RESUMEN
Acute myeloid leukemia (AML) with the FLT3 internal tandem duplication (FLT3-ITD AML) accounts for 20-30% of AML cases. This subtype usually responds poorly to conventional therapies, and might become resistant to FLT3 tyrosine kinase inhibitors (TKIs) due to molecular bypass mechanisms. New therapeutic strategies focusing on resistance mechanisms are therefore urgently needed. Pim kinases are FLT3-ITD oncogenic targets that have been implicated in FLT3 TKI resistance. However, their precise biological function downstream of FLT3-ITD requires further investigation. We performed high-throughput transcriptomic and proteomic analyses in Pim2-depleted FLT3-ITD AML cells and found that Pim2 predominantly controlled apoptosis through Bax expression and mitochondria disruption. We identified ribosomal protein S6 kinase A3 (RSK2), a 90 kDa serine/threonine kinase involved in the mitogen-activated protein kinase cascade encoded by the RPS6KA3 gene, as a novel Pim2 target. Ectopic expression of an RPS6KA3 allele rescued the viability of Pim2-depleted cells, supporting the involvement of RSK2 in AML cell survival downstream of Pim2. Finally, we showed that RPS6KA3 knockdown reduced the propagation of human AML cells in vivo in mice. Our results point to RSK2 as a novel Pim2 target with translational therapeutic potential in FLT3-ITD AML.
Asunto(s)
Duplicación de Gen , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Secuencias Repetidas en Tándem , Tirosina Quinasa 3 Similar a fms/genética , Animales , Apoptosis , Caspasas/metabolismo , Línea Celular Tumoral , Supervivencia Celular/genética , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Leucemia Mieloide Aguda/patología , Ratones , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Transcriptoma , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Alterations in metabolic activities are cancer hallmarks that offer a wide range of new therapeutic opportunities. Here we decipher the interplay between mTORC1 activity and glucose metabolism in acute myeloid leukemia (AML). We show that mTORC1 signaling that is constantly overactivated in AML cells promotes glycolysis and leads to glucose addiction. The level of mTORC1 activity determines the sensitivity of AML cells to glycolysis inhibition as switch-off mTORC1 activity leads to glucose-independent cell survival that is sustained by an increase in mitochondrial oxidative phosphorylation. Metabolic analysis identified the pentose phosphate pathway (PPP) as an important pro-survival pathway for glucose metabolism in AML cells with high mTORC1 activity and provided a clear rational for targeting glucose-6-phosphate dehydrogenase (G6PD) in AML. Indeed, our analysis of the cancer genome atlas AML database pinpointed G6PD as a new biomarker in AML, as its overexpression correlated with an adverse prognosis in this cohort. Targeting the PPP using the G6PD inhibitor 6-aminonicotinamide induces in vitro and in vivo cytotoxicity against AML cells and synergistically sensitizes leukemic cells to chemotherapy. Our results demonstrate that high mTORC1 activity creates a specific vulnerability to G6PD inhibition that may work as a new AML therapy.
Asunto(s)
Glucosafosfato Deshidrogenasa/antagonistas & inhibidores , Leucemia Mieloide Aguda/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Glucosa/metabolismo , Glucólisis , Humanos , Leucemia Mieloide Aguda/enzimología , Leucemia Mieloide Aguda/patología , Fosforilación OxidativaRESUMEN
Sepsis can provoke kidney injury, which increases mortality. Human and animal studies have documented increased renal oxidative injury and mitochondrial damage during sepsis. However, few studies have attempted to dissect specific renal targets and/or types of oxidative injury using the cecal ligation and puncture (CLP) murine model of sepsis. The purpose of this short communication is to examine the extent of lipid peroxidation within renal mitochondria using CLP and blue native gel electrophoresis which separates intact mitochondrial respiratory complexes. Our results show that CLP induced increased 4-hydroxy-nonenal protein adduction (marker of lipid peroxidation) in renal homogenates and mitochondrial fractions. Blue native gel electrophoresis revealed that respiratory complex III was selectively targeted within mitochondrial fractions. This supports our prior report showing renal complex III inactivation following CLP. Future studies will identify specific renal proteins within complex III that are modified during sepsis to provide mechanistic insight on how mitochondrial respiration is inhibited during sepsis.
