RESUMEN

The mammalian target of rapamycin (mTOR) pathway plays a key role in determining immune cells function through modulation of their metabolic status. By specific deletion of Rictor in CD11c+ myeloid cells (referred to here as CD11cRicΔ/Δ), we investigated the role of mTOR complex 2 (mTORC2) signaling in dendritic cells (DCs) function in mice. We showed that upon dextran sulfate sodium-induced colitis, the lack of mTORC2 signaling CD11c+ cells diminishes the colitis score and abrogates DC migration to the mesenteric lymph nodes, thereby diminishing the infiltration of T helper 17 cells in the lamina propria and subsequent inflammation. These findings corroborate with the abrogation of cytoskeleton organization and the decreased activation of Rac1 and Cdc42 GTPases observed in CD11c+-mTORC2-deficient cells. Meta-analysis on colonic samples from ulcerative colitis patients revealed increased gene expression of proinflammatory cytokines, which coincided with augmented expression of the mTOR pathway, a positive correlation between the DC marker ITGAX and interleukin-6, the expression of RICTOR, and CDC42. Together, this work proposes that targeting mTORC2 on DCs offers a key to hamper inflammatory responses, and this way, ameliorates the progression and severity of intestinal inflammatory diseases.

Asunto(s)

Movimiento Celular , Colitis , Células Dendríticas , Sulfato de Dextran , Diana Mecanicista del Complejo 2 de la Rapamicina , Células Mieloides , Transducción de Señal , Animales , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Ratones , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Colitis/patología , Colitis/inducido químicamente , Colitis/inmunología , Células Mieloides/metabolismo , Células Mieloides/inmunología , Sulfato de Dextran/toxicidad , Proteína Asociada al mTOR Insensible a la Rapamicina/metabolismo , Proteína Asociada al mTOR Insensible a la Rapamicina/genética , Antígeno CD11c/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Humanos , Proteína de Unión al GTP rac1/metabolismo , Ratones Endogámicos C57BL , Modelos Animales de Enfermedad , Ratones Noqueados , Neuropéptidos , Antígenos CD11

RESUMEN

TOR proteins, also known as targets of rapamycin, are serine/threonine kinases involved in various signaling pathways that regulate cell growth. The protozoan parasite Giardia lamblia is the causative agent of giardiasis, a neglected infectious disease in humans. In this study, we used a bioinformatics approach to examine the structural features of GTOR, a G. lamblia TOR-like protein, and predict functional associations. Our findings confirmed that it shares significant similarities with functional TOR kinases, including a binding domain for the FKBP-rapamycin complex and a kinase domain resembling that of phosphatidylinositol 3-kinase-related kinases. In addition, it can form multiprotein complexes such as TORC1 and TORC2. These results provide valuable insights into the structure-function relationship of GTOR, highlighting its potential as a molecular target for controlling G. lamblia cell proliferation. Furthermore, our study represents a step toward rational drug design for specific anti-giardiasis therapeutic agents.

Asunto(s)

Giardia lamblia , Giardiasis , Humanos , Sirolimus/farmacología , Giardia lamblia/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Transducción de Señal , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo

RESUMEN

CD8+ T lymphocytes are one of the main effector cells of the immune system, they protect the organism against intracellular threats such as viruses and bacteria, as well as neoplasms. It is currently well established that CD8+ T cells have distinct immune responses, given by their phenotypes Tc1, Tc2, Tc17, and TcReg. The cellular plasticity of such phenotypes depends on the presence of different combinations of cytokines in the extracellular medium. It is known that metabolic imbalances play an important role in immune response, but the precise role of metabolic disturbances on the differentiation and function of CD8+ T cells, however, has not been explored. In this work, we used a computational model to explore the potential effect of metabolic alterations such as hyperglycemia, high alcohol consumption, dyslipidemia, and diabetes on CD8+ T cell differentiation. Our model predicts that metabolic alterations preclude the effector function of all CD8+ T cell phenotypes except for TcReg cells. It also suggests that such inhibition originates from the increase of reactive oxygen species in response to metabolic stressors. Finally, we simulated the outcome of treating metabolic-inhibited CD8+ T cells with drugs targeting key molecules such as mTORC1, mTORC2, Akt, and others. We found that overstimulation of mTORC2 may restore cell differentiation and functions of all effector phenotypes, even in diabetic patients. These findings highlight the importance of our predictive model to find potential targets to strengthen immunosuppressed patients in chronic diseases, like diabetes.

