RESUMEN
Sphingomyelin (SM) is an abundant plasma membrane and plasma lipoprotein sphingolipid. We previously reported that ATP-binding cassette family A protein 1 (ABCA1) deficiency in humans and mice decreases plasma SM levels. However, overexpression, induction, downregulation, inhibition, and knockdown of ABCA1 in human hepatoma Huh7 cells did not decrease SM efflux. Using unbiased siRNA screening, here, we identified that ABCA7 plays a role in the biosynthesis and efflux of SM without affecting cellular uptake and metabolism. Since loss of function mutations in the ABCA7 gene exhibit strong associations with late-onset Alzheimer's disease across racial groups, we also studied the effects of ABCA7 deficiency in the mouse brain. Brains of ABCA7-deficient (KO) mice, compared with WT, had significantly lower levels of several SM species with long chain fatty acids. In addition, we observed that older KO mice exhibited behavioral deficits in cognitive discrimination in the active place avoidance task. Next, we performed synaptic transmission studies in brain slices obtained from older mice. We found anomalies in synaptic plasticity at the intracortical synapse in layer II/III of the lateral entorhinal cortex but not in the hippocampal CA3-CA1 synapses in KO mice. These synaptic abnormalities in KO brain slices were rescued with extracellular SM supplementation but not by supplementation with phosphatidylcholine. Taken together, these studies identify a role of ABCA7 in brain SM metabolism and the importance of SM in synaptic plasticity and cognition, as well as provide a possible explanation for the association between ABCA7 and late-onset Alzheimer's disease.
Asunto(s)
Enfermedad de Alzheimer , Cognición , Corteza Entorrinal , Plasticidad Neuronal , Esfingomielinas , Animales , Humanos , Ratones , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Corteza Entorrinal/metabolismo , Esfingomielinas/biosíntesis , Ratones NoqueadosRESUMEN
Sphingosine 1-phosphate (S1P) is a well-defined bioactive lipid molecule derived from membrane sphingolipid metabolism. In the past decades, a series of key enzymes involved in generation of S1P have been identified and characterized in detail, as well as enzymes degrading S1P. S1P requires transporter to cross the plasma membrane and carrier to deliver to its cognate receptors and therefore transduces signaling in autocrine, paracrine, or endocrine fashions. The essential roles in regulation of development, metabolism, inflammation, and many other aspects of life are mainly executed when S1P binds to receptors provoking the downstream signaling cascades in distinct cells. This chapter will review the synthesis, degradation, transportation, and signaling of S1P and try to provide a comprehensive view of the biology of S1P, evoking new enthusiasms and ideas into the field of the fascinating S1P.
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Lisofosfolípidos , Esfingosina , Lisofosfolípidos/metabolismo , Transducción de Señal/fisiología , Esfingosina/análogos & derivados , Esfingosina/metabolismoRESUMEN
Angiogenesis has a great impact on human health, owing to its participation in development, wound healing and the pathogenesis of several diseases. It has been reported that let-7a is a tumour suppressor, but whether it plays a role in angiogenesis is unclear. Here we showed that let-7a, a microRNA conserved in vertebrates, regulated angiogenesis by concomitantly down-regulating TGFBR3. Overexpression of let-7a or knockdown of TGFBR3 in cell culture inhibited the tube formation and reduced migration rate. Moreover, xenograft experiments showed that overexpression of let-7a or knockdown of TGFBR3 had smaller tumour size. Downstream genes, such as VEGFC and MMP9, were also down-regulated in let-7a overexpression or TGFBR3 knockdown groups. Therefore, our results revealed a novel mechanism that let-7a regulate angiogenesis through post-transcriptional regulation of TGFBR3.
