RESUMEN
The downstream signaling of the interleukin-7 (IL-7) receptor (IL-7R) plays important physiological and pathological roles, including the differentiation of lymphoid cells and proliferation of acute lymphoblastic leukemia cells. Gain-of-function mutations in the IL-7Rα chain, the specific component of the receptor for IL-7, result in constitutive, IL-7-independent signaling and trigger acute lymphoblastic leukemia. Here, we show that the loss of the phosphoinositide 5-phosphatase INPP5K is associated with increased levels of the INPP5K substrate phosphatidylinositol 4,5-bisphosphate (PtdIns[4,5]P2) and causes an altered dynamic structure of the IL-7 receptor. We discovered that the IL-7Rα chain contains a very conserved positively charged polybasic amino acid sequence in its cytoplasmic juxtamembrane region; this region establish stronger ionic interactions with negatively charged PtdIns(4,5)P2 in the absence of INPP5K, freezing the IL-7Rα chain structure. This dynamic structural alteration causes defects in IL-7R signaling, culminating in decreased expressions of EBF1 and PAX5 transcription factors, in microdomain formation, cytoskeletal reorganization, and bone marrow B-cell differentiation. Similar alterations after the reduced INPP5K expression also affected mutated, constitutively activated IL-7Rα chains that trigger leukemia development, leading to reduced cell proliferation. Altogether, our results indicate that the lipid 5-phosphatase INPP5K hydrolyzes PtdIns(4,5)P2, allowing the requisite conformational changes of the IL-7Rα chain for optimal signaling.
Asunto(s)
Interleucina-7 , Leucemia-Linfoma Linfoblástico de Células Precursoras , Humanos , Interleucina-7/genética , Interleucina-7/metabolismo , Fosfatidilinositol 4,5-Difosfato , Receptores de Interleucina-7/genética , Receptores de Interleucina-7/metabolismo , Transducción de Señal/genéticaRESUMEN
Cell apoptosis is an important process that occurs during development or in response to stress stimuli such as oxidative stress. The serine-threonine kinase Akt enhances survival and suppress apoptosis. SHIP2 is known as a negative regulator of Akt. In addition to its lipid 5'-phosphatase activity, SHIP2 interacts and signals as a scaffolding complex with several proteins. Several findings have pointed out a possible role of SHIP2 in apoptosis regulation. However, the molecular mechanisms behind remain unknown. Using embryonic fibroblast lacking the lipid 5'-phosphatase domain as a genetic model system and human liver cancer cells treated with SHIP2 inhibitor (AS1949490), as a pharmacological model system. We provide the first evidence that SHIP2 regulates apoptosis independently of its 5'-phosphates activity. Indeed, absence of the 5'-phosphatase domain of SHIP2 did not prevent H2O2-induced apoptosis in fibroblasts. Whereas chemical inactivation or RNAi knockdown of SHIP2 blocked H2O2-induced apoptosis in HepG2 cells. We found that suppression of apoptosis upon SHIP2 inhibition is PI3K/Akt independent but rather MAP kinase dependent. In addition, we found that AS1949490 altered both 5'-phosphatase and scaffolding function of SHIP2. Indeed, AS1949490 mediated SHIP2 inhibition promotes protein complex formation of SHIP2 together with non-receptor tyrosine kinase SRC and ABL which in turn enhances PI3K/Akt and MAP kinase pathways activation. Dual inhibition of SRC/ABL blocked activation of both pathways upon SHIP2 inhibition and H2O2 treatment. Altogether, these findings indicate that SHIP2 protein play a determinant role in H2O2-induced apoptosis.
Asunto(s)
Apoptosis , Estrés Oxidativo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas/metabolismo , Apoptosis/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Células Hep G2 , Humanos , Peróxido de Hidrógeno/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Estrés Oxidativo/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Tiofenos/farmacologíaRESUMEN
The transcriptional architecture of intracellular circadian clocks is similar across phyla, but in mammals interneuronal mechanisms confer a higher level of circadian integration. The suprachiasmatic nucleus (SCN) is a unique model to study these mechanisms, as it operates as a â¼24 h clock not only in the living animal, but also when isolated in culture. This "clock in a dish" can be used to address fundamental questions, such as how intraneuronal mechanisms are translated by SCN neurons into circuit-level emergent properties and how the circuit decodes, and responds to, light input. This review addresses recent developments in understanding the relationship between electrical activity, [Ca(2+)]i, and intracellular clocks. Furthermore, optogenetic and chemogenetic approaches to investigate the distinct roles of neurons and glial cells in circuit encoding of circadian time will be discussed, as well as the epigenetic and circuit-level mechanisms that enable the SCN to translate light input into coherent daily rhythms.