Asunto(s)

Linfocitos T CD8-positivos , Activación de Linfocitos , Diferenciación Celular , Citocinas/metabolismo , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo

RESUMEN

Deletion of mechanistic target of rapamycin complex 2 (mTORC2) essential component rapamycin insensitive companion of mTOR (Rictor) by a Cre recombinase under control of the broad, nonadipocyte-specific aP2/FABP4 promoter impairs thermoregulation and brown adipose tissue (BAT) glucose uptake on acute cold exposure. We investigated herein whether adipocyte-specific mTORC2 deficiency affects BAT and inguinal white adipose tissue (iWAT) signaling, metabolism, and thermogenesis in cold-acclimated mice. For this, 8-wk-old male mice bearing Rictor deletion and therefore mTORC2 deficiency in adipocytes (adiponectin-Cre) and littermates controls were either kept at thermoneutrality (30 ± 1°C) or cold-acclimated (10 ± 1°C) for 14 days and evaluated for BAT and iWAT signaling, metabolism, and thermogenesis. Cold acclimation inhibited mTORC2 in BAT and iWAT, but its residual activity is still required for the cold-induced increases in BAT adipocyte number, total UCP-1 content and mRNA levels of proliferation markers Ki67 and cyclin 1 D, and de novo lipogenesis enzymes ATP-citrate lyase and acetyl-CoA carboxylase. In iWAT, mTORC2 residual activity is partially required for the cold-induced increases in multilocular adipocytes, mitochondrial mass, and uncoupling protein 1 (UCP-1) content. Conversely, BAT mTORC1 activity and BAT and iWAT glucose uptake were upregulated by cold independently of mTORC2. Noteworthy, the impairment in BAT and iWAT total UCP-1 content and thermogenic capacity induced by adipocyte mTORC2 deficiency had no major impact on whole body energy expenditure in cold-acclimated mice due to a compensatory activation of muscle shivering. In conclusion, adipocyte mTORC2 deficiency impairs, through different mechanisms, BAT and iWAT total UCP-1 content and thermogenic capacity in cold-acclimated mice, without affecting glucose uptake and whole body energy expenditure.NEW & NOTEWORTHY BAT and iWAT mTORC2 is inhibited by cold acclimation, but its residual activity is required for cold-induced increases in total UCP-1 content and thermogenic capacity, but not glucose uptake and mTORC1 activity. The impaired BAT and iWAT total UCP-1 content and thermogenic capacity induced by adipocyte mTORC2 deficiency are compensated by activation of muscle shivering in cold-acclimated mice.

Asunto(s)

Aclimatación/fisiología , Adipocitos/metabolismo , Tejido Adiposo Pardo/fisiología , Tejido Adiposo Blanco/fisiología , Metabolismo Energético/fisiología , Glucosa/metabolismo , Diana Mecanicista del Complejo 2 de la Rapamicina/deficiencia , Termogénesis/genética , Animales , Frío , Eliminación de Gen , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/fisiología , Masculino , Diana Mecanicista del Complejo 2 de la Rapamicina/genética , Ratones , Ratones Endogámicos C57BL , Proteína Desacopladora 1

RESUMEN

The Hippo and mTOR signaling cascades are major regulators of cell growth and division. Aberrant regulation of these pathways has been demonstrated to contribute to gliomagenesis and result in enhanced glioblastoma proliferation and invasive characteristics. Several crosstalk mechanisms have been described between these two pathways, although a complete picture of these signaling interactions is lacking and is required for effective therapeutic targeting. Here we report the ability of mTORC2 to directly phosphorylate YAP at serine 436 (Ser436) positively regulating YAP activity. We show that mTORC2 activity enhances YAP transcriptional activity and the induction of YAP-dependent target gene expression while its ablation via genetic or pharmacological means has the opposite affects on YAP function. mTORC2 interacts with YAP via Sin1 and mutational analysis of serine 436 demonstrates that this phosphorylation event affects several properties of YAP leading to enhanced transactivation potential. Moreover, YAP serine 436 mutants display altered glioblastoma growth, migratory capacity and invasiveness both in vitro and in xenograft experiments. We further demonstrate that mTORC2 is able to regulate a Hippo pathway resistant allele of YAP suggesting that mTORC2 can regulate YAP independent of Hippo signaling. Correlative associations between the expression of these components in GBM patient samples also supported the presence of this signaling relationship. These results advance a direct mTORC2/YAP signaling axis driving GBM growth, motility and invasiveness.

