond to peroxisome proliferative and mitogenic effects of PPARα agonist Wy-14,643. Although PPARα(Δ)ob/ob mice are severely obese, there was no significant increase in liver tumor incidence, even when maintained on a diet containing Wy-14,643. We conclude that sustained PPARα activation-related increase in FAO in fatty livers of obese ob/ob mice increases liver cancer risk, whereas deletion of PPARα in ob/ob mice aggravates obesity and hepatic steatosis. However, it does not lead to liver tumor development because of reduction in FAO and energy burning.
Asunto(s)
Ácidos Grasos/metabolismo , Neoplasias Hepáticas/metabolismo , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Obesidad/metabolismo , PPAR alfa/deficiencia , Animales , Modelos Animales de Enfermedad , Immunoblotting , Neoplasias Hepáticas/etiología , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Enfermedad del Hígado Graso no Alcohólico/etiología , Obesidad/complicaciones , Oxidación-Reducción , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Spermatogenesis is a highly coordinated process. Signaling from nuclear hormone receptors, like those for retinoic acid (RA), is important for normal spermatogenesis. However, the mechanisms regulating these signals are poorly understood. Mediator complex subunit 1 (MED1) is a transcriptional enhancer that directly modulates transcription from nuclear hormone receptors. MED1 is present in male germ cells throughout mammalian development, but its function during spermatogenesis is unknown. To determine its role, we generated mice lacking Med1 specifically in their germ cells beginning just before birth. Conditional Med1 knockout males are fertile, exhibiting normal testis weights and siring ordinary numbers of offspring. RA-responsive gene products stimulated by RA gene 8 (Stra8) and synaptonemal complex protein 3 (Sycp3) are first detected in knockout spermatogonia at the expected time points during the first wave of spermatogenesis, and persist with normal patterns of cellular distribution in adult knockout testes. Meiotic progression, however, is altered in the absence of Med1. At postnatal day 7 (P7), zygotene-stage knockout spermatocytes are already detected, unlike in control testes, with fewer pre-leptotene-stage cells and more leptotene spermatocytes observed in the knockouts. At P9, Med1 knockout spermatocytes prematurely enter pachynema. Once formed, greater numbers of knockout spermatocytes remain in pachynema relative to the other stages of meiosis throughout testis development and its maintenance in the adult. Meiotic exit is not inhibited. We conclude that MED1 regulates the temporal progression of primary spermatocytes through meiosis, with its absence resulting in abbreviated pre-leptotene, leptotene, and zygotene stages, and a prolonged pachytene stage.
Asunto(s)
Subunidad 1 del Complejo Mediador/metabolismo , Meiosis/genética , Espermatogénesis/genética , Animales , Masculino , Subunidad 1 del Complejo Mediador/genética , Ratones , Ratones Noqueados , Fase Paquiteno/genética , Testículo/metabolismoRESUMEN
MED1 (Mediator complex subunit 1) is a co-activator of various transcription factors that function in multiple transcriptional pathways. We have already established keratinocyte-specific MED1 null mice (Med1(epi-/-)) that develop epidermal hyperplasia. Herein, to investigate the function(s) of MED1 in skin wound healing, full-thickness skin wounds were generated in Med1(epi-/-) and age-matched wild-type mice and the healing process was analyzed. Macroscopic wound closure and the re-epithelialization rate were accelerated in 8-week-old Med1(epi-/-) mice compared with age-matched wild-type mice. Increased lengths of migrating epithelial tongues and numbers of Ki67-positive cells at the wounded epidermis were observed in 8-week-old Med1(epi-/-) mice, whereas wound contraction and the area of α-SMA-positive myofibroblasts in the granulation tissue were unaffected. Migration was enhanced in Med1(epi-/-) keratinocytes compared with wild-type keratinocytes in vitro. Immunoblotting revealed that the expression of follistatin was significantly decreased in Med1(epi-/-) keratinocytes. Moreover, the mitogen-activated protein kinase pathway was enhanced before and after treatment of Med1(epi-/-) keratinocytes with activin A in vitro. Cell-cycle analysis showed an increased ratio of S phase cells after activin A treatment of Med1(epi-/-) keratinocytes compared with wild-type keratinocytes. These findings indicate that the activin-follistatin system is involved in this acceleration of skin wound healing in 8-week-old Med1(epi-/-) mice. On the other hand, skin wound healing in 6-month-old Med1(epi-/-) mice was significantly delayed with decreased numbers of Ki67-positive cells at the wounded epidermis as well as BrdU-positive label retaining cells in hair follicles compared with age-matched wild-type mice. These results agree with our previous observation that hair follicle bulge stem cells are reduced in older Med1(epi-/-) mice, indicating a decreased contribution of hair follicle stem cells to epidermal regeneration after wounding in 6-month-old Med1(epi-/-) mice. This study sheds light on the novel function of MED1 in keratinocytes and suggests a possible new therapeutic approach for skin wound healing and aging.