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MicroARNs/genética , Neovascularización Patológica/genética , Proteoglicanos/genética , ARN Mensajero/genética , Receptores de Factores de Crecimiento Transformadores beta/genética , Animales , Línea Celular , Regulación hacia Abajo/genética , Regulación Neoplásica de la Expresión Génica/genética , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana , Humanos , Ratones , Ratones Desnudos , Neovascularización Patológica/patología , Procesamiento Postranscripcional del ARN/genéticaRESUMEN
The RNA-binding protein Elavl1 (also known as HuR) regulates gene expression at the posttranscriptional level. Early embryonic lethality of the mouse knockout challenges investigation into hematopoietic functions for Elavl1. We identified 2 zebrafish elavl1 genes, designated elavl1a (the predominant isoform during embryogenesis) and elavl1b. Knockdown of Elavl1a using specific morpholinos resulted in a striking loss of primitive embryonic erythropoiesis. Transcript levels for early hematopoietic regulatory genes including lmo2 and scl are unaltered, but levels of gata1 transcripts, encoding a key erythroid transcription factor, are significantly reduced in elavl1a morphants. Other mesoderm markers are mostly unchanged by depletion of Elav1a. The 3'-untranslated region (UTR) of gata1 contains putative Elavl1a-binding sites that support robust expression levels when fused to a transfected luciferase reporter gene, and Elavl1a binds the gata1 3'-UTR sequences in a manner dependent on these sites. Moreover, expression of a transgenic reporter specifically in developing embryonic erythroid cells is enhanced by addition of the gata1 3'UTR with intact Elavl1-binding sites. Injection of gata1 messenger RNA partially rescues the erythropoiesis defect caused by Elavl1 knockdown. Our study reveals a posttranscriptional regulatory mechanism by which RNA-binding protein Elavl1a regulates embryonic erythropoiesis by maintaining appropriate levels of gata1 expression.
Asunto(s)
Proteínas ELAV/fisiología , Eritropoyesis/genética , Factor de Transcripción GATA1/genética , Procesamiento Postranscripcional del ARN/genética , Proteínas de Pez Cebra/genética , Pez Cebra/embriología , Pez Cebra/genética , Regiones no Traducidas 3'/genética , Animales , Animales Modificados Genéticamente , Sitios de Unión/genética , Línea Celular Tumoral , Embrión no Mamífero , Factor de Transcripción GATA1/metabolismo , Regulación del Desarrollo de la Expresión Génica , Células HEK293 , Humanos , Ratones , Proteínas de Pez Cebra/metabolismoRESUMEN
BACKGROUND: Sepsis is characterized by an inappropriate systemic inflammatory response and bacteremia that promote multiorgan failure and mortality. Sphingosine 1-phosphate receptor 2 (S1PR2) modulates endotoxin-induced inflammation in endothelium. However, as a highly expressed S1P receptor in macrophages, its role in regulating macrophage response to bacterial infection remains unclear. METHODS: Cecal ligation and puncture or intratracheal instillation of Escherichia coli was induced in wild-type or S1pr2-deficient mice. The antibacterial ability of cell-specific S1PR2 was tested in bone marrow reconstitution mice or mice with macrophage-specific deletion. Signaling molecules responsible for S1PR2-mediated phagocytosis were also measured in the bone marrow-derived macrophages. In addition, S1PR2 expression levels and its correlation with severity of sepsis were determined in critically ill patients (n = 25). RESULTS: Both genetic deletion and pharmaceutical inhibition of S1PR2 significantly limited bacterial burden, reduced lung damage, and improved survival (genetic deletion, 0% in S1pr2 vs. 78.6% in S1pr2, P < 0.001; pharmaceutical inhibition, 9.1% in vehicle vs. 22.2% in S1PR2 antagonist, P < 0.05). This protection was attributed to the enhanced phagocytic function of S1PR2-deficient macrophages (mean fluorescent intensity, 2035.2 ± 202.1 vs. 407.8 ± 71.6, P < 0.001). Absence of S1PR2 in macrophage inhibits RhoA-dependent cell contraction and promotes IQGAP1-Rac1-dependent lamellipodial protrusion, whose signaling pathways depend on extracellular stimulators. In septic patients, increased S1PR2 levels in peripheral blood mononuclear cells were positively correlated with the severity of sepsis (r = 0.845, P < 0.001). CONCLUSIONS: This study implies that S1PR2, as a critical receptor in macrophage, impairs phagocytosis and antimicrobial defense in the pathogenesis of sepsis. Interventions targeting S1PR2 signaling may serve as promising therapeutic approaches for sepsis.
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Especificidad del Huésped/fisiología , Macrófagos/metabolismo , Fagocitosis/fisiología , Receptores de Lisoesfingolípidos/deficiencia , Sepsis/metabolismo , Transducción de Señal/fisiología , Animales , Células Cultivadas , Femenino , Macrófagos/inmunología , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Sepsis/inmunología , Receptores de Esfingosina-1-FosfatoRESUMEN
Regulation of microsomal triglyceride transfer protein (MTP) expression mainly occurs at the transcriptional level. We have previously shown that MTTP gene expression was repressed in nondifferentiated intestinal cells by nuclear receptor 2 family 1 (NR2F1). However, mechanisms involved in the repression of MTP by NR2F1 were not elucidated. Here, we show that MTP expression requires hepatic nuclear factor (HNF)-4α transcription factor. Different HNF-1 proteins synergistically enhance MTP promoter activity along with HNF-4α by binding to different cis elements. NR2F1 does not alter individual effects of HNF-4α and HNF-1 proteins on the MTTP gene promoter. However, NR2F1 suppresses synergistic activation of the MTP promoter by HNF-4α/HNF-1α by binding to a direct repeat 1 (DR1) element. This suppression is further enhanced in the presence of nuclear receptor corepressor 1. In short, these studies identified a novel mechanism of MTP repression that involves binding of NR2F1 to the DR1 element and recruitment of corepressors. In this mechanism, NR2F1 does not affect activities of individual transcription factors; instead, it abrogates synergistic activation by HNF-4α and HNF-1 proteins.