Asunto(s)

Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Carga Tumoral/fisiología , Proteínas Señalizadoras YAP/metabolismo , Animales , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Femenino , Glioblastoma/genética , Glioblastoma/patología , Vía de Señalización Hippo/fisiología , Humanos , Diana Mecanicista del Complejo 2 de la Rapamicina/genética , Ratones , Ratones SCID , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Fosforilación/fisiología , Ensayos Antitumor por Modelo de Xenoinjerto/métodos , Proteínas Señalizadoras YAP/genética

RESUMEN

SCOPE: Evidence gathered in the last decades suggests that lipotoxicity and inflammation are the main factors connecting adipose tissue dysfunction to the development of metabolic diseases such as insulin resistance, nonalcoholic fatty liver disease (NAFLD), cardiovascular disease, and certain types of cancer, among others. The mechanistic target of rapamycin (mTOR) is a serine threonine kinase that functions as the catalytic entity of two multiprotein complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). These complexes are important components of signaling pathways activated by nutrients, growth factors, and inflammatory mediators and are therefore directly involved in the regulation of adipocyte and macrophage metabolism and function. METHODS AND RESULTS: In this article, studies that evaluate the involvement of mTORC1 and 2 in the regulation of macrophage and adipocyte function and their implication in the development of metabolic-disease-associated adipose tissue dysfunction are reviewed. CONCLUSION: In adipocytes, optimal levels of mTORC1 activity are required for its pro-lipogenic actions, while in macrophages, mTORC1 regulates features of both M1 and M2 polarization. mTORC2, on the other hand, promotes glucose uptake and de novo lipogenesis in adipocytes and counteracts macrophage inflammatory response.

Asunto(s)

Adipocitos/metabolismo , Tejido Adiposo Blanco/patología , Macrófagos/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Tejido Adiposo Blanco/metabolismo , Animales , Humanos , Mediadores de Inflamación/metabolismo , Enfermedades Metabólicas/metabolismo , Obesidad/metabolismo , Obesidad/patología

RESUMEN

mTOR, a serine/threonine protein kinase that is involved in a series of critical cellular processes, can be found in two functionally distinct complexes, mTORC1 and mTORC2. In contrast to mTORC1, little is known about the mechanisms that regulate mTORC2. Here we show that mTORC2 activity is reduced in mice with a hypomorphic mutation of the Ric-8B gene. Ric-8B is a highly conserved protein that acts as a non-canonical guanine nucleotide exchange factor (GEF) for heterotrimeric Gαs/olf type subunits. We found that Ric-8B hypomorph embryos are smaller than their wild type littermates, fail to close the neural tube in the cephalic region and die during mid-embryogenesis. Comparative transcriptome analysis revealed that signaling pathways involving GPCRs and G proteins are dysregulated in the Ric-8B mutant embryos. Interestingly, this analysis also revealed an unexpected impairment of the mTOR signaling pathway. Phosphorylation of Akt at Ser473 is downregulated in the Ric-8B mutant embryos, indicating a decreased activity of mTORC2. Knockdown of the endogenous Ric-8B gene in cultured cell lines leads to reduced phosphorylation levels of Akt (Ser473), further supporting the involvement of Ric-8B in mTORC2 activity. Our results reveal a crucial role for Ric-8B in development and provide novel insights into the signals that regulate mTORC2.

Asunto(s)

Factores de Intercambio de Guanina Nucleótido/genética , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Animales , Células Cultivadas , Regulación hacia Abajo/genética , Embrión de Mamíferos , Desarrollo Embrionario/genética , Femenino , Eliminación de Gen , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de Señal/genética

RESUMEN

Tubule-interstitial injury (TII) is a critical step in the progression of renal disease. It has been proposed that changes in proximal tubule (PT) albumin endocytosis plays an important role in the development of TII. Some reports have shown protective effects of lithium on kidney injury animal models that was correlated to proteinuria. We tested the hypothesis that lithium treatment ameliorates the development of TII due to changes in albumin endocytosis. Two experimental models were used: (1) TII induced by albumin overload in an animal model; (2) LLC-PK1 cells, a PT cell line. Lithium treatment ameliorates TII induced by albumin overload measured by (1) proteinuria; (2) collagen deposition; (3) area of tubule-interstitial space, and (4) macrophage infiltration. Lithium treatment increased mTORC2 activity leading to the phosphorylation of protein kinase B (PKB) at Ser473 and its activation. This mechanism enhanced albumin endocytosis in PT cells, which decreased the proteinuria observed in TII induced by albumin overload. This effect did not involve changes in the expression of megalin, a PT albumin receptor. In addition, activation of this pathway decreased apoptosis in LLC-PK1 cells, a PT cell line, induced by higher albumin concentration, similar to that found in pathophysiologic conditions. Our results indicate that the protective role of lithium treatment on TII induced by albumin overload involves an increase in PT albumin endocytosis due to activation of the mTORC2/PKB pathway. These results open new possibilities in understanding the effects of lithium on the progression of renal disease.

