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Class I PI3K enzymes play critical roles in B cell activation by phosphorylating plasma membrane lipids to generate two distinct phosphoinositide (PI) products, PI(3,4,5)P3 and PI(3,4)P2. These PIs each bind distinct but overlapping sets of intracellular proteins that control cell survival, cytoskeletal reorganization, and metabolic activity. The tandem PH domain containing proteins (TAPPs) bind with high specificity to PI(3,4)P2, and their genetic uncoupling from PI(3,4)P2 in TAPP knock in (KI) mice was previously found to cause chronic B cell activation, abnormal germinal centers (GCs), and autoimmunity. In this article, we find that TAPPs provide feedback regulation affecting PI3K signaling and metabolic activation of B cells. Upon activation, TAPP KI B cells show enhanced metabolic activity associated with increased extracellular acidification rate, increased expression of glucose transporter GLUT1, and increased glucose uptake. TAPP KI B cells show markedly increased activation of the PI3K-regulated kinases Akt, GSK3ß, and p70-S6K. Conversely, overexpression of the C-terminal TAPP PH domains in B cells can inhibit Akt phosphorylation by a mechanism requiring the TAPP PI(3,4)P2-binding pocket. Inhibition of the PI3K pathway in TAPP KI B cells reduced GLUT1 expression and glucose uptake, whereas inhibition of Akt alone was not sufficient to normalize these responses. TAPP KI GC B cells also show increased GLUT1 and glucose uptake, and treatment with the inhibitor of glycolysis 2-deoxy-D-glucose reduced chronic GC responses and autoantibody production within these mice. Our findings show that TAPP-PI(3,4)P2 interaction controls activation of glycolysis and highlights the significance of this pathway for B cell activation, GC responses, and autoimmunity.
Asunto(s)
Autoinmunidad/inmunología , Linfocitos B/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de Señal/fisiología , Animales , Linfocitos B/inmunología , Células Cultivadas , Femenino , Centro Germinal/inmunología , Centro Germinal/metabolismo , Transportador de Glucosa de Tipo 1/metabolismo , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Fosfatidilinositol 3-Quinasas/inmunología , Fosfatos de Fosfatidilinositol/metabolismo , Fosforilación/inmunología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismoRESUMEN
The epigenetic factor Methyl-CpG-Binding Protein 2 (MeCP2) is a nuclear protein that binds methylated DNA molecules (both 5-methylcytosine and 5-hydroxymethylcytosine) and controls gene transcription. MeCP2 is an important transcription factor that acts in a dose-dependent manner in the brain; thus, its optimal expression level in brain cells is important. As such, its deregulated expression, as well as gain- or loss-of-function mutation, lead to impaired neurodevelopment, and compromised structure and function of brain cells, particularly in neurons. Studies from others and us have characterized two well-recognized MeCP2 isoforms: MeCP2E1 and MeCP2E2. We have reported that in Daoy medulloblastoma brain cells, MeCP2E2 overexpression leads to MeCP2E1 protein degradation. Whether MeCP2 isoforms regulate the Mecp2 promoter regulatory elements remains unexplored. We previously showed that in Daoy cells, metformin (an anti-diabetic drug) induces MECP2E1 transcripts. However, possible impact of metformin on the Mecp2 promoter activity was not studied. Here, we generated stably transduced Daoy cell reporters to express EGFP driven by the Mecp2 promoter. Transduced cells were sorted into four EGFP-expressing groups (R4-to-R7) with different intensities of EGFP expression. Our results confirm that the Mecp2 promoter is active in Daoy cells, and that overexpression of either isoform inhibits the Mecp2 promoter activity, as detected by flow cytometry and luciferase reporter assays. Interestingly, metformin partially relieved the inhibitory effect of MeCP2E1 on the Mecp2 promoter, detected by flow cytometry. Taken together, our data provide important insight towards the regulation of MeCP2 isoforms at the promoter level, which might have biological relevance to the neurobiology of the brain.
Asunto(s)
Neoplasias Cerebelosas , Metformina , Humanos , Proteína 2 de Unión a Metil-CpG/genética , Proteína 2 de Unión a Metil-CpG/metabolismo , Retroalimentación , Metformina/farmacología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMEN
Background Unlike T-wave alternans (TWA), the relation between QRS alternans (QRSA) and ventricular arrhythmia (VA) risk has not been evaluated in hypertrophic cardiomyopathy (HCM). We assessed microvolt QRSA/TWA in relation to HCM risk factors and late VA outcomes in HCM. Methods and Results Prospectively enrolled patients with HCM (n=130) with prophylactic implantable cardioverter-defibrillators underwent digital 12-lead ECG recordings during ventricular pacing (100-120 beats/min). QRSA/TWA was quantified using the spectral method. Patients were categorized as QRSA+ and/or TWA+ if sustained alternans was present in ≥2 precordial leads. The VA end point was appropriate implantable cardioverter-defibrillator therapy over 5 years of follow-up. QRSA+ and TWA+ occurred together in 28% of patients and alone in 7% and 7% of patients, respectively. QRSA magnitude increased with pacing rate (1.9±0.6 versus 6.2±2.0 µV; P=0.006). Left ventricular thickness was greater in QRSA+ than in QRSA- patients (22±7 versus 20±6 mm; P=0.035). Over 5 years follow-up, 17% of patients had VA. The annual VA rate was greater in QRSA+ versus QRSA- patients (5.8% versus 2.0%; P=0.006), with the QRSA+/TWA- subgroup having the greatest rate (13.3% versus 2.6%; P<0.001). In those with <2 risk factors, QRSA- patients had a low annual VA rate compared QRSA+ patients (0.58% versus 7.1%; P=0.001). Separate Cox models revealed QRSA+ (hazard ratio [HR], 2.9 [95% CI, 1.2-7.0]; P=0.019) and QRSA+/TWA- (HR, 7.9 [95% CI, 2.9-21.7]; P<0.001) as the most significant VA predictors. TWA and HCM risk factors did not predict VA. Conclusions In HCM, microvolt QRSA is a novel, rate-dependent phenomenon that can exist without TWA and is associated with greater left ventricular thickness. QRSA increases VA risk 3-fold in all patients, whereas the absence of QRSA confers low VA risk in patients with <2 risk factors. Registration URL: https://www.clinicaltrials.gov; Unique identifier: NCT02560844.
