RESUMEN
BACKGROUND: Exosomes and other extracellular vesicles (EVs) have emerged as an important mechanism of cell-to-cell communication. However, previous studies either did not fully resolve what genetic materials were shuttled by exosomes or only focused on a specific set of miRNAs and mRNAs. A more systematic method is required to identify the genetic materials that are potentially transferred during cell-to-cell communication through EVs in an unbiased manner. RESULTS: In this work, we present a novel next generation of sequencing (NGS) based approach to identify EV mediated mRNA exchanges between co-cultured adipocyte and macrophage cells. We performed molecular and genomic profiling and jointly considered data from RNA sequencing (RNA-seq) and genotyping to track the "sequence varying mRNAs" transferred between cells. We identified 8 mRNAs being transferred from macrophages to adipocytes and 21 mRNAs being transferred in the opposite direction. These mRNAs represented biological functions including extracellular matrix, cell adhesion, glycoprotein, and signal peptides. CONCLUSIONS: Our study sheds new light on EV mediated RNA communications between adipocyte and macrophage cells, which may play a significant role in developing insulin resistance in diabetic patients. This work establishes a new method that is applicable to examining genetic material exchanges in many cellular systems and has the potential to be extended to in vivo studies as well.
Asunto(s)
Comunicación Celular , Vesículas Extracelulares/metabolismo , ARN Mensajero/metabolismo , Adipocitos/metabolismo , Línea Celular , Técnicas de Cocultivo , Expresión Génica , Técnicas de Genotipaje , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Macrófagos/metabolismo , Transporte de ARN , Análisis de Secuencia de ARNRESUMEN
Adipose tissue lipogenesis is paradoxically impaired in human obesity, promoting ectopic triglyceride (TG) deposition, lipotoxicity, and insulin resistance. We previously identified mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4), a sterile 20 protein kinase reported to be upstream of c-Jun NH2-terminal kinase (JNK) signaling, as a novel negative regulator of insulin-stimulated glucose transport in adipocytes. Using full-genome microarray analysis we uncovered a novel role for Map4k4 as a suppressor of lipid synthesis. We further report here the surprising finding that Map4k4 suppresses adipocyte lipogenesis independently of JNK. Thus, while Map4k4 silencing in adipocytes enhances the expression of lipogenic enzymes, concomitant with increased conversion of (14)C-glucose and (14)C-acetate into TGs and fatty acids, JNK1 and JNK2 depletion causes the opposite effects. Furthermore, high expression of Map4k4 fails to activate endogenous JNK, while Map4k4 depletion does not attenuate JNK activation by tumor necrosis factor α. Map4k4 silencing in cultured adipocytes elevates both the total protein expression and cleavage of sterol-regulated element binding protein-1 (Srebp-1) in a rapamycin-sensitive manner, consistent with Map4k4 signaling via mechanistic target of rapamycin complex 1 (mTORC1). We show Map4k4 depletion requires Srebp-1 upregulation to increase lipogenesis and further show that Map4k4 promotes AMP-protein kinase (AMPK) signaling and the phosphorylation of mTORC1 binding partner raptor (Ser792) to inhibit mTORC1. Our results indicate that Map4k4 inhibits adipose lipogenesis by suppression of Srebp-1 in an AMPK- and mTOR-dependent but JNK-independent mechanism.
Asunto(s)
Adipocitos/metabolismo , Lipogénesis , Sistema de Señalización de MAP Quinasas , Proteínas Serina-Treonina Quinasas/fisiología , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Células 3T3-L1 , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Activación Enzimática , Expresión Génica , Técnicas de Silenciamiento del Gen , Ratones , Obesidad/enzimología , Serina-Treonina Quinasas TOR/metabolismo , Activación Transcripcional , Triglicéridos/biosíntesis , Quinasa de Factor Nuclear kappa BRESUMEN
The receptor peroxisome proliferator-activated receptor gamma (PPARgamma) is considered a master regulator of adipocyte differentiation and promotes glucose and lipid metabolism in mature adipocytes. We recently identified the yeast Sterile 20 (Ste20) protein kinase ortholog, Map4k4, in an RNA interference-based screen as an inhibitor of PPARgamma expression in cultured adipocytes. Here, we show that RNA interference-mediated silencing of Map4k4 elevates the levels of both PPARgamma1 and PPARgamma2 proteins in 3T3-L1 adipocytes without affecting PPARgamma mRNA levels, suggesting that Map4k4 regulates PPARgamma at a post-transcriptional step. PPARgamma degradation rates are remarkably rapid as measured in the presence of cycloheximide (t(1/2) = 2 h), but silencing Map4k4 had no effect on PPARgamma degradation. However, depletion of Map4k4 significantly enhances [(35)S]methionine/cysteine incorporation into proteins, suggesting that Map4k4 signaling decreases protein translation. We show a function of Map4k4 is to inhibit rapamycin-sensitive mammalian target of rapamycin (mTOR) activity, decreasing 4E-BP1 phosphorylation. In addition, our results show mTOR and 4E-BP1 are required for the increased PPARgamma protein expression upon Map4k4 knockdown. Consistent with this concept, adenovirus-mediated expression of Map4k4 decreased PPARgamma protein levels and mTOR phosphorylation. These data show that Map4k4 negatively regulates PPARgamma post-transcriptionally, by attenuating mTOR signaling and a 4E-BP1-dependent mechanism.