Asunto(s)

Túbulos Renales Proximales/efectos de los fármacos , Carbonato de Litio/farmacología , Diana Mecanicista del Complejo 2 de la Rapamicina/genética , Sustancias Protectoras/farmacología , Proteinuria/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-akt/genética , Albúminas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Línea Celular , Movimiento Celular/efectos de los fármacos , Endocitosis/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/patología , Regulación de la Expresión Génica , Humanos , Túbulos Renales Proximales/lesiones , Túbulos Renales Proximales/metabolismo , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Diana Mecanicista del Complejo 2 de la Rapamicina/agonistas , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Ratones , Ratones Endogámicos BALB C , Proteinuria/metabolismo , Proteinuria/fisiopatología , Proteínas Proto-Oncogénicas c-akt/agonistas , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos

RESUMEN

Maternal diabetes programs cardiovascular alterations in the adult offspring but the mechanisms involved remain unclarified. Here, we addresed whether maternal diabetes programs cardiac alterations related to extracellular matrix remodeling in the adult offspring, as well as the role of forkhead box transcription factor 1 (FOXO1) in the induction of these alterations. The heart from adult offspring from control and streptozotocin-induced diabetic rats was evaluated. Increased glycemia, triglyceridemia and insulinemia and markers of cardiomyopathy were found in the offspring from diabetic rats. In the heart, an increase in active FOXO1 and mRNA levels of its target genes, Mmp-2 and Ctgf, genes related to an altered extracellular matrix remodeling, together with an increase in collagen deposition and a decrease in the connexin43 levels, were found in the offspring from diabetic rats. Altogether, these results suggest an important role of FOXO1 activation in the cardiac alterations induced by intrauterine programming in maternal diabetes.

Asunto(s)

Enfermedades Cardiovasculares/metabolismo , Diabetes Gestacional/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Biomarcadores/metabolismo , Peso Corporal , Colágeno/metabolismo , Conexina 43/metabolismo , Matriz Extracelular/metabolismo , Femenino , Metaloproteinasa 2 de la Matriz/metabolismo , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Miocardio/metabolismo , Miocardio/patología , Tamaño de los Órganos , Fosforilación , Embarazo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas Wistar , Serina-Treonina Quinasas TOR/metabolismo

RESUMEN

OBJECTIVES: After bariatric surgery, modifications to signaling pathway networks including those of the metabolic regulator called mammalian or mechanistic target of rapamycin (mTOR) may lead to molecular alterations related to energy source availability, systemic nutrients, and catabolic and anabolic cellular processes. This study aimed to identify gene expression changes with regard to the mTOR complex 2 subunit signaling pathway in obese patients before and after bariatric surgery. METHODS: The experimental group included 13 obese women who were examined before (preoperative) and 6 mo after (postoperative) Roux-en-Y gastric bypass (RYGB) surgery. The control group included nine apparently eutrophic women matched by age and without any other metabolic diseases (i.e., no diabetes and no liver or kidney diseases). Peripheral blood mononuclear cell samples were collected for RNA extraction and subsequent microarray analysis. RESULTS: After this methodological procedure, we identified 47 000 differentially expressed genes. A subsequent bioinformatic analysis showed that three diferentially expressed genes (rapamycin-insensitive companion of mTOR [RICTOR], phosphoinositide-3-kinase regulatory subunit 1 [PIK3 R1], and hypoxia inducible factor 1 alpha subunit 1A [HIF1 A]) participated in the mTOR signaling pathway. Real-time quantitative polymerase chain reaction revealed that RICTOR, PIK3 R1, and HIF1 A were upregulated 6 mo after RYGB surgery (P <0.05). In addition, patients in the experimental group lost weight significantly and presented significant improvement in biochemical/metabolic variables. CONCLUSIONS: The weight loss that was induced by RYGB surgery alters the mTOR signaling pathway and specifically the mTOR complex 2 subunit. The increased expression of genes that act in this pathway such as RICTOR, PIK3 R1, and HIF1 A reflects the induced weight loss and improved metabolic indicators (e.g., insulin resistance and lipolysis) that are evidenced in this study.