Asunto(s)
Arritmias Cardíacas , Cardiomiopatía Hipertrófica , Arritmias Cardíacas/epidemiología , Cardiomiopatía Hipertrófica/fisiopatología , Humanos , Factores de RiesgoRESUMEN
Medulloblastoma is a common pediatric brain tumor and one of the main types of solid cancers in children below the age of 10. Recently, cholesterol-lowering "statin" drugs have been highlighted for their possible anti-cancer effects. Clinically, statins are reported to have promising potential for consideration as an adjuvant therapy in different types of cancers. However, the anti-cancer effects of statins in medulloblastoma brain tumor cells are not currently well-defined. Here, we investigated the cell death mechanisms by which simvastatin mediates its effects on different human medulloblastoma cell lines. Simvastatin is a lipophilic drug that inhibits HMG-CoA reductase and has pleotropic effects. Inhibition of HMG-CoA reductase prevents the formation of essential downstream intermediates in the mevalonate cascade, such as farnesyl pyrophosphate (FPP) and gernaylgerany parophosphate (GGPP). These intermediates are involved in the activation pathway of small Rho GTPase proteins in different cell types. We observed that simvastatin significantly induces dose-dependent apoptosis in three different medulloblastoma brain tumor cell lines (Daoy, D283, and D341 cells). Our investigation shows that simvastatin-induced cell death is regulated via prenylation intermediates of the cholesterol metabolism pathway. Our results indicate that the induction of different caspases (caspase 3, 7, 8, and 9) depends on the nature of the medulloblastoma cell line. Western blot analysis shows that simvastatin leads to changes in the expression of regulator proteins involved in apoptosis, such as Bax, Bcl-2, and Bcl-xl. Taken together, our data suggests the potential application of a novel non-classical adjuvant therapy for medulloblastoma, through the regulation of protein prenylation intermediates that occurs via inhibition of the mevalonate pathway.
RESUMEN
Rett syndrome (RTT) is a severe and rare neurological disorder that is caused by mutations in the X-linked MECP2 (methyl CpG-binding protein 2) gene. MeCP2 protein is an important epigenetic factor in the brain and in neurons. In Mecp2-deficient neurons, nucleoli structures are compromised. Nucleoli are sites of active ribosomal RNA (rRNA) transcription and maturation, a process mainly controlled by nucleolin and mechanistic target of rapamycin (mTOR)-P70S6K signaling. Currently, it is unclear how nucleolin-rRNA-mTOR-P70S6K signaling from RTT cellular model systems translates into human RTT brain. Here, we studied the components of nucleolin-rRNA-mTOR-P70S6K signaling in the brain of RTT patients with common T158M and R255X mutations. Immunohistochemical examination of T158M brain showed disturbed nucleolin subcellular localization, which was absent in Mecp2-deficient homozygous male or heterozygote female mice, compared to wild type (WT). We confirmed by Western blot analysis that nucleolin protein levels are altered in RTT brain, but not in Mecp2-deficient mice. Further, we studied the expression of rRNA transcripts in Mecp2-deficient mice and RTT patients, as downstream molecules that are controlled by nucleolin. By data mining of published ChIP-seq studies, we showed MeCP2-binding at the multi-copy rRNA genes in the mouse brain, suggesting that rRNA might be a direct MeCP2 target gene. Additionally, we observed compromised mTOR-P70S6K signaling in the human RTT brain, a molecular pathway that is upstream of rRNA-nucleolin molecular conduits. RTT patients showed significantly higher phosphorylation of active mTORC1 or mTORC2 complexes compared to age- and sex-matched controls. Correlational analysis of mTORC1/2-P70S6K signaling pathway identified multiple points of deviation from the control tissues that may result in abnormal ribosome biogenesis in RTT brain. To our knowledge, this is the first report of deregulated nucleolin-rRNA-mTOR-P70S6K signaling in the human RTT brain. Our results provide important insight toward understanding the molecular properties of human RTT brain.
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Cardiovascular disease leading to heart failure (HF) remains a leading cause of morbidity and mortality worldwide. Improved pharmacological and interventional coronary procedures have led to improved outcomes following acute myocardial infarction. This success has translated into an unforeseen increased incidence in HF. This review summarizes the signaling pathways implicated in the transition to HF following cardiac injury. In addition, we provide an update on cell death signaling and discuss recent advances in cardiac fibrosis as an independent event leading to HF. Finally, we discuss cell-based therapies and their possible use to avert the deteriorating nature of HF. © 2019 American Physiological Society. Compr Physiol 9:75-125, 2019.