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Factor de Transcripción COUP I/metabolismo , Proteínas Portadoras/genética , Factor Nuclear 1-alfa del Hepatocito/metabolismo , Factor Nuclear 4 del Hepatocito/metabolismo , Activación Transcripcional/genética , Secuencia de Bases , Células CACO-2 , Células HEK293 , Humanos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , Secuencias Repetidas en Tándem/genéticaRESUMEN
Background: The sphingosine 1-phosphate (S1P)/S1P receptor (S1pr) 1 signaling plays an essential role in regulating vascular integrity and angiogenesis. We have previously shown that cell-surface expression of endoglin (Eng) is sustained by S1P/S1pr1 signaling in endothelial cells (ECs). However, whether S1pr1 mediates Eng signaling, or vice versa, remains unknown. Methods: S1pr1 inhibitors were used to study whether pharmacological inhibition induces basal vascular leakage in vivo. An acute respiratory distress syndrome (ARDS) model was used to study whether S1pr1 inhibition evoked greater inflammation in lungs. A S1pr1 inhibitor, a bone morphogenetic protein 9 (BMP9) blocking antibody, or lentivirus-mediated expression of soluble extracellular domain of Eng (sEng) were used to test whether blocking both S1P/S1pr1 and BMP9/Eng signaling axes would impose any interaction in retinal angiogenesis. To clarify whether S1P and BMP9 function in a linear pathway, a study of trans-endothelial electrical resistance (TEER) measurement was carried out using a mouse islet EC line MS1; time course studies were executed to exam downstream effectors of S1P and BMP9 signaling pathways in ECs; two stable MS1 cell lines were generated, one with overexpression of human S1PR1 and the other with knockdown of Eng, to validate S1pr1 and Eng were the key players for the crosstalk. Inhibitor of extracellular regulated protein kinases (ERK) was used to check whether this signaling was involved in S1P-induced cell-surface localization of Eng. Results: The present study elucidated that S1pr1 and Eng are both pivotal for angiogenesis in the postnatal mouse retina, and that the activation of S1pr1 or Eng increases vascular barrier function. Activation of S1pr1 enhanced the phosphorylation of Smad family members 1, 5, and 8 (pSmad1/5/8), while the inhibition of S1pr1 reduced the levels of pSma1/5/8 induced by BMP9 treatment. Activation or loss of Eng did not affect S1pr1 signaling. Moreover, activation of ERK was involved in promoting EC-surface expression of Eng by S1pr1. Conclusions: Our data demonstrates for the first time that there exists a linear pathway of S1pr1-Eng signaling axis in ECs, which governs vascular homeostasis. Functional BMP9/Eng signaling requires S1P/S1pr1 activation, and S1pr1 signaling acts as a vascular protection mechanism upstream of Eng.
RESUMEN
OBJECTIVE: Our aim was to elucidate mechanisms involved in the acquisition of lipid transport properties during enterocyte differentiation. METHODS AND RESULTS: We show that lipid mobilization via apolipoprotein B lipoproteins is dependent on the expression of microsomal triglyceride transfer protein (MTP) during differentiation of Caco-2 cells into enterocyte-like cells. Mechanistic studies showed that binding of the nuclear receptor family 2 group F member 1 (NR2F1) to the DR1 element in the MTTP promoter suppresses MTTP expression in undifferentiated cells. During cellular differentiation, NR2F1 expression and its binding to MTTP promoter decline and MTP induction ensues. Moreover, undifferentiated cells express inositol-requiring enzyme 1beta (IRE1beta), a protein that posttranscriptionally degrades MTP mRNA, and its expression substantially decreases during differentiation, contributing to MTP induction. Immunohistochemical studies revealed a significant negative relationship between the expressions of MTP and NR2F1/IRE1beta in undifferentiated and differentiated Caco-2 cells, as well as in crypt-villus and jejunum-colon axes of mouse intestine. CONCLUSIONS: We propose that transcriptional and posttranscriptional mechanisms involving NR2F1 and IRE1beta ensure low MTP expression in undifferentiated intestinal cells and avoid apolipoprotein B lipoprotein biosynthesis.