Asunto(s)

Derivación Gástrica , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Obesidad/genética , Transducción de Señal/genética , Pérdida de Peso/genética , Adulto , Femenino , Humanos , Leucocitos Mononucleares/metabolismo , Estudios Longitudinales , Persona de Mediana Edad , Obesidad/cirugía , Periodo Posoperatorio , Periodo Preoperatorio , Resultado del Tratamiento

RESUMEN

OBJECTIVE: Temporal lobe epilepsy (TLE) is a chronic epilepsy syndrome defined by seizures and progressive neurological disabilities, including cognitive impairments, anxiety, and depression. Here, human TLE specimens were investigated focusing on the mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) and complex 2 (mTORC2) activities in the brain, given that both pathways may represent unique targets for treatment. METHODS: Surgically resected hippocampal and temporal lobe samples from therapy-resistant TLE patients were analyzed by western blotting to quantify the expression of established mTORC1 and mTORC2 activity markers and upstream or downstream signaling pathways involving the two complexes. Histological and immunohistochemical techniques were used to assess hippocampal and neocortical structural abnormalities and cell-specific expression of individual biomarkers. Samples from patients with focal cortical dysplasia (FCD) type II served as positive controls. RESULTS: We found significantly increased expression of phospho-mTOR (Ser2448), phospho-S6 (Ser235/236), phospho-S6 (Ser240/244), and phospho-Akt (Ser473) in TLE samples compared to controls, consistent with activation of both mTORC1 and mTORC2. Our work identified the phosphoinositide 3-kinase and Ras/extracellular signal-regulated kinase signaling pathways as potential mTORC1 and mTORC2 upstream activators. In addition, we found that overactive mTORC2 signaling was accompanied by induction of two protein kinase B-dependent prosurvival pathways, as evidenced by increased inhibitory phosphorylation of forkhead box class O3a (Ser253) and glycogen synthase kinase 3 beta (Ser9). INTERPRETATION: Our data demonstrate that mTOR signaling is significantly dysregulated in human TLE, offering new targets for pharmacological interventions. Specifically, clinically available drugs that suppress mTORC1 without compromising mTOR2 signaling, such as rapamycin and its analogs, may represent a new group of antiepileptogenic agents in TLE patients. Ann Neurol 2018;83:311-327.

Asunto(s)

Epilepsia del Lóbulo Temporal/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Transducción de Señal/fisiología , Adulto , Encéfalo/metabolismo , Epilepsia del Lóbulo Temporal/fisiopatología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Serina-Treonina Quinasas TOR/metabolismo , Adulto Joven

RESUMEN

BACKGROUND: Little is known about how new-generation adenosine triphosphate-competitive mechanistic target of rapamycin (mTOR) kinase inhibitors affect immunity and allograft rejection. METHODS: mTOR complex (C) 1 and 2 signaling in dendritic cells and T cells was analyzed by Western blotting, whereas immune cell populations in normal and heart allograft recipient mice were analyzed by flow cytometry. Alloreactive T cell proliferation was quantified in mixed leukocyte reaction; intracellular cytokine production and serum antidonor IgG levels were determined by flow analysis and immunofluorescence staining used to detect IgG in allografts. RESULTS: The novel target of rapamycin kinase inhibitor AZD2014 impaired dendritic cell differentiation and T cell proliferation in vitro and depressed immune cells and allospecific T cell responses in vivo. A 9-day course of AZD2014 (10 mg/kg, intraperitoneally, twice daily) or rapamycin (RAPA) (1 mg/kg, intraperitoneally, daily) prolonged median heart allograft survival time significantly (25 days for AZD2014, 100 days for RAPA, 9.5 days for control). Like RAPA, AZD2014 suppressed graft mononuclear cell infiltration, increased regulatory T cell to effector memory T cell ratios and reduced T follicular helper and B cells 7 days posttransplant. By 21 days (10 days after drug withdrawal), however, T follicular helper and B cells and donor-specific IgG1 and IgG2c antibody titers were significantly lower in RAPA-treated compared with AZD2014-treated mice. Elevated regulatory T cell to effector memory T cell ratios were maintained after RAPA, but not AZD2014 withdrawal. CONCLUSIONS: Immunomodulatory effects of AZD2014, unlike those of RAPA, were not sustained after drug withdrawal, possibly reflecting distinct pharmacokinetics or/and inhibitory effects of AZD2014 on mTORC2.