Asunto(s)
Queratinocitos/metabolismo , Subunidad 1 del Complejo Mediador/metabolismo , Piel/metabolismo , Cicatrización de Heridas/fisiología , Actinas/metabolismo , Activinas/metabolismo , Animales , Movimiento Celular/fisiología , Folistatina/metabolismo , Folículo Piloso/metabolismo , Antígeno Ki-67/metabolismo , Ratones , Ratones Noqueados , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Repitelización/fisiología , Fase S/fisiología , Transducción de Señal/fisiología , Células Madre/metabolismoRESUMEN
Several nuclear receptors regulate diverse metabolic functions that impact on critical biological processes, such as development, differentiation, cellular regeneration, and neoplastic conversion. In the liver, some members of the nuclear receptor family, such as peroxisome proliferator-activated receptors (PPARs), constitutive androstane receptor (CAR), farnesoid X receptor (FXR), liver X receptor (LXR), pregnane X receptor (PXR), glucocorticoid receptor (GR), and others, regulate energy homeostasis, the formation and excretion of bile acids, and detoxification of xenobiotics. Excess energy burning resulting from increases in fatty acid oxidation systems in liver generates reactive oxygen species, and the resulting oxidative damage influences liver regeneration and liver tumor development. These nuclear receptors are important sensors of exogenous activators as well as receptor-specific endogenous ligands. In this regard, gene knockout mouse models revealed that some lipid-metabolizing enzymes generate PPARα-activating ligands, while others such as ACOX1 (fatty acyl-CoA oxidase1) inactivate these endogenous PPARα activators. In the absence of ACOX1, the unmetabolized ACOX1 substrates cause sustained activation of PPARα, and the resulting increase in energy burning leads to hepatocarcinogenesis. Ligand-activated nuclear receptors recruit the multisubunit Mediator complex for RNA polymerase II-dependent gene transcription. Evidence indicates that the Med1 subunit of the Mediator is essential for PPARα, PPARγ, CAR, and GR signaling in liver. Med1 null hepatocytes fail to respond to PPARα activators in that these cells do not show induction of peroxisome proliferation and increases in fatty acid oxidation enzymes. Med1-deficient hepatocytes show no increase in cell proliferation and do not give rise to liver tumors. Identification of nuclear receptor-specific coactivators and Mediator subunits should further our understanding of the complexities of metabolic diseases associated with increased energy combustion in liver.
Asunto(s)
Carcinogénesis/genética , Metabolismo Energético , Regeneración Hepática , Subunidad 1 del Complejo Mediador/fisiología , Receptores Citoplasmáticos y Nucleares/metabolismo , Animales , Humanos , Subunidad 1 del Complejo Mediador/genéticaRESUMEN
Cholesterol is catabolized to bile acids by peroxisomal ß-oxidation in which the side chain of C27-bile acid intermediates is shortened by three carbon atoms to form mature C24-bile acids. Knockout mouse models deficient in AMACR (α-methylacyl-CoA racemase) or MFE-2 (peroxisomal multifunctional enzyme type 2), in which this ß-oxidation pathway is prevented, display a residual C24-bile acid pool which, although greatly reduced, implies the existence of alternative pathways of bile acid synthesis. One alternative pathway could involve Mfe-1 (peroxisomal multifunctional enzyme type 1) either with or without Amacr. To test this hypothesis, we generated a double knockout mouse model lacking both Amacr and Mfe-1 activities and studied the bile acid profiles in wild-type, Mfe-1 and Amacr single knockout mouse line and Mfe-1 and Amacr double knockout mouse lines. The total bile acid pool was decreased in Mfe-1-/- mice compared with wild-type and the levels of mature C24-bile acids were reduced in the double knockout mice when compared with Amacr-deficient mice. These results indicate that Mfe-1 can contribute to the synthesis of mature bile acids in both Amacr-dependent and Amacr-independent pathways.