Asunto(s)
Factor de Transcripción COUP I/metabolismo , Proteínas Portadoras/metabolismo , Diferenciación Celular , Endorribonucleasas/metabolismo , Mucosa Intestinal/metabolismo , Lipoproteínas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Apolipoproteínas B/metabolismo , Secuencia de Bases , Células CACO-2 , Colon/citología , Colon/metabolismo , Enterocitos/citología , Enterocitos/metabolismo , Factor Nuclear 1 del Hepatocito/genética , Factor Nuclear 1 del Hepatocito/metabolismo , Factor Nuclear 4 del Hepatocito/genética , Factor Nuclear 4 del Hepatocito/metabolismo , Humanos , Yeyuno/citología , Yeyuno/metabolismo , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Datos de Secuencia MolecularRESUMEN
Hepatic apolipoprotein B (apoB) lipoprotein production is metabolically regulated via the phosphoinositide 3-kinase cascade; however, the role of the key negative regulator of this pathway, the tumor suppressor phosphatase with tensin homology (PTEN), is unknown. Here, we demonstrate that hepatic protein levels of apoB100 and microsomal triglyceride transfer protein (MTP) are significantly down-regulated (73% and 36%, respectively) in the liver of PTEN liver-specific knockout (KO) mice, and this is accompanied by increased triglyceride (TG) accumulation and lipogenic gene expression, and reduced hepatic apoB secretion in freshly isolated hepatocytes. MTP protein mass and lipid transfer activity were also significantly reduced in liver of PTEN KO mice. Overexpression of the dominant negative mutant PTEN C/S124 (adenovirus expressing PTEN C/S mutant [AdPTENC/S]) possessing constitutive phospoinositide 3-kinase activity in HepG2 cells led to significant reductions in both secreted apoB100 and cellular MTP mass (76% and 34%, respectively), and increased messenger RNA (mRNA) levels of sterol regulatory element binding protein 1c (SREBP-1c), fatty acid synthase (FAS), and acetyl-CoA carboxylase (ACC). Reduced apoB100 secretion induced by AdPTENC/S was associated with increased degradation of newly-synthesized cellular apoB100, in a lactacystin-sensitive manner, suggesting enhanced proteasomal degradation. AdPTENC/S also reduced apoB-lipoprotein production in McA-RH7777 and primary hamster hepatocytes. Our findings suggest a link between PTEN expression and hepatic production of apoB-containing lipoproteins. We postulate that perturbations in PTEN not only may influence hepatic insulin signaling and hepatic lipogenesis, but also may alter hepatic apoB-lipoprotein production and the MTP stability. On loss of PTEN activity, increased lipid substrate availability in the face of reduced hepatic lipoprotein production capacity can rapidly lead to hepatosteatosis and fatty liver.
Asunto(s)
Apolipoproteínas B/metabolismo , Proteínas Portadoras/metabolismo , Hígado Graso/metabolismo , Lipogénesis/fisiología , Fosfohidrolasa PTEN/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Acetil-CoA Carboxilasa/metabolismo , Animales , Apolipoproteína B-100/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Ácido Graso Sintasas/metabolismo , Hígado Graso/patología , Insulina/metabolismo , Hígado/metabolismo , Hígado/patología , Ratones , Ratones Noqueados , Fosfatidilinositol 3-Quinasas/metabolismo , Receptores de LDL/metabolismo , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Triglicéridos/metabolismoRESUMEN
Elavl1 (also known as HuR), an RNA binding protein highly conserved between zebrafish and human, regulates gene expression by stabilizing target mRNA. Our previous studies have uncovered that the predominant isoform elavl1a is required for zebrafish embryonic erythropoiesis. However, the exact mechanism of how elav11 spatiotemporally stabilizes target mRNAs to regulate specific erythropoiesis is not yet understood. Here we show that phosphorylation of elavl1a at Ser219 and Ser316 by PKC is necessarily required for cytosolic shuttling from the nucleus to stabilize gata1 mRNA and thus promotes erythropoiesis. Knockdown of elavl1a resulted in the hindrance of erythropoiesis and Hemin-induced erythroid differentiation of human myeloid leukemia K562 cells. Interestingly, inhibition of PKC reproduced the phenotype seen during zebrafish embryogenesis and erythroid differentiation of myeloid leukemia. Mechanistically, Hemin induced elavl1a export from nuclear to cytoplasmic space in K562 cells in a manner dependent on phosphorylation on Ser219 and Ser316, as overexpression of elavl1a with mutations on Ser219 and Ser316 resulted in erythropoiesis failure. Additionally, co-administration of low doses of elavl1a morpholino (MO) and three PKC inhibitors showed a combined effect in zebrafish embryonic erythropoiesis dysplasia. In conclusion, our study reveals that PKC-mediated phosphorylation of elavl1a at Ser219 and Ser316 sites controls its nucleo-cytoplasmic translocation in zebrafish, thereby regulating embryonic erythropoiesis.