Asunto(s)

Adenosina Trifosfato/química , Rechazo de Injerto , Trasplante de Corazón , Sistema Inmunológico/efectos de los fármacos , Diana Mecanicista del Complejo 1 de la Rapamicina/antagonistas & inhibidores , Morfolinas/farmacología , Animales , Benzamidas , Proliferación Celular , Células Dendríticas/citología , Supervivencia de Injerto/efectos de los fármacos , Inmunoglobulina G/química , Inmunosupresores/farmacología , Masculino , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas , Sirolimus/farmacología , Linfocitos T/citología , Trasplante Homólogo

RESUMEN

The lack of a successful treatment for triple-negative breast cancer demands the study of the heterogeneity of cells that constitute these tumors. With this aim, two clones from triple negative breast MDA-MB-231 cancer cells were isolated: One with fibroblast-like appearance (F) and another with semi-epithelial (SE) morphology. Cells of the F clone have a higher migration and tumorigenesis capacity than SE cells, suggesting that these cells are in a more advanced stage of epithelial to mesenchymal transformation. In agreement, F cells have a diminished expression of the tight junction proteins claudins 1 and 4, and an increased content of ß-catenin. The latter is due to an augmented activity of the canonical Wnt route and of the EGFR/PI3K/mTORC2/AKT pathway favoring the cytoplasmic accumulation of ß-catenin and its transcriptional activity. In addition, F cells display increased phosphorylation of ß-catenin at Tyr654 by Src. These changes favor in F cells, the over-expression of Snail that promotes EMT. Finally, we observe that both F and SE cells display markers of cancer stem cells, which are more abundant in the F clone.

Asunto(s)

Receptores ErbB/metabolismo , Complejos Multiproteicos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Animales , Apoptosis , Western Blotting , Proliferación Celular , Quimiotaxis , Transición Epitelial-Mesenquimal , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Diana Mecanicista del Complejo 2 de la Rapamicina , Ratones , Ratones Desnudos , Células Madre Neoplásicas/citología , Células Madre Neoplásicas/metabolismo , Fosforilación , Neoplasias de la Mama Triple Negativas/patología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto

Asunto(s)

Inmunosupresores/uso terapéutico , Trasplante de Riñón/efectos adversos , Complejos Multiproteicos/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/uso terapéutico , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Descubrimiento de Drogas , Humanos , Inmunosupresores/efectos adversos , Diana Mecanicista del Complejo 1 de la Rapamicina , Diana Mecanicista del Complejo 2 de la Rapamicina , Terapia Molecular Dirigida , Complejos Multiproteicos/metabolismo , Inhibidores de Proteínas Quinasas/efectos adversos , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , Resultado del Tratamiento

RESUMEN

Since the discovery of Rapamycin (RAPA) and its immunosuppressive properties, enormous progress has been made in characterizing the mechanistic target of rapamycin (mTOR). Use of RAPA and its analogues (rapalogs) as anti-rejection agents has been accompanied by extensive investigation of how targeting of mTOR complex 1 (mTORC1), the principal target of RAPA, and more recently mTORC2, affects the function of immune cells, as well as vascular endothelial cells, that play crucial roles in regulation of allograft rejection. While considerable knowledge has accumulated on the function of mTORC1 and 2 in T cells, understanding of the differential roles of these complexes in antigen-presenting cells, NK cells and B cells/plasma cells is only beginning to emerge. Immune cell-specific targeting of mTORC1 or mTORC2, together with use of novel, second generation, dual mTORC kinase inhibitors (TORKinibs) have started to play an important role in elucidating the roles of these complexes and their potential for targeting in transplantation. Much remains unknown about the role of mTOR complexes and the consequences of mTOR targeting on immune reactivity in clinical transplantation. Here we address recent advances in understanding and evolving perspectives of the role of mTOR complexes and mTOR targeting in immunity, with extrapolation to transplantation.

Asunto(s)

Sistema Inmunológico/fisiología , Complejos Multiproteicos/fisiología , Serina-Treonina Quinasas TOR/fisiología , Trasplante , Animales , Humanos , Inmunosupresores/administración & dosificación , Diana Mecanicista del Complejo 1 de la Rapamicina , Diana Mecanicista del Complejo 2 de la Rapamicina

RESUMEN

The phosphoinositide-3-kinase (PI3K)/protein kinase B (Akt) axis plays a central role in attenuating inflammation upon macrophage stimulation with toll-like receptor (TLR) ligands. The mechanistic target of rapamycin complex 2 (mTORC2) relays signal from PI3K to Akt but its role in modulating inflammation in vivo has never been investigated. To evaluate the role of mTORC2 in the regulation of inflammation in vivo, we have generated a mouse model lacking Rictor, an essential mTORC2 component, in myeloid cells. Primary macrophages isolated from myeloid-specific Rictor null mice exhibited an exaggerated response to TLRs ligands, and expressed high levels of M1 genes and lower levels of M2 markers. To determine whether the loss of Rictor similarly affected inflammation in vivo, mice were either fed a high fat diet, a situation promoting chronic but low-grade inflammation, or were injected with lipopolysaccharide (LPS), which mimics an acute, severe septic inflammatory condition. Although high fat feeding contributed to promote obesity, inflammation, macrophage infiltration in adipose tissue and systemic insulin resistance, we did not observe a significant impact of Rictor loss on these parameters. However, mice lacking Rictor exhibited a higher sensitivity to septic shock when injected with LPS. Altogether, these results indicate that mTORC2 is a key negative regulator of macrophages TLR signalling and that its role in modulating inflammation is particularly important in the context of severe inflammatory challenges. These observations suggest that approaches aimed at modulating mTORC2 activity may represent a possible therapeutic approach for diseases linked to excessive inflammation.