Asunto(s)
Ácidos y Sales Biliares/biosíntesis , Complejos Multienzimáticos/fisiología , Racemasas y Epimerasas/fisiología , Animales , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Animales , Complejos Multienzimáticos/deficiencia , Complejos Multienzimáticos/genética , Racemasas y Epimerasas/deficiencia , Racemasas y Epimerasas/genética , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
BACKGROUND: In renal Fanconi's syndrome, dysfunction in proximal tubular cells leads to renal losses of water, electrolytes, and low-molecular-weight nutrients. For most types of isolated Fanconi's syndrome, the genetic cause and underlying defect remain unknown. METHODS: We clinically and genetically characterized members of a five-generation black family with isolated autosomal dominant Fanconi's syndrome. We performed genomewide linkage analysis, gene sequencing, biochemical and cell-biologic investigations of renal proximal tubular cells, studies in knockout mice, and functional evaluations of mitochondria. Urine was studied with the use of proton nuclear magnetic resonance ((1)H-NMR) spectroscopy. RESULTS: We linked the phenotype of this family's Fanconi's syndrome to a single locus on chromosome 3q27, where a heterozygous missense mutation in EHHADH segregated with the disease. The p.E3K mutation created a new mitochondrial targeting motif in the N-terminal portion of EHHADH, an enzyme that is involved in peroxisomal oxidation of fatty acids and is expressed in the proximal tubule. Immunocytofluorescence studies showed mistargeting of the mutant EHHADH to mitochondria. Studies of proximal tubular cells revealed impaired mitochondrial oxidative phosphorylation and defects in the transport of fluids and a glucose analogue across the epithelium. (1)H-NMR spectroscopy showed elevated levels of mitochondrial metabolites in urine from affected family members. Ehhadh knockout mice showed no abnormalities in renal tubular cells, a finding that indicates a dominant negative nature of the mutation rather than haploinsufficiency. CONCLUSIONS: Mistargeting of peroxisomal EHHADH disrupts mitochondrial metabolism and leads to renal Fanconi's syndrome; this indicates a central role of mitochondria in proximal tubular function. The dominant negative effect of the mistargeted protein adds to the spectrum of monogenic mechanisms of Fanconi's syndrome. (Funded by the European Commission Seventh Framework Programme and others.).
Asunto(s)
Síndrome de Fanconi/genética , Túbulos Renales Proximales/metabolismo , Mitocondrias/metabolismo , Mutación Missense , Enzima Bifuncional Peroxisomal/genética , Secuencia de Aminoácidos , Animales , Población Negra , Cromosomas Humanos Par 3 , Modelos Animales de Enfermedad , Síndrome de Fanconi/etnología , Femenino , Ligamiento Genético , Humanos , Masculino , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Linaje , Enzima Bifuncional Peroxisomal/química , Enzima Bifuncional Peroxisomal/metabolismo , Fenotipo , Análisis de Secuencia de ADNRESUMEN
UNLABELLED: Growth arrest and DNA damage-inducible beta (GADD45b) plays an important role in many intracellular events, such as cell cycle arrest, DNA repair, cell survival, apoptosis, and senescence. However, its mechanism of transcriptional regulation remains unclear. In this study the mechanism of peroxisome proliferator-activated receptor α (PPARα) ligand induction of the Gadd45b gene in mouse liver was investigated. Gadd45b messenger RNA (mRNA) was markedly induced by the PPARα agonist Wy-14,643 in wild-type mice but not in Ppara-null mice. Signal transducer and activator of transcription 3 (STAT3) was found to be a repressor of the Gadd45b gene through binding to upstream regulatory elements. The role of STAT3 in control of Gadd45b was confirmed using liver-specific Stat3-null mice. Wy-14,643 treatment stimulated STAT3 ubiquitination leading to activation of the Gadd45b gene as a result of loss of Gadd45b repression by STAT3. STAT3 degradation was induced by forced overexpression of the PPARα target gene-encoded enzyme ACOX1, which produces increased H(2)O(2) as a byproduct of fatty acid ß-oxidation. H(2)O(2) also stimulated expression of Gadd45b in cultured cells. CONCLUSION: PPARα indirectly induces the Gadd45b gene in liver through promoting degradation of the repressor STAT3 as a result of elevated oxidative stress.