Asunto(s)
Adipocitos/metabolismo , Proteínas Portadoras/metabolismo , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , PPAR gamma/antagonistas & inhibidores , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/fisiología , Transducción de Señal , Células 3T3-L1 , Proteínas Adaptadoras Transductoras de Señales , Adipocitos/citología , Animales , Proteínas de Ciclo Celular , Factores Eucarióticos de Iniciación , Regulación de la Expresión Génica , Ratones , PPAR gamma/biosíntesis , Fosforilación , Estabilidad Proteica , Serina-Treonina Quinasas TOR , Quinasa de Factor Nuclear kappa BRESUMEN
The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand-dependent transcription factor that acts as a primary regulator of adipogenesis and controls adipocyte metabolism and insulin action. Increased expression of tumor necrosis factor (TNFalpha) in adipose tissue of obese subjects potently suppresses the expression of PPARgamma and attenuates adipocyte functions. Here we show that PPARgamma is a substrate of caspase-3 and caspase-6 during TNFalpha receptor signaling in adipocytes, and the consequent PPARgamma cleavage disrupts its nuclear localization. TNFalpha treatment of 3T3-L1 adipocytes decreases full-length PPARgamma while increasing the level of a 45-kDa immunoreactive PPARgamma fragment. Specific inhibitors of caspase-3 and caspase-6 attenuate the cleavage of PPARgamma protein in response to TNFalpha in cultured adipocytes. Incubation of nuclear fractions with recombinant caspase-3 and caspase-6 also generates a 45-kDa PPARgamma cleavage product. Dispersion of nuclear PPARgamma to the cytoplasm in response to TNFalpha treatment occurs in parallel with detection of activated caspase-3. We suggest that activation of the caspase cascade by TNFalpha down-regulates PPARgamma protein and PPARgamma-mediated metabolic processes in adipose cells.
Asunto(s)
Adipocitos/efectos de los fármacos , Adipocitos/metabolismo , Caspasas/metabolismo , PPAR gamma/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Células 3T3-L1 , Animales , Caspasa 3/metabolismo , Caspasa 6/metabolismo , Caspasa 8/metabolismo , Cinética , Ratones , Modelos Biológicos , PPAR gamma/química , PPAR gamma/genética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/metabolismoRESUMEN
Tumor necrosis factor alpha (TNFalpha) is a cytokine secreted by macrophages and adipocytes that contributes to the low grade inflammation and insulin resistance observed in obesity. TNFalpha signaling decreases peroxisome proliferator-activated receptor gamma and glucose transporter isoform 4 (GLUT4) expression in adipocytes, impairing insulin action, and this is mediated in part by the yeast Ste20 protein kinase ortholog Map4k4. Here we show that Map4k4 expression is selectively up-regulated by TNFalpha, whereas the expression of the protein kinases JNK1/2, ERK1/2, p38 stress-activated protein kinase, and mitogen-activated protein kinase kinases 4/7 shows little or no response. Furthermore, the cytokines interleukin 1beta (IL-1beta) and IL-6 as well as lipopolysaccharide fail to increase Map4k4 mRNA levels in cultured adipocytes under conditions where TNFalpha elicits a 3-fold effect. Using agonistic and antagonistic antibodies and small interfering RNA (siRNA) against TNFalpha receptor 1 (TNFR1) and TNFalpha receptor 2 (TNFR2), we show that TNFR1, but not TNFR2, mediates the increase in Map4k4 expression. TNFR1, but not TNFR2, also mediates a potent effect of TNFalpha on the phosphorylation of JNK1/2 and p38 stress-activated protein kinase and their downstream transcription factor substrates c-Jun and activating transcription factor 2 (ATF2). siRNA-based depletion of c-Jun and ATF2 attenuated TNFalpha action on Map4k4 mRNA expression. Consistent with this concept, the phosphorylation of ATF2 along with the expression and phosphorylation of c-Jun by TNFalpha signaling was more robust and prolonged compared with that of IL-1beta, which failed to modulate Map4k4. These data reveal that TNFalpha selectively stimulates the expression of a key component of its own signaling pathway, Map4k4, through a TNFR1-dependent mechanism that targets the transcription factors c-Jun and ATF2.