Asunto(s)

Proteínas Portadoras/genética , Eliminación de Gen , Macrófagos Peritoneales/patología , Obesidad/patología , Animales , Proteínas Portadoras/inmunología , Dieta Alta en Grasa , Fibroblastos/inmunología , Fibroblastos/patología , Regulación de la Expresión Génica , Inflamación/inducido químicamente , Inflamación/genética , Inflamación/inmunología , Inflamación/patología , Resistencia a la Insulina , Lipopolisacáridos , Macrófagos Peritoneales/inmunología , Diana Mecanicista del Complejo 2 de la Rapamicina , Ratones , Ratones Noqueados , Complejos Multiproteicos/genética , Complejos Multiproteicos/inmunología , Obesidad/inducido químicamente , Obesidad/genética , Obesidad/inmunología , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/inmunología , Cultivo Primario de Células , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/inmunología , Proteína Asociada al mTOR Insensible a la Rapamicina , Transducción de Señal , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/inmunología , Receptores Toll-Like/genética , Receptores Toll-Like/inmunología

RESUMEN

Diverse G protein-coupled receptors depend on Gßγ heterodimers to promote cell polarization and survival via direct activation of PI3Kγ and potentially other effectors. These events involve full activation of AKT via its phosphorylation at Ser473, suggesting that mTORC2, the kinase that phosphorylates AKT at Ser473, is activated downstream of Gßγ. Thus, we tested the hypothesis that Gßγ directly contributes to mTOR signaling. Here, we demonstrate that endogenous mTOR interacts with Gßγ. Cell stimulation with serum modulates Gßγ interaction with mTOR. The carboxyl terminal region of mTOR, expressed as a GST-fusion protein, including the serine/threonine kinase domain, binds Gßγ heterodimers containing different Gß subunits, except Gß4. Both, mTORC1 and mTORC2 complexes interact with Gß1γ2 which promotes phosphorylation of their respective substrates, p70S6K and AKT. In addition, chronic treatment with rapamycin, a condition known to interfere with assembly of mTORC2, reduces the interaction between Gßγ and mTOR and the phosphorylation of AKT; whereas overexpression of Gαi interfered with the effect of Gßγ as promoter of p70S6K and AKT phosphorylation. Altogether, our results suggest that Gßγ positively regulates mTOR signaling via direct interactions and provide further support to emerging strategies based on the therapeutical potential of inhibiting different Gßγ signaling interfaces.

Asunto(s)

Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Western Blotting , Activación Enzimática/efectos de los fármacos , Subunidades beta de la Proteína de Unión al GTP/genética , Subunidades gamma de la Proteína de Unión al GTP/genética , Células HEK293 , Humanos , Inmunoprecipitación , Diana Mecanicista del Complejo 1 de la Rapamicina , Diana Mecanicista del Complejo 2 de la Rapamicina , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Transducción de Señal/efectos de los fármacos , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/genética , Técnicas del Sistema de Dos Híbridos

RESUMEN

Epithelial-mesenchymal transition (EMT), proliferation and migration of RPE cells characterize the development of proliferative vitreoretinopathy (PVR) and other fibro-proliferative eye diseases leading to blindness. A common event in these pathologies is the alteration of the BRB which allows the interaction of RPE cells with thrombin, a pro-inflammatory protease contained in serum. Thrombin promotion of cytoskeletal reorganization, proliferation, and migration has been reported in different cell types, although the molecular mechanisms involved in these processes remain poorly understood. Our previous work demonstrated that thrombin promotes RPE cell proliferation, cytoskeletal remodeling and migration, hallmark processes in the development of PVR. Thrombin induction of RPE cell proliferation requires PI3K, PDK1, and Akt/PKB (Akt) signaling leading to cyclin D1 gene expression. Since Akt functions as an upstream activator of mechanistic target of rapamycin complex 1 (mTORC1) and is also a downstream target for mTORC2, the aim of this work was to determine whether mTOR is involved in thrombin-induced RPE cell proliferation by regulating cyclin D1 expression in immortalized rat RPE-J cell line. Results demonstrate that thrombin-induced cyclin D1 expression and cell proliferation require Akt-independent phosphorylation/activation of mTOR at Ser 2448 mediated by PI3K/PKC-ζ/ERK1/2 signaling, concomitant to Akt-dependent activation of p70S6K carried by mTORC1.