Asunto(s)
Relojes Circadianos/genética , Relojes Circadianos/fisiología , Ritmo Circadiano/genética , Ritmo Circadiano/fisiología , Neuronas/fisiología , Núcleo Supraquiasmático/citología , Núcleo Supraquiasmático/fisiología , Animales , Péptidos y Proteínas de Señalización del Ritmo Circadiano/genética , Expresión Génica/fisiología , Mamíferos/fisiología , Neuroglía/fisiología , Fotoperiodo , TiempoRESUMEN
Although circadian rhythms in mammalian physiology and behavior are dependent upon a biological clock in the suprachiasmatic nuclei (SCN) of the hypothalamus, the molecular mechanism of this clock is in fact cell autonomous and conserved in nearly all cells of the body. Thus, the SCN serves in part as a "master clock," synchronizing "slave" clocks in peripheral tissues, and in part directly orchestrates circadian physiology. In this chapter, we first consider the detailed mechanism of peripheral clocks as compared to clocks in the SCN and how mechanistic differences facilitate their functions. Next, we discuss the different mechanisms by which peripheral tissues can be entrained to the SCN and to the environment. Finally, we look directly at how peripheral oscillators control circadian physiology in cells and tissues.
Asunto(s)
Relojes Circadianos/fisiología , Ritmo Circadiano/fisiología , Animales , Alimentos , Humanos , Núcleo Supraquiasmático/fisiología , TemperaturaRESUMEN
Circadian clock controls daily behavior and physiology. The activity of various signaling pathways affects clock gene expression. Here, we show that the core circadian clock gene CRY1 is a direct target of the Hippo pathway effector YAP. YAP binds to TEADs and occupies the proximal promoter regions of CRY1, positively regulating its transcription. Interestingly, we further identified that CRY1 acts in a feedback loop to fine-tune Hippo pathway activation by modulating the expression of YAP and MOB1. Indeed, loss of CRY1 results in enhanced YAP activation. Consistently, we found that YAP levels and activity control clock gene expression and oscillation in synchronized cells. Furthermore, in breast cancer cells, CRY1 downregulation causes YAP/TAZ hyperactivation and enhanced DNA damage. Together, our findings provide a direct mechanistic link between the Hippo pathway and the circadian clock, where CRY1 and Hippo components form an orchestrated signaling network that influences cell growth and circadian rhythm.
RESUMEN
Cell polarity is a common feature of many living cells, especially epithelial cells, and plays important roles in development, tissue homeostasis, and diseases. Therefore, the signaling pathways involved in establishing and maintaining cell polarity are tightly controlled. Protein S-palmitoylation has been recently recognized as an important posttranslational modification involved in cell polarity, via dynamic covalent attachment of fatty acyl groups to the cysteine residues of cell polarity proteins. Here, we describe the methods to study the function and regulation of S-palmitoylation of cell polarity proteins.
Asunto(s)
Polaridad Celular , Lipoilación , Cisteína/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas/metabolismoRESUMEN
Phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P3) is required for protein kinase B (AKT) activation. The level of PI(3,4,5)P3 is constantly regulated through balanced synthesis by phosphoinositide 3-kinase (PI3K) and degradation by phosphoinositide phosphatases phosphatase and tensin homologue (PTEN) and SH2-domain containing phosphatidylinositol-3,4,5-trisphosphate 5-phosphatase 2 (SHIP2), known as negative regulators of AKT. Here, I show that SHIP2 inhibition in cervical cancer cell lines alters H2 O2 -mediated AKT and mitogen-activated protein kinase/extracellular signal-regulated kinase pathway activation. In addition, SHIP2 inhibition enhances reactive oxygen species generation. Interestingly, I found that SHIP2 inhibition and H2 O2 treatment enhance lipid and protein phosphatase activity of PTEN. Pharmacological targeting or RNA interference(RNAi) mediated knockdown of PTEN rescues extracellular signal-regulated kinase and AKT activation. Using a series of pharmacological and biochemical approaches, I provide evidence that crosstalk between SHIP2 and PTEN occurs upon an increase in oxidative stress to modulate the activity of mitogen-activated protein kinase and phosphoinositide 3/ATK pathways.