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Proteína 1 Similar a ELAV/metabolismo , Eritropoyesis/genética , Eritropoyesis/fisiología , Animales , Diferenciación Celular , Línea Celular Tumoral , Proteína 1 Similar a ELAV/fisiología , Factor de Transcripción GATA1/genética , Factor de Transcripción GATA1/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Hemina/farmacología , Humanos , Células K562 , Fosforilación , Proteína Quinasa C/metabolismo , Proteína Quinasa C/fisiología , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
The endothelial cell (EC) barrier disruption has been implicated in vascular leakage and pulmonary edema. Many reports have shown that the EC barrier dysfunction is regulated by the sphingosine-1-phophate (S1P)/S1P receptor-1 (S1PR1) axis. Identifying downstream effectors for the S1P/S1PR1 axis in pulmonary vasculature has been limited by mixed populations in vitro cultures that do not retain physiological EC phenotype and complex of tedious proteomics. In this study, we used a combination of in vivo biotinylation and liquid chromatograph tandem mass spectrometry on three mouse models of S1pr1 expression, namely normal, knockout (KO) and high, to identify EC membrane proteins whose cell-surface expression is S1pr1-dependent. EC-specific KO of S1pr1 caused severe pulmonary vascular disruption and reduction of many membrane proteins on ECs. Using the MaxQuant software we were able to identify novel membrane targets of S1pr1, for instance, Cd105 and Plvap, by comparison with their membrane expressions among the three EC model systems. Moreover, regulation of Cd105 and Plvap by S1pr1 were validated with Western blot and immunostaining in vivo and in vitro. Our data suggest that S1pr1 dictates cell-surface localization of several apical membrane proteins in ECs. Our results are insightful for development of novel therapeutics to specifically target EC barrier function.
Asunto(s)
Membrana Celular/metabolismo , Endoglina/metabolismo , Células Endoteliales/metabolismo , Regulación de la Expresión Génica , Modelos Biológicos , Receptores de Esfingosina-1-Fosfato/metabolismo , Animales , Línea Celular , Membrana Celular/genética , Endoglina/genética , Células Endoteliales/citología , Ratones , Ratones Noqueados , Receptores de Esfingosina-1-Fosfato/genéticaRESUMEN
Reserpine is widely used for treatment of hypertension and schizophrenia. As a specific inhibitor of monoamine transporters, reserpine is known to deplete monoamine neurotransmitters and cause decreased movement symptoms. However, how zebrafish larvae respond to reserpine treatment is not well studied. Here we show that swimming distance and average velocity are significantly reduced after reserpine exposure under various stimulatory conditions. Using liquid chromatograph-mass spectrometer analysis, decreased levels of monoamines (e.g. dopamine, noradrenaline, and serotonin) were detected in reserpine-treated larvae. Moreover, reserpine treatment significantly reduced the number of dopaminergic neurons, which was identified with th (Tyrosine Hydroxylase) in situ hybridization in the preoptic area. Interestingly, dopaminergic neuron development-associated genes, such as otpa, otpb, wnt1, wnt3, wnt5 and manf, were downregulated in reserpine treated larvae. Our data indicates that 2â¯mg/L reserpine exposure induces dopaminergic neuron damage in the brain, demonstrating a chemical induced depression-like model in zebrafish larvae for future drug development.