Asunto(s)
Factor de Transcripción Activador 2/metabolismo , Proteínas Quinasas Activadas por Mitógenos/biosíntesis , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteínas Proto-Oncogénicas c-jun/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Transducción de Señal , Factor de Necrosis Tumoral alfa/metabolismo , Regulación hacia Arriba , Células 3T3 , Adipocitos/metabolismo , Animales , Transportador de Glucosa de Tipo 4/metabolismo , Inflamación/metabolismo , Resistencia a la Insulina , Interleucina-1beta/metabolismo , Interleucina-1beta/farmacología , Interleucina-6/metabolismo , Interleucina-6/farmacología , Lipopolisacáridos/farmacología , Macrófagos/metabolismo , Ratones , PPAR gamma/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-jun/antagonistas & inhibidores , ARN Interferente Pequeño/farmacología , Receptores Tipo I de Factores de Necrosis Tumoral/agonistas , Receptores Tipo II del Factor de Necrosis Tumoral/agonistas , Receptores Tipo II del Factor de Necrosis Tumoral/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Regulación hacia Arriba/efectos de los fármacos , Quinasa de Factor Nuclear kappa BRESUMEN
BACKGROUND: The TOR (target of rapamycin) ser/thr protein kinase is the central component of a eukaryotic signaling pathway that regulates growth and is the direct target of the clinically useful drug rapamycin. Recent efforts have identified at least two multiprotein complexes that contain TOR, but little is known in higher eukaryotes about the genes downstream of TOR that control growth. RESULTS: By combining the use of a small molecule inhibitor (rapamycin), transcriptional profiling, and RNA interference in Drosophila tissue culture cells, we identified genes whose expression responds to Drosophila TOR (dTOR) inhibition and that regulate cell size. Several of the dTOR-regulated genes that function in cell size control have additional roles in cell division. Most of these genes are conserved in mammals and several are linked to human disease. This set of genes is highly enriched for regulators of ribosome biogenesis, which emphasizes the importance of TOR-dependent transcription in building the protein synthesis machinery in higher eukaryotes. In addition, we identify two dTOR-regulated genes, CG3071 and CG6677, whose human orthologs, SAW and ASH2L, are also under TOR-dependent transcriptional control and encode proteins with conserved functional roles in growth. CONCLUSIONS: We conclude that combining RNA interference with genomic analysis approaches, such as transcriptional profiling, is an effective way to identify genes functioning in a particular biological process. Moreover, this strategy, if applied in model systems with simpler genomes, can identify genes with conserved functions in mammals.
Asunto(s)
División Celular/genética , Procesos de Crecimiento Celular/genética , Proteínas de Drosophila/fisiología , Drosophila/genética , Genes del Desarrollo/fisiología , Fosfatidilinositol 3-Quinasas/fisiología , Animales , Antibióticos Antineoplásicos , Aumento de la Célula , Línea Celular , Regulación hacia Abajo , Proteínas de Drosophila/genética , Perfilación de la Expresión Génica , Proteínas Nucleares , Proteínas Quinasas , Interferencia de ARN , Ribosomas/metabolismo , Sirolimus , Serina-Treonina Quinasas TOR , Factores de Transcripción/metabolismo , Regulación hacia ArribaRESUMEN
Antennal proteins of the male fire ant (Solenopsis invicta) were analyzed by two-dimensional gel electrophoresis, with the objective of identifying pheromone-binding proteins, which have not previously been found in ant antennae. The major low-molecular weight protein found in the male fire ant antenna was subjected to Edman degradation to determine the N-terminal amino acid sequence. Degenerate PCR primers based on this sequence were used to obtain a cDNA sequence corresponding to the full-length protein sequence. In-gel trypsin digestion followed by MALDI-TOF mass spectrometry and HPLC-ESI/MS/MS demonstrated that the protein gel spot contained only the protein corresponding to the cDNA sequence obtained by PCR. The sequence is similar to apolipophorin-III, an exchangeable lipid-binding protein. Fire ant apolipophorin-III is expressed in the antenna as well as the head, thorax and abdomen.
Asunto(s)
Hormigas/genética , Hormigas/metabolismo , Apolipoproteínas/metabolismo , Órganos de los Sentidos/metabolismo , Secuencia de Aminoácidos , Animales , Apolipoproteínas/genética , Secuencia de Bases , Cartilla de ADN , Electroforesis en Gel Bidimensional , Masculino , Espectrometría de Masas , Datos de Secuencia Molecular , Compuestos Organofosforados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
mTOR and raptor are components of a signaling pathway that regulates mammalian cell growth in response to nutrients and growth factors. Here, we identify a member of this pathway, a protein named GbetaL that binds to the kinase domain of mTOR and stabilizes the interaction of raptor with mTOR. Like mTOR and raptor, GbetaL participates in nutrient- and growth factor-mediated signaling to S6K1, a downstream effector of mTOR, and in the control of cell size. The binding of GbetaL to mTOR strongly stimulates the kinase activity of mTOR toward S6K1 and 4E-BP1, an effect reversed by the stable interaction of raptor with mTOR. Interestingly, nutrients and rapamycin regulate the association between mTOR and raptor only in complexes that also contain GbetaL. Thus, we propose that the opposing effects on mTOR activity of the GbetaL- and raptor-mediated interactions regulate the mTOR pathway.