Asunto(s)
Fosfohidrolasa PTEN/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas/metabolismo , Neoplasias del Cuello Uterino/metabolismo , Femenino , Células HEK293 , Humanos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , Fosfohidrolasa PTEN/fisiología , Fosfatidilinositol 3-Quinasa/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas/fisiología , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Neoplasias del Cuello Uterino/genéticaRESUMEN
Opsismodysplasia (OPS) is a rare but severe autosomal recessive skeletal chondrodysplasia caused by inactivating mutations in the Inppl1/Ship2 gene. The molecular mechanism leading from Ship2 gene inactivation to OPS is currently unknown. Here, we used our Ship2Δ/Δ mouse expressing reduced amount of a catalytically-inactive SHIP2 protein and a previously reported SHIP2 inhibitor to investigate growth plate development and mineralization in vivo, ex vivo and in vitro. First, as observed in OPS patients, catalytic inactivation of SHIP2 in mouse leads to reduced body length, shortening of long bones, craniofacial dysmorphism, reduced height of the hyperthrophic chondrocyte zone and to defects in growth plate mineralization. Second, intrinsic Ship2Δ/Δ bone defects were sufficient to induce the characteristic OPS alterations in bone growth, histology and mineralization ex vivo. Third, expression of osteocalcin was significantly increased in SHIP2-inactivated chondrocyte cultures whereas production of mineralized nodules was markedly decreased. Targeting osteocalcin mRNA with a specific shRNA increased the production of mineralized nodules. Fourth, levels of p-MEK and p-Erk1/2 were significantly increased in SHIP2-inactivated chondrocytes in response to serum and IGF-1, but not to FGF2, as compared to control chondrocytes. Treatment of chondrocytes and bones in culture with a MEK inhibitor partially rescued the production of mineralized nodules, the size of the hypertrophic chondrocyte zone and bone growth, raising the possibility of a treatment that could partially reduce the phenotype of this severe condition. Altogether, our results indicate that Ship2Δ/Δ mice represent a relevant model for human OPS. They also highlight the important role of SHIP2 in chondrocytes during endochondral ossification and its different differentiation steps. Finally, we identified a role of osteocalcin in mineralized nodules production and for the MEK-Erk1/2 signaling pathway in the OPS phenotype.
Asunto(s)
Condrocitos/metabolismo , Quinasas Quinasa Quinasa PAM/genética , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/genética , Osteocalcina/genética , Osteocondrodisplasias/genética , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas/genética , Aminoacetonitrilo/análogos & derivados , Aminoacetonitrilo/farmacología , Animales , Calcificación Fisiológica/genética , Diferenciación Celular , Condrocitos/patología , Modelos Animales de Enfermedad , Factor 2 de Crecimiento de Fibroblastos/farmacología , Regulación de la Expresión Génica , Placa de Crecimiento/metabolismo , Placa de Crecimiento/patología , Humanos , Factor I del Crecimiento Similar a la Insulina/farmacología , Quinasas Quinasa Quinasa PAM/antagonistas & inhibidores , Quinasas Quinasa Quinasa PAM/metabolismo , Ratones , Ratones Noqueados , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Osteocalcina/antagonistas & inhibidores , Osteocalcina/metabolismo , Osteocondrodisplasias/metabolismo , Osteocondrodisplasias/patología , Osteogénesis/genética , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas/antagonistas & inhibidores , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas/deficiencia , Fosforilación/efectos de los fármacos , Cultivo Primario de Células , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Tiofenos/farmacologíaRESUMEN
A circadian clock governs most aspects of mammalian behavior. Although its properties are in part genetically determined, altered light-dark environment can change circadian period length through a mechanism requiring de novo DNA methylation. We show here that this mechanism is mediated not via cell-autonomous clock properties, but rather through altered networking within the suprachiasmatic nuclei (SCN), the circadian "master clock," which is DNA methylated in region-specific manner. DNA methylation is necessary to temporally reorganize circadian phasing among SCN neurons, which in turn changes the period length of the network as a whole. Interruption of neural communication by inhibiting neuronal firing or by physical cutting suppresses both SCN reorganization and period changes. Mathematical modeling suggests, and experiments confirm, that this SCN reorganization depends upon GABAergic signaling. Our results therefore show that basic circadian clock properties are governed by dynamic interactions among SCN neurons, with neuroadaptations in network function driven by the environment.