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Neuronas Dopaminérgicas/efectos de los fármacos , Larva/efectos de los fármacos , Síndromes de Neurotoxicidad/etiología , Reserpina/toxicidad , Pez Cebra , Animales , Monoaminas Biogénicas/metabolismo , Neuronas Dopaminérgicas/patología , Embrión no Mamífero/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Larva/metabolismo , Luz , Locomoción/efectos de los fármacos , Factores de Crecimiento Nervioso/genética , Sonido , Proteína Wnt-5a/genética , Proteína Wnt1/genética , Proteína Wnt3A/genética , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
Using MDCK cells as a model system, evidence is presented demonstrating that the signaling pathways mammalian target of rapamycin (mTOR) and phosphoinositide 3-kinase (PI 3-kinase) play important roles in the regulation of epithelial tubule formation. Incubation of cells with collagen gel overlays induced early (4-8 h) reorganization of cells (epithelial remodeling) into three-dimensional multicellular tubular structures over 24 h. An MDCK cell line stably expressing the PH domain of Akt, a PI 3-kinase downstream effector, coupled to green fluorescent protein (GFP-Akt-PH) was used to determine the distribution of phosphatidyl inositol-3,4,5-P(3) (PIP(3)), a product of PI 3-kinase. GFP-Akt-PH was associated with lateral membranes in control cells. After incubation with collagen gel overlays, GFP-Akt-PH redistributed into the lamellipodia of migrating cells suggesting that PIP(3) plays a role in epithelial remodeling. Using the small molecule inhibitor LY-294002 that inhibits both mTOR and PI 3-kinase, we demonstrated that kinase activity was required for epithelial remodeling, disruption of cell junctions and subsequent modulation of tubule formation. Since the mTOR signaling pathway is downstream of PI 3-kinase, the effects of rapamycin, a specific mTOR inhibitor, on tubule formation were assessed. Rapamycin did not affect epithelial remodeling or GFP-Akt-PH redistribution but inhibited elongated tubule formation that occurred later (24 h) in morphogenesis. These results were further supported by using RNA interference to down-regulate mTOR and inhibit tubule formation. Our studies demonstrate that PI 3-kinase regulates early epithelial remodeling stages while mTOR modulates latter stages of tubule development.
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Epitelio , Morfogénesis , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Quinasas/metabolismo , Transducción de Señal/fisiología , Animales , Biomarcadores/metabolismo , Línea Celular , Cromonas/metabolismo , Perros , Inhibidores Enzimáticos/metabolismo , Epitelio/anatomía & histología , Epitelio/embriología , Uniones Intercelulares/metabolismo , Morfolinas/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Proteínas Quinasas/genética , Interferencia de ARN , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Sirolimus/metabolismo , Serina-Treonina Quinasas TORRESUMEN
AIMS: To investigate whether there exists a cardio-protective effect of Fasudil, a selective Rho kinase (ROCK) inhibitor, in an experimental murine model of acute viral myocarditis. METHODS: Male BALB/c mice were randomly assigned to three groups: control, myocarditis treated with placebo and myocarditis treated with Fasudil (n = 40 animals per group). Myocarditis was established by intraperitoneal injection with coxsackievirus B3 (CVB3). Twenty-four hours after infection, Fasudil was intraperitoneally administered for 14 consecutive days. Twenty mice were randomly selected from each group to monitor a 14-day survival rate. On day 7 and day 14, eight surviving mice from each group were sacrificed and their hearts and blood were obtained to perform serological and histological examinations. Expression of ROCKs, IL-17, IL-1b, TNFα, RORgt, and Foxp3 were quantified with RT-PCR. Plasma levels of TNF alpha, IL-1 beta, and IL-17 were measured by ELISA. In addition, protein levels of IL-17 and ROCK2 in cardiac tissues were analyzed with Western blot. RESULTS: Fasudil treatment significantly increased survival, attenuated myocardial necrotic lesions, reduced CVB3 replication and expression of ROCK2 and IL-17 in the infected hearts. This treatment also imposed a T-cell subpopulation shift, from Th17 to Treg, in cardiac tissues. CONCLUSIONS: ROCK pathway inhibition was cardio-protective in viral myocarditis with increased survival, decreased viral replication, and inflammatory response. These findings suggest that Fasudil might be a potential therapeutic agent for patients with viral myocarditis.