RESUMEN
Sepsis-induced acute kidney injury (SAKI) is a frequent complication of infant sepsis that approximately doubles the mortality rate. The poor prognosis of these patients is a result of care that is mainly supportive, nontargeted, and usually begun only after symptoms of the systemic inflammatory response syndrome are observed. Preclinical studies from relevant rodent models of SAKI suggest that mitochondria-targeted antioxidants may be a new mode of therapy that could promote recovery.
Asunto(s)
Lesión Renal Aguda/tratamiento farmacológico , Antioxidantes/uso terapéutico , Mitocondrias/efectos de los fármacos , Compuestos Organofosforados/uso terapéutico , Piperidinas/uso terapéutico , Sepsis/complicaciones , Animales , Modelos Animales de Enfermedad , Humanos , Lactante , Ratones , Mitocondrias/metabolismo , RatasRESUMEN
The mTORC1 signaling pathway is constitutively activated in almost all acute myelogenous leukemia (AML) patients. We conducted a phase Ib trial combining RAD001 (everolimus), an allosteric inhibitor of mTORC1, and conventional chemotherapy, in AML patients under 65 years of age at first relapse (clinical trial NCT 01074086). Increasing doses of RAD001 from 10-70 mg were administrated orally on days 1 and 7 (d1 and d7) of a 3+7 daunorubicin+cytarabine conventional induction chemotherapy regimen. Twenty-eight patients were enrolled in this trial. The treatment was well tolerated with <10% toxicity, mainly involving the gastrointestinal tract and lungs. In this phase Ib trial, the RAD001 maximum tolerated dose was not reached at 70 mg. Sixty-eight percent of patients achieved CR, of which 14 received a double induction. Eight subsequently were intensified with allogeneic-stem cell transplant. Strong plasma inhibition of P-p70S6K was observed after RAD001 administration, still detectable at d7 (d7)at the 70 mg dosage. CR rates in patients with RAD001 areas under or above the curve median were 53% versus 85%. A 70 mg dose of RAD001 at d1 and d7 of an induction chemotherapy regimen for AML has acceptable toxicity and may improve treatment.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Citarabina/administración & dosificación , Daunorrubicina/administración & dosificación , Leucemia Mieloide Aguda/tratamiento farmacológico , Sirolimus/análogos & derivados , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Adulto , Anciano , Antibióticos Antineoplásicos/administración & dosificación , Antibióticos Antineoplásicos/efectos adversos , Antineoplásicos/administración & dosificación , Antineoplásicos/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Citarabina/efectos adversos , Daunorrubicina/efectos adversos , Everolimus , Femenino , Humanos , Masculino , Persona de Mediana Edad , Recurrencia , Transducción de Señal/efectos de los fármacos , Sirolimus/administración & dosificación , Sirolimus/efectos adversos , Resultado del Tratamiento , Adulto JovenAsunto(s)
Antimetabolitos Antineoplásicos/uso terapéutico , Azacitidina/uso terapéutico , Leucemia Mieloide Aguda/tratamiento farmacológico , Síndromes Mielodisplásicos/tratamiento farmacológico , Neoplasias Primarias Secundarias/tratamiento farmacológico , Receptor fas/genética , Biomarcadores , Islas de CpG , Metilación de ADN , Humanos , Regiones Promotoras Genéticas , Modelos de Riesgos Proporcionales , Riesgo , Receptor fas/análisisRESUMEN
The serine/threonine kinase mammalian target of rapamycin (mTOR) is crucial for cell growth and proliferation, and is constitutively activated in primary acute myeloid leukemia (AML) cells, therefore representing a major target for drug development in this disease. We show here that the specific mTOR kinase inhibitor AZD8055 blocked mTORC1 and mTORC2 signaling in AML. Particularly, AZD8055 fully inhibited multisite eIF4E-binding protein 1 phosphorylation, subsequently blocking protein translation, which was in contrast to the effects of rapamycin. In addition, the mTORC1-dependent PI3K/Akt feedback activation was fully abrogated in AZD8055-treated AML cells. Significantly, AZD8055 decreased AML blast cell proliferation and cell cycle progression, reduced the clonogenic growth of leukemic progenitors and induced caspase-dependent apoptosis in leukemic cells but not in normal immature CD34+ cells. Interestingly, AZD8055 strongly induced autophagy, which may be either protective or cell death inducing, depending on concentration. Finally, AZD8055 markedly increased the survival of AML transplanted mice through a significant reduction of tumor growth, without apparent toxicity. Our current results strongly suggest that AZD8055 should be tested in AML patients in clinical trials.