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Antígenos de Diferenciación/metabolismo , Hígado/metabolismo , Estrés Oxidativo/fisiología , PPAR alfa/metabolismo , Factor de Transcripción STAT3/metabolismo , Animales , Células Cultivadas , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Hepatocitos/patología , Peróxido de Hidrógeno/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Animales , PPAR alfa/deficiencia , PPAR alfa/genética , Proliferadores de Peroxisomas/farmacología , Pirimidinas/farmacología , ARN Mensajero/metabolismo , Factor de Transcripción STAT3/deficiencia , Factor de Transcripción STAT3/genéticaRESUMEN
Peroxisome proliferator-activated receptor-α (PPARα) modulates the activities of all three interlinked hepatic fatty acid oxidation systems, namely mitochondrial and peroxisomal ß-oxidation and microsomal ω-oxidation pathways. Hyperactivation of PPARα, by both exogenous and endogenous activators up-regulates hepatic fatty acid oxidation resulting in excess energy burning in liver contributing to the development of liver cancer in rodents. Sustained PPARα signaling disproportionately increases H2O2-generating fatty acid metabolizing enzymes as compared to H2O2-degrading enzymes in liver leading to enhanced generation of DNA damaging reactive oxygen species, progressive endoplasmic reticulum stress and inflammation. These alterations also contribute to increased liver cell proliferation with changes in apoptosis. Thus, reactive oxygen species, oxidative stress and hepatocellular proliferation are likely the main contributing factors in the pathogenesis of hepatocarcinogenesis, mediated by sustained PPARα activation-related energy burning in liver. Furthermore, the transcriptional co-activator Med1, a key subunit of the Mediator complex, is essential for PPARα signaling in that both PPARα-null and Med1-null hepatocytes are unresponsive to PPARα activators and fail to give rise to liver tumors when chronically exposed to PPARα activators.
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Metabolismo Energético , Neoplasias Hepáticas/inducido químicamente , PPAR alfa/metabolismo , Proliferadores de Peroxisomas/efectos adversos , Peroxisomas/fisiología , Animales , Proliferación Celular , Ácidos Grasos/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Subunidad 1 del Complejo Mediador/fisiología , Ratones , Ratones Noqueados , MicroARNs/fisiología , Oxidación-Reducción , Estrés OxidativoRESUMEN
PRIP-Interacting protein with methyl transferase domain (PIMT) serves as a molecular bridge between CREB-binding protein (CBP)/ E1A binding protein p300 (Ep300) -anchored histone acetyl transferase and the Mediator complex sub-unit1 (Med1) and modulates nuclear receptor transcription. Here, we report that ERK2 phosphorylates PIMT at Ser(298) and enhances its ability to activate PEPCK promoter. We observed that PIMT is recruited to PEPCK promoter and adenoviral-mediated over-expression of PIMT in rat primary hepatocytes up-regulated expression of gluconeogenic genes including PEPCK. Reporter experiments with phosphomimetic PIMT mutant (PIMT(S298D)) suggested that conformational change may play an important role in PIMT-dependent PEPCK promoter activity. Overexpression of PIMT and Med1 together augmented hepatic glucose output in an additive manner. Importantly, expression of gluconeogenic genes and hepatic glucose output were suppressed in isolated liver specific PIMT knockout mouse hepatocytes. Furthermore, consistent with reporter experiments, PIMT(S298D) but not PIMT(S298A) augmented hepatic glucose output via up-regulating the expression of gluconeogenic genes. Pharmacological blockade of MAPK/ERK pathway using U0126, abolished PIMT/Med1-dependent gluconeogenic program leading to reduced hepatic glucose output. Further, systemic administration of T4 hormone to rats activated ERK1/2 resulting in enhanced PIMT ser(298) phosphorylation. Phosphorylation of PIMT led to its increased binding to the PEPCK promoter, increased PEPCK expression and induction of gluconeogenesis in liver. Thus, ERK2-mediated phosphorylation of PIMT at Ser(298) is essential in hepatic gluconeogenesis, demonstrating an important role of PIMT in the pathogenesis of hyperglycemia.