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1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , Antiinflamatorios/farmacología , Antivirales/farmacología , Infecciones por Coxsackievirus/prevención & control , Enterovirus Humano B/efectos de los fármacos , Miocarditis/prevención & control , Miocardio/enzimología , Inhibidores de Proteínas Quinasas/farmacología , Quinasas Asociadas a rho/antagonistas & inhibidores , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Animales , Infecciones por Coxsackievirus/enzimología , Infecciones por Coxsackievirus/patología , Infecciones por Coxsackievirus/virología , Citocinas/sangre , Modelos Animales de Enfermedad , Enterovirus Humano B/crecimiento & desarrollo , Mediadores de Inflamación/sangre , Masculino , Ratones Endogámicos BALB C , Miocarditis/enzimología , Miocarditis/patología , Miocarditis/virología , Miocardio/patología , Necrosis , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/metabolismo , Linfocitos T Reguladores/virología , Células TH1/efectos de los fármacos , Células TH1/metabolismo , Células TH1/virología , Carga Viral , Replicación Viral/efectos de los fármacos , Quinasas Asociadas a rho/metabolismoRESUMEN
BACKGROUND: In multiple myeloma (MM), increased neoangiogenesis contributes to tumor growth and disease progression. Increased levels of endothelial progenitor cells (EPCs) contribute to neoangiogenesis in MM, and, importantly, covary with disease activity and response to treatment. In order to understand the mechanisms responsible for increased EPC levels and neoangiogenic function in MM, we investigated whether these cells were clonal by determining X-chromosome inactivation (XCI) patterns in female patients by a human androgen receptor assay (HUMARA). In addition, EPCs and bone marrow cells were studied for the presence of clonotypic immunoglobulin heavy-chain (IGH) gene rearrangement, which indicates clonality in B cells; thus, its presence in EPCs would indicate a close genetic link between tumor cells in MM and endothelial cells that provide tumor neovascularization. METHODS: A total of twenty-three consecutive patients who had not received chemotherapy were studied. Screening in 18 patients found that 11 displayed allelic AR in peripheral blood mononuclear cells, and these patients were further studied for XCI patterns in EPCs and hair root cells by HUMARA. In 2 patients whose EPCs were clonal by HUMARA, and in an additional 5 new patients, EPCs were studied for IGH gene rearrangement using PCR with family-specific primers for IGH variable genes (VH). RESULTS: In 11 patients, analysis of EPCs by HUMARA revealed significant skewing (> or = 77% expression of a single allele) in 64% (n = 7). In 4 of these patients, XCI skewing was extreme (> or = 90% expression of a single allele). In contrast, XCI in hair root cells was random. Furthermore, PCR amplification with VH primers resulted in amplification of the same product in EPCs and bone marrow cells in 71% (n = 5) of 7 patients, while no IGH rearrangement was found in EPCs from healthy controls. In addition, in patients with XCI skewing in EPCs, advanced age was associated with poorer clinical status, unlike patients whose EPCs had random XCI. CONCLUSION: Our results suggest that EPCs in at least a substantial subpopulation of MM patients are related to the neoplastic clone and that this is an important mechanism for upregulation of tumor neovascularization in MM.
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Células Endoteliales/fisiología , Mieloma Múltiple/genética , Mieloma Múltiple/fisiopatología , Células Madre Multipotentes/fisiología , Neovascularización Patológica/fisiopatología , Factores de Edad , Células Clonales , Femenino , Reordenamiento Génico , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Pronóstico , Receptores Androgénicos/análisis , Inactivación del Cromosoma XRESUMEN
Ischemia/reperfusion (I/R)-induced autophagy increases the severity of cardiomyocyte injury. The aim of this study was to investigate the effects of berberine, a natural extract from Rhizoma coptidis, on the I/R-induced excessive autophagy in in vitro and in vivo models. Autophagy was increased both in H9c2 myocytes during hypoxia/reoxygenation (H/R) injury and in mouse hearts exposed to I/R. And the expression level of p-AMPK and p-mTORC2 (Ser2481) were increased during H/R period. In addition, the increased autophagy level was correlated with reduced cell survival in H9c2 myocytes and increased infarct size in mouse hearts. However, berberine treatment significantly enhanced the H/R-induced cell viability and reduced I/R-induced myocardial infarct size, which was accompanied by improved cardiac function. The beneficial effect of berberine is associated with inhibiting the cellular autophagy level, due to decreasing the expression level of autophagy-related proteins such as SIRT1, BNIP3, and Beclin-1. Furthermore, both the level of p-AMPK and p-mTORC2 (Ser2481) in H9c2 myocytes exposed to H/R were decreased by berberine. In summary, berberine protects myocytes during I/R injury through suppressing autophagy activation. Therefore, berberine may be a promising agent for treating I/R-induced cardiac myocyte injury.