Asunto(s)
Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/prevención & control , Morfolinas/farmacología , Proteínas/antagonistas & inhibidores , Factores de Transcripción/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Western Blotting , Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Humanos , Técnicas para Inmunoenzimas , Inmunoprecipitación , Leucemia Mieloide Aguda/mortalidad , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones Desnudos , Complejos Multiproteicos , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfoproteínas/metabolismo , Fosforilación/efectos de los fármacos , Proteínas/metabolismo , Tasa de Supervivencia , Serina-Treonina Quinasas TOR , Factores de Transcripción/metabolismo , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Mammalian target of rapamycin (mTOR) is a protein kinase implicated in the regulation of various cellular processes, including those required for tumor development, such as the initiation of mRNA translation, cell-cycle progression and cellular proliferation. In a wide range of hematological malignancies, the mTORC1 signaling pathway has been found to be deregulated and has been designed as a major target for tumor therapy. Given that pre-clinical studies have clearly established the therapeutic value of mTORC1 inhibition, numerous clinical trials of rapamycin and its derivates (rapalogs) are ongoing for treatment of these diseases. At this time, although disease stabilization and tumor regression have been observed, objective responses in some tumor types have been modest. Nevertheless, some of the mechanisms underlying cancer-cell resistance to rapamycin have now been described, thereby leading to the development of new strategy to efficiently target mTOR signaling in these diseases. In this review, we discuss the rationale for using mTOR inhibitors as novel therapies for a variety of hematological, malignancies with a focus on promising new perspectives for these approaches.
Asunto(s)
Neoplasias Hematológicas/tratamiento farmacológico , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Ensayos Clínicos como Asunto , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Serina-Treonina Quinasas TORRESUMEN
Spontaneous apoptosis of bone marrow erythroid precursors accounts for the anemia that characterizes most low-grade myelodysplastic syndromes (MDS). We have shown that death of these precursors involved the Fas-dependent activation of caspase-8. To explore the pathway leading from caspase-8 activation to apoptosis, we transduced MDS bone marrow CD34(+) cells with a lentivirus encoding wild-type (WT) or endoplasmic reticulum (ER)-targeted Bcl-2 protein before inducing their erythroid differentiation. Both WT-Bcl-2 and ER-targeted Bcl-2 prevented spontaneous and Fas-dependent apoptosis in MDS erythroid precursors. ER-targeted Bcl-2 inhibited mitochondrial membrane depolarization and cytochrome c release in MDS erythroid precursors undergoing apoptosis, indicating a role for the ER in the death pathway, upstream of the mitochondria. MDS erythroid precursors demonstrated elevated ER Ca(2+) stores and these stores remained unaffected by ER-targeted Bcl-2. The ER-associated protein Bcl-2-associated protein (BAP) 31 was cleaved by caspase-8 in MDS erythroid precursors undergoing apoptosis. The protective effect of ER-targeted Bcl-2 toward spontaneous and Fas-induced apoptosis correlated with inhibition of BAP31 cleavage. A protective effect of erythropoietin against Fas-induced BAP31 cleavage and apoptosis was observed. We propose that apoptosis of MDS erythroid precursors involves the ER, downstream of Fas and upstream of the mitochondria, through the cleavage of the ER-associated BAP31 protein.