Asunto(s)

Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Complejos Multiproteicos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , Trombina/farmacología , Animales , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular , Proliferación Celular/efectos de los fármacos , Ciclina D1/antagonistas & inhibidores , Ciclina D1/genética , Ciclina D1/metabolismo , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina , Diana Mecanicista del Complejo 2 de la Rapamicina , Fosfoproteínas/antagonistas & inhibidores , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora , Interferencia de ARN , ARN Interferente Pequeño , Proteína Asociada al mTOR Insensible a la Rapamicina , Ratas , Proteína Reguladora Asociada a mTOR , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/metabolismo , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo

RESUMEN

OBJECTIVES: Insulin is recognized to increase renal salt reabsorption in the distal nephron and hyperinsulinemic states have been shown to be associated with increased expression of the renal NaCl cotransporter (NCC). However, the effect of insulin on NCC functional activity has not been reported. METHODS: Using a heterologous expression system of Xenopus laevis oocytes, a mouse distal convoluted cell line, mDCT15 cells, endogenously expressing NCC, and an ex-vivo kidney perfusion technique, we assessed the effect of insulin on the activity and phosphorylation of NCC. The signaling pathway involved was analyzed. RESULTS: In Xenopus oocytes insulin increases the activity of NCC together with its phosphorylation at threonine residue 58. Activation of NCC by insulin was also observed in mDCT15 cells. Additionally, insulin increased the NCC phosphorylation in kidney under the ex-vivo perfusion technique. In oocytes and mDCT15 cells, insulin effect on NCC was prevented with inhibitors of phosphatidylinositol 3-kinase (PI3K), mTORC2, and AKT1 kinases, but not by inhibitors of MAP or mTORC1 kinases, suggesting that PI3K-mTORC2-AKT1 is the intracellular pathway required. Additionally, activation of NCC by insulin was not affected by wild-type or mutant versions of with no lysine kinase 1, with no lysine kinase 4, or serum glucocorticoid kinase 1, but it was no longer observed in the presence of wild-type or the dominant negative, catalytically inactive with no lysine kinase 3, implicating this kinase in the process. CONCLUSION: Insulin induces activation and phosphorylation of NCC. This effect could play an important role in arterial hypertension associated with hyperinsulinemic states, such as obesity, metabolic syndrome, or type 2 diabetes mellitus.

Asunto(s)

Insulina/farmacología , Riñón/efectos de los fármacos , Simportadores del Cloruro de Sodio/metabolismo , Animales , Western Blotting , Células Cultivadas , Riñón/metabolismo , Diana Mecanicista del Complejo 2 de la Rapamicina , Ratones , Complejos Multiproteicos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Xenopus laevis

RESUMEN

Resveratrol, a phenolic compound found in various plants, including grapes, berries, and peanuts, shows promise for the treatment of cancer, aging, type 2 diabetes, and cardiovascular diseases. Resveratrol can promote transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf2) activation, increase the expression level of SIRT-1, which is a sirtuin family protein, and reduce mTOR pathway signaling. This compound has anti-inflammatory properties in that it inhibits or antagonizes the nuclear factor-κB (NF-κB) activity, which is a redox-sensitive transcription factor that coordinates the inflammatory response. Inflammation and oxidative stress, which are common features in patients with chronic kidney disease (CKD), are interrelated and associated with cardiovascular disease and the progression of CKD itself. Because of the modulation of the mechanisms involved in the inflammatory-oxidative stress cycle, resveratrol could play an important role in controlling CKD-related metabolic derangements. Although resveratrol supplementation in theory is a promising therapy in this patient group, there are no studies evaluating its effects. Thus, the present review aims to describe the role of resveratrol in inflammation and oxidative stress modulation and its possible benefits to patients with CKD.

Asunto(s)

Insuficiencia Renal Crónica/tratamiento farmacológico , Estilbenos/uso terapéutico , Humanos , Inflamación/patología , Diana Mecanicista del Complejo 1 de la Rapamicina , Diana Mecanicista del Complejo 2 de la Rapamicina , Complejos Multiproteicos/metabolismo , Estrés Oxidativo/efectos de los fármacos , Resveratrol , Estilbenos/química , Estilbenos/farmacología , Serina-Treonina Quinasas TOR/metabolismo