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Autofagia/efectos de los fármacos , Berberina/farmacología , Citoprotección/efectos de los fármacos , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Daño por Reperfusión Miocárdica/patología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/patología , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Apoptosis/efectos de los fármacos , Berberina/uso terapéutico , Línea Celular , Masculino , Diana Mecanicista del Complejo 2 de la Rapamicina , Ratones , Ratones Endogámicos C57BL , Complejos Multiproteicos/metabolismo , Daño por Reperfusión Miocárdica/metabolismo , Daño por Reperfusión Miocárdica/fisiopatología , Miocitos Cardíacos/metabolismo , Fosforilación/efectos de los fármacos , Recuperación de la Función/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Microsomal triglyceride transfer protein (MTP) is a heterodimeric protein that transfers neutral lipids between membranes in vitro. Absence of this lipid transfer activity in the microsomes of abetalipoproteinemia patients established its pivotal function in lipoprotein assembly. Recent studies indicate that the lipid transfer activity is involved in importing triglycerides into the lumen of the endoplasmic reticulum. In addition to its lipid transfer activity, MTP physically interacts with apoB. This led to speculation that MTP may act as a chaperone. It remains to be determined whether the binding of MTP to apoB plays a role in either proper folding or net lipidation of nascent apoB. Both functions, lipid transfer and apoB binding, may be involved in the initial step of lipidation of nascent apoB resulting in the synthesis of primordial lipoprotein particles. Furthermore, it has been shown that MTP stably associates with lipid vesicles. The lipid-associated MTP may be important in core expansion of primordial lipoproteins. In summary, three independent functions (lipid transfer, apoB binding and membrane association) of MTP have been identified. Here, we propose these functions are carried out by a combination of different structural motifs. Based on sequence homology with lipovitellin, the M subunit of MTP is predicted to contain three beta-sheets (A, C, and N) and one alpha-helical domain. The A- and C-sheets may be involved in lipid transfer, the N-sheet and the helical domain in apoB binding, and the N- and A-sheets in membrane association. It is also speculated that MTP may function in physiologic processes beyond lipoprotein assembly.
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Proteínas Portadoras/fisiología , Animales , Proteínas Portadoras/química , Humanos , Conformación ProteicaRESUMEN
To understand how the Bcl6 transcriptional repressor functions in the immune system, we disrupted its RD2 repression domain in mice. Bcl6RD2(MUT) mice exhibit a complete loss of germinal center (GC) formation but retain normal extrafollicular responses. Bcl6RD2(MUT) antigen-engaged B cells migrate to the interfollicular zone and interact with cognate T helper cells. However, these cells fail to complete early GC-commitment differentiation and coalesce as nascent GC aggregates. Bcl6 directly binds and represses trafficking receptors S1pr1 and Gpr183 by recruiting Hdac2 through the RD2 domain. Deregulation of these genes impairs B cell migration and may contribute to GC failure in Bcl6RD2(MUT) mice. The development of functional GC-TFH cells was partially impaired in Bcl6RD2(MUT) mice. In contrast to Bcl6(-/-) mice, Bcl6RD2(MUT) animals experience no inflammatory disease or macrophage deregulation. These results reveal an essential role for RD2 repression in early GC commitment and striking biochemical specificity in Bcl6 control of humoral and innate immune-cell phenotypes.
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Linfocitos B/metabolismo , Proteínas de Unión al ADN/metabolismo , Centro Germinal/citología , Animales , Linfocitos B/inmunología , Linfocitos B/fisiología , Movimiento Celular , Citocinas/genética , Citocinas/metabolismo , Proteínas de Unión al ADN/química , Centro Germinal/inmunología , Histona Desacetilasa 2/metabolismo , Activación de Linfocitos , Ratones , Mutación , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-bcl-6 , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Lisoesfingolípidos/metabolismo , Receptores de Esfingosina-1-Fosfato , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Colaboradores-Inductores/metabolismoRESUMEN
PURPOSE OF REVIEW: This review summarizes recent advances about the role of microsomal triglyceride transfer protein in plasma and tissue lipid homeostasis. RECENT FINDINGS: Microsomal triglyceride transfer protein emerged as a phospholipid transfer protein and acquired triacylglycerol transfer activity during evolution from invertebrates to vertebrates. These activities are proposed to participate in 'nucleation' and 'desorption' steps during the biosynthesis of primordial apoB-containing lipoproteins. Microsomal triglyceride transfer protein also transfers phospholipids to the glycolipid antigen presentation molecule CD1d. Under physiologic conditions, plasma apoB-containing lipoproteins and microsomal triglyceride transfer protein expression exhibit diurnal variations synchronized by food and light. Microsomal triglyceride transfer protein is regulated at the transcriptional level. HNF4alpha is critical for its transcription. Other transcription factors along with coactivators and corepressors modulate microsomal triglyceride transfer protein expression. Reductions in microsomal triglyceride transfer protein mRNA and activity are related to steatosis in HCV-3 infected patients. CCl4 induces steatosis by enhancing proteasomal degradation of microsomal triglyceride transfer protein and can be partially avoided by inhibiting this degradation. Chemical antagonists cause hepatosteatosis, but this was not seen in the absence of fatty acid binding protein. SUMMARY: Microsomal triglyceride transfer protein is a target to lower plasma lipids and to reduce inflammation in certain immune disorders. More knowledge is required, however, regarding its regulation and its role in the biosynthesis of apoB-containing lipoproteins and CD1d.