Asunto(s)
Apoptosis , Retículo Endoplásmico/fisiología , Células Precursoras Eritroides/fisiología , Síndromes Mielodisplásicos/sangre , Receptor fas/fisiología , Anemia/etiología , Calcio/metabolismo , Inhibidores de Caspasas , Células Precursoras Eritroides/química , Eritropoyetina/uso terapéutico , Humanos , Proteínas de la Membrana/metabolismo , Mitocondrias/fisiología , Síndromes Mielodisplásicos/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Proteínas Proto-Oncogénicas c-bcl-2/fisiologíaRESUMEN
The phosphatidylinositol 3-kinase (PI3K)/Akt and mammalian target of rapamycin complex 1 (mTORC1) signaling pathways are frequently activated in acute myelogenous leukemia (AML). mTORC1 inhibition with RAD001 induces PI3K/Akt activation and both pathways are activated independently, providing a rationale for dual inhibition of both pathways. PI-103 is a new potent PI3K/Akt and mTOR inhibitor. In human leukemic cell lines and in primary blast cells from AML patients, PI-103 inhibited constitutive and growth factor-induced PI3K/Akt and mTORC1 activation. PI-103 was essentially cytostatic for cell lines and induced cell cycle arrest in the G1 phase. In blast cells, PI-103 inhibited leukemic proliferation, the clonogenicity of leukemic progenitors and induced mitochondrial apoptosis, especially in the compartment containing leukemic stem cells. In contrast, apoptosis was not induced with RAD001 and IC87114 association, which specifically inhibits mTORC1 and p110delta activity, respectively. PI-103 had additive proapoptotic effects with etoposide in blast cells and in immature leukemic cells. Interestingly, PI-103 did not induce apoptosis in normal CD34(+) cells and had moderate effects on their clonogenic and proliferative properties. Here, we demonstrate that multitargeted therapy against PI3K/Akt and mTOR with PI-103 may be of therapeutic value in AML.
Asunto(s)
Antineoplásicos/farmacología , Furanos/farmacología , Leucemia Mieloide Aguda/tratamiento farmacológico , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/efectos de los fármacos , Piridinas/farmacología , Pirimidinas/farmacología , Apoptosis/efectos de los fármacos , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/patología , Furanos/uso terapéutico , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Leucemia Mieloide Aguda/patología , Células Madre Neoplásicas/efectos de los fármacos , Inhibidores de Proteínas Quinasas/uso terapéutico , Piridinas/uso terapéutico , Pirimidinas/uso terapéutico , Serina-Treonina Quinasas TOR , Células Tumorales CultivadasRESUMEN
In a 70 year old man with primary glomerulonephritis, severe anemia occurred after 4 years on hemodialysis and rHu-EPO. The usual mechanisms of EPO-resistance were excluded. A bone marrow sample showed red all aplasia. No circulating EPO could be detected; the serum inhibited the growth of erythroid precursors in bone marrow cultures. Immunoprecipitation identified an IgG anti-EPO, still active against deglycosylated EPO, i.e. directed against the peptidic matrix. Its high neutralising capacity and the absence of any immune abnormality rule out an auto-antibody. Anti-rHu EPO immunisation is a very rare occurrence, made severe by transfusion-dependence and the risk of hemosiderosis. An immuno-modulating treatment can therefore be justified.
Asunto(s)
Anemia/tratamiento farmacológico , Eritropoyetina/inmunología , Inmunoglobulina G/inmunología , Isoanticuerpos/inmunología , Anciano , Anemia/sangre , Anemia/etiología , Angiodisplasia/complicaciones , Transfusión Sanguínea , Médula Ósea/patología , División Celular/efectos de los fármacos , Terapia Combinada , Sistema Digestivo/irrigación sanguínea , Resistencia a Medicamentos/inmunología , Células Precursoras Eritroides/patología , Eritropoyetina/uso terapéutico , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Hematemesis/tratamiento farmacológico , Hematemesis/etiología , Hematemesis/terapia , Humanos , Inmunoglobulina G/biosíntesis , Isoanticuerpos/biosíntesis , Fallo Renal Crónico/complicaciones , Fallo Renal Crónico/terapia , Leucemia Eritroblástica Aguda/patología , Masculino , Melena/tratamiento farmacológico , Melena/etiología , Melena/terapia , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/uso terapéutico , Diálisis Renal , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