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Gluconeogénesis/fisiología , Hepatocitos/metabolismo , Hígado/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína D-Aspartato-L-Isoaspartato Metiltransferasa/metabolismo , Animales , Línea Celular , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Gluconeogénesis/efectos de los fármacos , Glucosa/biosíntesis , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Ratones Noqueados , Modelos Biológicos , Fosforilación/efectos de los fármacos , Regiones Promotoras Genéticas , Unión Proteica , Proteína D-Aspartato-L-Isoaspartato Metiltransferasa/genética , Proteínas Serina-Treonina Quinasas/genética , Ratas , Especificidad por Sustrato , Hormonas Tiroideas/farmacología , Transcripción Genética , Activación TranscripcionalRESUMEN
Specific metabolic pathways are activated by different nutrients to adapt the organism to available resources. Although essential, these mechanisms are incompletely defined. Here, we report that medium-chain fatty acids contained in coconut oil, a major source of dietary fat, induce the liver ω-oxidation genes Cyp4a10 and Cyp4a14 to increase the production of dicarboxylic fatty acids. Furthermore, these activate all ω- and ß-oxidation pathways through peroxisome proliferator activated receptor (PPAR) α and PPARγ, an activation loop normally kept under control by dicarboxylic fatty acid degradation by the peroxisomal enzyme L-PBE. Indeed, L-pbe(-/-) mice fed coconut oil overaccumulate dicarboxylic fatty acids, which activate all fatty acid oxidation pathways and lead to liver inflammation, fibrosis, and death. Thus, the correct homeostasis of dicarboxylic fatty acids is a means to regulate the efficient utilization of ingested medium-chain fatty acids, and its deregulation exemplifies the intricate relationship between impaired metabolism and inflammation.
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Ácidos Grasos/metabolismo , Hígado/enzimología , Peroxisomas/metabolismo , Animales , Aceite de Coco , Sistema Enzimático del Citocromo P-450/metabolismo , Familia 4 del Citocromo P450 , Grasas de la Dieta/administración & dosificación , Grasas de la Dieta/farmacocinética , Ácidos Grasos/química , Hígado/metabolismo , Fallo Hepático Agudo/enzimología , Fallo Hepático Agudo/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Oxidación-Reducción , PPAR alfa/metabolismo , PPAR gamma/metabolismo , Aceites de Plantas/administración & dosificación , Aceites de Plantas/farmacocinética , Transducción de SeñalRESUMEN
Mediator, a large multisubunit protein complex, plays a pivotal role in gene transcription by linking gene-specific transcription factors with the preinitiation complex and RNA polymerase II. In the liver, the key subunit of the Mediator complex, Med1, interacts with several nuclear receptors and transcription factors to direct gene-specific transcription. Conditional knock-out of Med1 in the liver showed that hepatocytes lacking Med1 did not regenerate following either partial hepatectomy or treatment with certain nuclear receptor activators and failed to give rise to tumors when challenged with carcinogens. We now report that the adenovirally driven overexpression of Med1 in mouse liver stimulates hepatocyte DNA synthesis with enhanced expression of DNA replication, cell cycle control, and liver-specific genes, indicating that Med1 alone is necessary and sufficient for liver cell proliferation. Importantly, we demonstrate that AMP-activated protein kinase (AMPK), an important cellular energy sensor, interacts with, and directly phosphorylates, Med1 in vitro at serine 656, serine 756, and serine 796. AMPK also phosphorylates Med1 in vivo in mouse liver and in cultured primary hepatocytes and HEK293 and HeLa cells. In addition, we demonstrate that PPARα activators increase AMPK-mediated Med1 phosphorylation in vivo. Inhibition of AMPK by compound C decreased hepatocyte proliferation induced by Med1 and also by the PPARα activators fenofibrate and Wy-14,643. Co-treatment with compound C attenuated PPARα activator-inducible fatty acid ß-oxidation in liver. Our results suggest that Med1 phosphorylation by its association with AMPK regulates liver cell proliferation and fatty acid oxidation, most likely as a downstream effector of PPARα and AMPK.