Asunto(s)
Potenciales de Acción/fisiología , Relojes Circadianos/genética , Metilación de ADN/genética , Luz , Neuronas/fisiología , Núcleo Supraquiasmático/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Animales , Relojes Circadianos/fisiología , Ritmo Circadiano , Masculino , Ratones , Modelos Teóricos , Neuronas/citología , Técnicas de Placa-Clamp , Proteínas Circadianas Period/genética , Núcleo Supraquiasmático/citología , Núcleo Supraquiasmático/fisiología , Tálamo/citología , Tálamo/metabolismoRESUMEN
The mammalian target of rapamycin complex 2 (mTORC2) contains the essential protein RICTOR and is activated by growth factors. mTORC2 in adipose tissue contributes to the regulation of glucose and lipid metabolism. In the perivascular adipose tissue, mTORC2 ensures normal vascular reactivity by controlling expression of inflammatory molecules. To assess whether RICTOR/mTORC2 contributes to blood pressure regulation, we applied a radiotelemetry approach in control and Rictor knockout (Rictor(aP2KO)) mice generated using adipocyte protein-2 gene promoter-driven CRE recombinase expression to delete Rictor. The 24-hour mean arterial pressure was increased in Rictor(aP2KO) mice, and the physiological decline in mean arterial pressure during the dark period was impaired. In parallel, heart rate and locomotor activity were elevated during the dark period with a pattern similar to blood pressure changes. This phenotype was associated with mild cardiomyocyte hypertrophy, decreased cardiac natriuretic peptides, and their receptor expression in adipocytes. Moreover, clock gene expression was reduced or phase-shifted in perivascular adipose tissue. No differences in clock gene expression were observed in the master clock suprachiasmatic nucleus, although Rictor gene expression was also lower in brain of Rictor(aP2KO) mice. Thus, this study highlights the importance of RICTOR/mTORC2 for interactions between vasculature, adipocytes, and brain to tune physiological outcomes, such as blood pressure and locomotor activity.
Asunto(s)
Tejido Adiposo/metabolismo , Presión Sanguínea/fisiología , Encéfalo/metabolismo , Proteínas CLOCK/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Eliminación de Gen , Animales , Proteínas CLOCK/genética , Expresión Génica , Frecuencia Cardíaca/fisiología , Hipertrofia , Insulina/metabolismo , Diana Mecanicista del Complejo 2 de la Rapamicina , Ratones , Ratones Noqueados , Modelos Animales , Actividad Motora/fisiología , Complejos Multiproteicos/metabolismo , Miocitos Cardíacos/patología , Proteína Asociada al mTOR Insensible a la Rapamicina , Serina-Treonina Quinasas TOR/metabolismo , Vasoconstricción/fisiologíaRESUMEN
The timing of daily circadian behavior can be highly variable among different individuals, and twin studies have suggested that about half of this variability is environmentally controlled. Similar plasticity can be seen in mice exposed to an altered lighting environment, for example, 22-h instead of 24-h, which stably alters the genetically determined period of circadian behavior for months. The mechanisms mediating these environmental influences are unknown. We found that transient exposure of mice to such lighting stably altered global transcription in the suprachiasmatic nucleus (SCN) of the hypothalamus (the master clock tissue regulating circadian behavior in mammals). In parallel, genome-wide methylation profiling revealed global alterations in promoter DNA methylation in the SCN that correlated with these changes. Behavioral, transcriptional and DNA methylation changes were reversible after prolonged re-entrainment to 24-h d. Notably, infusion of a methyltransferase inhibitor to the SCN suppressed period changes. We conclude that the SCN utilizes DNA methylation as a mechanism to drive circadian clock plasticity.
Asunto(s)
Péptidos y Proteínas de Señalización del Ritmo Circadiano/genética , Péptidos y Proteínas de Señalización del Ritmo Circadiano/metabolismo , Ritmo Circadiano/genética , Metilación de ADN/genética , Plasticidad Neuronal/fisiología , Fotoperiodo , Actigrafía , Animales , Conducta Animal/fisiología , Ratones , Ratones Endogámicos C57BL , Núcleo Supraquiasmático/metabolismo , Transcriptoma/genéticaRESUMEN
Histone modifications are important epigenetic marks that influence chromatin structure and consequently play a role in the control of eukaryotic transcription. Several histone modifying enzymes have been characterized in Schistosoma mansoni and it has been suggested that the regulation of gene transcription in schistosomes may require the action of these enzymes. However, the influence of chromatin structure on gene transcription in schistosomes has never been investigated. Chromatin immunoprecipitation (ChIP) is the technique of choice to study the relationship between histone modifications and gene expression. Although this technique has been widely used with cultured cells from model organisms and with many unicellular organisms, it remains challenging to apply this technique to non-conventional organisms that undergo complex life cycles. In this work, we describe a native ChIP procedure that is applicable to all the stages of the S. mansoni life cycle and does not require expensive equipment. Immunoprecipitated DNA was analysed on a whole-genome scale using massively parallel sequencing (ChIP-Sequencing or ChIP-Seq). We show that ChIP-Seq and conventional quantitative PCR deliver comparable results for a life-cycle regulated locus, smRHO, that encodes a guanine-protein coupled receptor. This is the first time that the ChIP-Seq procedure has been applied to a parasite. This technique opens new ways for analyzing epigenetic mechanisms in S. mansoni at a whole-genome scale and on the level of individual loci.