We recently reported that nitrotyrosine and acetaminophen (APAP)-cysteine protein adducts colocalize in the hepatic centrilobular cells following a toxic dose of APAP to mice. Whereas APAP-adducts are formed by reaction of the metabolite N-acetyl-p-benzoquinone imine with cysteine, nitrotyrosine residues are formed by reaction of tyrosine with peroxynitrite. Peroxynitrite is formed from nitric oxide (NO) and superoxide. This manuscript examines APAP (300 mg/kg) hepatotoxicity in mice lacking inducible nitric oxide synthase activity (NOS2 null or knockout mice; C57BL/6-Nos2(tm1Lau)) and in the wildtype mice. In a time course the ALT levels in the exposed NOS2 null mice were approximately 50% of the wildtype mice; however, histological examination of liver sections indicated similar levels of centrilobular hepatic necrosis in both wild-type and NOS2 null mice. Serum nitrate plus nitrite levels (NO synthesis) were identical in saline-treated NOS2 null and wild-type mice (53 +/- 2 microM). APAP increased NO synthesis in wild-type mice only. The increases paralleled the increases in ALT levels with peak levels of serum nitrate plus nitrite at 6 h (168 +/- 27 microM). In wild-type mice hepatic tyrosine nitration was greatly increased relative to saline treated controls. Tyrosine nitration increased in NOS2 null mice also, but the increase was much less. APAP increased hepatic malonaldehyde levels (lipid peroxidation) in NOS2 null mice only. The results suggest the presence of multiple pathways to APAP-mediated hepatic necrosis, one via nitrotyrosine, as in the wild-type mice, and another that is not dependent upon inducible nitric oxide synthase activity, but which may involve increased superoxide.
Asunto(s)
Acetaminofén/toxicidad , Analgésicos no Narcóticos/toxicidad , Hígado/efectos de los fármacos , Óxido Nítrico Sintasa/deficiencia , Tirosina/análogos & derivados , Alanina Transaminasa/sangre , Animales , Aspartato Aminotransferasas/sangre , Eliminación de Gen , Técnicas para Inmunoenzimas , Peroxidación de Lípido , Hígado/enzimología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Nitratos/metabolismo , Óxido Nítrico/biosíntesis , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo II , Nitritos/metabolismo , Tirosina/metabolismoRESUMEN
During the cell transformation processes leading to erythroleukemia, erythroid progenitors often become erythropoietin (Epo)-independent for their proliferation. The biochemical events that could lead an erythroleukemic cell to growth factor-independence were investigated using spi-1 transgenic poerythroblasts. Spi-1/PU.1 is a myeloid and B-cell transcription factor of the ETS family and is activated by insertional mutagenesis during Friend erythroleukemia. Its overexpression in proerythroblasts induces their differentiation arrest without altering their erythropoietin requirement for proliferation (HS1 cells). At a later step, genetic alterations most probably occur allowing spi-1 transgenic poerythroblasts to proliferate in the absence of erythropoietin (HS2 cells). The signaling transduction pathways in HS1 and HS2 proerythroblasts were analyzed. The authors have previously shown that the Jak/STAT pathway was not activated in Epo-independent cells, but remained sensitive to Epo stimulation. In the present study, it is shown that the Epo-independent proliferation of HS2 cells requires active phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways. In these cells, PI3K was constitutively associated with the molecular adapters Grb2 and Gab1, and with the phosphatases SHP-2 and SHIP. Moreover, PI3K activity was correlated with the constitutive phosphorylation of serine-threonine protein kinase (AKT) in HS2 cells. Lastly, a constitutive activation of the MAPKs extracellular signal-regulated kinases (ERK1/2) in HS2 cells was observed that occurs in a PI3K-independent manner, but depends strictly on the activity of the protein kinase C (PKC). These results suggest that constitutive activations of PI3K/AKT and PKC/MAPK pathways can act in synergy to lead a proerythroblast to proliferate without Epo.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Eritroblastos/fisiología , Eritropoyetina/farmacología , Sistema de Señalización de MAP Quinasas/fisiología , Proteínas Quinasas Activadas por Mitógenos/sangre , Fosfatidilinositol 3-Quinasas/sangre , Receptores de Eritropoyetina/genética , Animales , Secuencia de Bases , División Celular/efectos de los fármacos , Células Cultivadas , Cartilla de ADN , ADN Complementario , Eritroblastos/efectos de los fármacos , Proteína Adaptadora GRB2 , Cinética , Ratones , Ratones Transgénicos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosforilación , Proteína Quinasa C/metabolismo , Subunidades de Proteína , Proteínas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Previous evidence suggests that both oxygen radicals and nitric oxide (NO) are important mediators of injury during renal ischemia-reperfusion (I-R) injury. However, the generation of reactive nitrogen species (RNS) has not been evaluated in this model at early time points. The purpose of these studies was to examine the development of oxidant stress and the formation of RNS during I-R injury. Male Sprague-Dawley rats were anesthetized and subjected to 40 min of bilateral renal ischemia followed by 0, 3, or 6 h of reperfusion. Control animals received a sham operation. Plasma urea nitrogen and creatinine levels were monitored as markers of renal injury. Glutathione (GSH) oxidation and 4-hydroxynonenal (4-HNE)-protein adducts were used as markers of oxidant stress. 3-Nitrotyrosine (3-NT) was used as a biomarker of RNS formation. Significant increases in plasma creatinine concentrations and urea nitrogen levels were found following both 3 and 6 h of reperfusion. Increases in GSH oxidation, 4-HNE-protein adduct levels, and 3-NT levels were observed following 40 min of ischemia with no reperfusion. Since these results suggested RNS generation during the 40 min of ischemia, a time course of RNS generation following 0, 5, 10, 20, and 40 min of ischemia was evaluated. Significant increases in 3-NT generation was detected as early as 10 min of ischemia and rose to values nearly 10-fold higher than Control at 40 min of ischemia. No additional increase was observed following reperfusion. The data clearly demonstrate that oxidative stress and RNS generation occur in the kidney during ischemia.
Asunto(s)
Riñón/irrigación sanguínea , Nitratos/metabolismo , Estrés Oxidativo , Daño por Reperfusión/metabolismo , Tirosina/análogos & derivados , Aldehídos/metabolismo , Animales , Nitrógeno de la Urea Sanguínea , Creatinina/sangre , Radicales Libres/metabolismo , Glutatión/metabolismo , Riñón/patología , Masculino , Oxidación-Reducción , Proteínas/metabolismo , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/sangre , Daño por Reperfusión/patología , Factores de Tiempo , Tirosina/metabolismoRESUMEN
In this study, we show that upon thrombopoietin (Tpo) stimulation the two adapter proteins Gab1 and Gab2 are strongly tyrosine phosphorylated and associated with Shc, SHP2, PI 3-kinase and Grb2 in mpl-expressing UT7 cells. Although Gab1 and Gab2 seem to mediate overlapping biological signals in many cells, only Gab1 is expressed and phosphorylated in response to Tpo in primary human megakaryocytic progenitors; furthermore, it associates with the same proteins. Although a low level of tyrosine phosphorylated IRS-2 protein is also detected in PI 3-kinase immunoprecipitates, Gab proteins are the essential proteins associated with PI 3-kinase after Tpo stimulation. We demonstrate that, albeit no association is detected between the Tpo receptor mpl and Gab proteins, Y112 located in the C-terminal cytoplasmic domain of mpl is required for Gab1/2 tyrosine phosphorylation. Gab proteins are not tyrosine phosphorylated after Tpo stimulation of UT-7 and Ba/F3 cells expressing a mpl mutant lacking Y112. Moreover, no activation of the PI 3-kinase/Akt pathway is observed in cells expressing this mpl mutant. Finally, we show that this mutant does not allow cell proliferation, thereby confirming that PI 3-kinase activation is required for Tpo-induced cell proliferation.
Asunto(s)
Fosfatidilinositol 3-Quinasas/metabolismo , Fosfoproteínas/fisiología , Proteínas Serina-Treonina Quinasas , Trombopoyetina/farmacología , Proteínas Adaptadoras Transductoras de Señales , Animales , División Celular/efectos de los fármacos , División Celular/fisiología , Activación Enzimática/efectos de los fármacos , Sangre Fetal/citología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Humanos , Megacariocitos/citología , Megacariocitos/efectos de los fármacos , Megacariocitos/metabolismo , Ratones , Fosfoproteínas/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Conejos , Proteínas Recombinantes/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Trombopoyetina/genética , Tirosina/metabolismoRESUMEN
ANG II exerts a biphasic effect on Na+ transport in the kidney through its effects on Na+-K+-ATPase activity. Beginning at 10(-13) M, ANG II increased Na+-K+-ATPase in freshly isolated rat proximal tubules to a maximum stimulation at 10(-11) M of 1.43 +/- 0.08-fold above control. Stimulation decreased progressively at concentrations >10(-10) M to a value of 0.96 +/- 0.1-fold at 10(-7) M. In the presence of additional L-arginine, the substrate for NO synthesis, the stimulatory effect of ANG II (10(-11) M) was lost. Conversely, N-monomethyl-L-arginine (L-NMMA), the nitric oxide (NO) synthase inhibitor, unmasked the stimulatory effect of ANG II at 10(-7) M (1.40 +/- 0.1-fold). 1H-[1,2,4]oxadiazole-[4,3-a]quinoxalin-1-one, the soluble guanylyl cyclase inhibitor, like L-NMMA, unmasked the stimulatory effect of ANG II at 10(-7) M (1.30 +/- 0.1-fold). The intracellular cGMP concentration was increased 1.58 +/- 0.28-fold at 10(-7) M ANG II. The ANG II AT(1) receptor antagonist SK&F 108566 blocked the stimulatory effect of ANG II at 10(-11) M. These data suggest that the NO/cGMP signaling pathway serves as a negative component in the regulation of Na+-K+-ATPase activity by ANG II.
Asunto(s)
Angiotensina II/fisiología , GMP Cíclico/fisiología , Túbulos Renales Proximales/enzimología , Óxido Nítrico/fisiología , Transducción de Señal/fisiología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Angiotensina II/farmacología , Antagonistas de Receptores de Angiotensina , Animales , Arginina/farmacología , Calcio/metabolismo , GMP Cíclico/biosíntesis , Inhibidores Enzimáticos/farmacología , Guanilato Ciclasa/antagonistas & inhibidores , Membranas Intracelulares/metabolismo , Túbulos Renales Proximales/metabolismo , Masculino , Oxadiazoles/farmacología , Quinoxalinas/farmacología , Ratas , Ratas Sprague-Dawley , Receptor de Angiotensina Tipo 1 , Receptor de Angiotensina Tipo 2 , Solubilidad , omega-N-Metilarginina/farmacologíaRESUMEN
Although the mechanisms involved in the activation of mitogen-activated protein kinases (MAPK) by receptor tyrosine kinases do not display an obvious role for phosphoinositide 3-kinases (PI3Ks), we have observed in the nontransformed cell line Vero stimulated with epidermal growth factor (EGF) that wortmannin and LY294002 nearly abolished MAPK activation. The effect was observed under strong stimulation and was independent of EGF concentration. In addition, three mutants of class Ia PI3Ks were found to inhibit MAPK activation to an extent similar to their effect on Akt/protein kinase B activation. To determine the importance of PI3K lipid kinase activity in MAPK activation, we have used the phosphatase PTEN and the pleckstrin homology domain of Tec kinase. Overexpression of these proteins, but not control mutants, was found to inhibit MAPK activation, suggesting that the lipid products of class Ia PI3K are necessary for MAPK signaling. We next investigated the location of PI3K in the MAPK cascade. Pharmacological inhibitors and dominant negative forms of PI3K were found to block the activation of Ras induced by EGF. Upstream from Ras, although association of Grb2 with its conventional effectors was independent of PI3K, we have observed that the recruitment of the tyrosine phosphatase SHP2 required PI3K. Because SHP2 was also essential for Ras activation, this suggested the existence of a PI3K/SHP2 pathway leading to the activation of Ras. In addition, we have observed that the docking protein Gab1, which is involved in PI3K activation during EGF stimulation, is also implicated in this pathway downstream of PI3K. Indeed, the association of Gab1 with SHP2 was blocked by PI3K inhibitors, and expression of Gab1 mutant deficient for binding to SHP2 was found to inhibit Ras stimulation without interfering with PI3K activation. These results show that, in addition to Shc and Grb2, a PI3K-dependent pathway involving Gab1 and SHP2 is essential for Ras activation under EGF stimulation.