RESUMEN
Aims: MicroRNAs (miRNAs) are crucial for the post-transcriptional control of protein-encoding genes and together with transcription factors (TFs) regulate gene expression; however, the regulatory activities of miRNAs during cardiac development are only partially understood. In this study, we tested the hypothesis that integrative computational approaches could identify miRNAs that experimentally could be shown to regulate cardiomyogenesis. Methods and results: We integrated expression profiles with bioinformatics analyses of miRNA and TF regulatory programs to identify candidate miRNAs involved with cardiac development. Expression profiling showed that miR-200c, which is not normally detected in adult heart, is progressively down-regulated both during cardiac development and in vitro differentiation of human embryonic stem cells (hESCs) to cardiomyocytes (CMs). We employed computational methodologies to predict target genes of both miR-200c and five key cardiac TFs to identify co-regulated gene networks. The inferred cardiac networks revealed that the cooperative action of miR-200c with these five key TFs, including three (GATA4, SRF and TBX5) targeted by miR-200c, should modulate key processes and pathways necessary for CM development and function. Experimentally, over-expression (OE) of miR-200c in hESC-CMs reduced the mRNA levels of GATA4, SRF and TBX5. Cardiac expression of Ca2+, K+ and Na+ ion channel genes (CACNA1C, KCNJ2 and SCN5A) were also significantly altered by knockdown or OE of miR-200c. Luciferase reporter assays validated miR-200c binding sites on the 3' untranslated region of CACNA1C. In hESC-CMs, elevated miR-200c increased beating frequency, and repressed both Ca2+ influx, mediated by the L-type Ca2+ channel and Ca2+ transients. Conclusions: Our analyses demonstrate that miR-200c represses hESC-CM differentiation and maturation. The integrative computation and experimental approaches described here, when applied more broadly, will enhance our understanding of the interplays between miRNAs and TFs in controlling cardiac development and disease processes.
Asunto(s)
Diferenciación Celular/genética , Biología Computacional/métodos , Redes Reguladoras de Genes , Células Madre Embrionarias Humanas/metabolismo , MicroARNs/genética , Miocitos Cardíacos/metabolismo , Transcriptoma , Canales de Calcio Tipo L/genética , Canales de Calcio Tipo L/metabolismo , Señalización del Calcio/genética , Línea Celular , Factor de Transcripción GATA4/genética , Factor de Transcripción GATA4/metabolismo , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Genotipo , Frecuencia Cardíaca/genética , Humanos , MicroARNs/metabolismo , Contracción Miocárdica/genética , Canal de Sodio Activado por Voltaje NAV1.5/genética , Canal de Sodio Activado por Voltaje NAV1.5/metabolismo , Fenotipo , Canales de Potasio de Rectificación Interna/genética , Canales de Potasio de Rectificación Interna/metabolismo , Reproducibilidad de los Resultados , Factor de Respuesta Sérica/genética , Factor de Respuesta Sérica/metabolismo , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Factores de TiempoRESUMEN
Reactive oxygen species (ROS) have been commonly accepted as inducers of autophagy, and autophagy in turn is activated to relieve oxidative stress. Yet, whether and how oxidative stress, generated in various human pathologies, regulates autophagy remains unknown. Here, we mechanistically studied the role of TRPM2 (transient receptor potential cation channel subfamily M member 2)-mediated Ca(2+) influx in oxidative stress-mediated autophagy regulation. On the one hand, we demonstrated that oxidative stress triggered TRPM2-dependent Ca(2+) influx to inhibit the induction of early autophagy, which renders cells more susceptible to death. On the other hand, oxidative stress induced autophagy (and not cell death) in the absence of the TRPM2-mediated Ca(2+) influx. Moreover, in response to oxidative stress, TRPM2-mediated Ca(2+) influx activated CAMK2 (calcium/calmodulin dependent protein kinase II) at levels of both phosphorylation and oxidation, and the activated CAMK2 subsequently phosphorylated BECN1/Beclin 1 on Ser295. Ser295 phosphorylation of BECN1 in turn decreased the association between BECN1 and PIK3C3/VPS34, but induced binding between BECN1 and BCL2. Clinically, acetaminophen (APAP) overdose is the most common cause of acute liver failure worldwide. We demonstrated that APAP overdose also activated ROS-TRPM2-CAMK2-BECN1 signaling to suppress autophagy, thereby causing primary hepatocytes to be more vulnerable to death. Inhibiting the TRPM2-Ca(2+)-CAMK2 cascade significantly mitigated APAP-induced liver injury. In summary, our data clearly demonstrate that oxidative stress activates the TRPM2-Ca(2+)-CAMK2 cascade to phosphorylate BECN1 resulting in autophagy inhibition.
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Beclina-1/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Estrés Oxidativo , Canales Catiónicos TRPM/metabolismo , Acetaminofén/química , Animales , Autofagia , Calcio/metabolismo , Señalización del Calcio , Línea Celular Tumoral , Sobredosis de Droga , Células HEK293 , Células HeLa , Hepatocitos/citología , Hepatocitos/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Mutagénesis , Fosforilación , Especies Reactivas de Oxígeno/metabolismo , Serina/química , Transducción de SeñalRESUMEN
Embryonic stem cells (ESCs) are promising resources for both scientific research and clinical regenerative medicine. With regards to the latter, ESCs are especially useful for treating several neurodegenerative disorders. Two significant characteristics of ESCs, which make them so valuable, are their capacity for self-renewal and their pluripotency, both of which are regulated by the integration of various signaling pathways. Intracellular Ca(2+) signaling is involved in several of these pathways. It is known to be precisely controlled by different Ca(2+) channels and pumps, which play an important role in a variety of cellular activities, including proliferation, differentiation and apoptosis. Here, we provide a review of the recent work conducted to investigate the function of Ca(2+) signaling in the self-renewal and the neural differentiation of ESCs. Specifically, we describe the role of intracellular Ca(2+) mobilization mediated by RyRs (ryanodine receptors); by cADPR (cyclic adenosine 5'-diphosphate ribose) and CD38 (cluster of differentiation 38/cADPR hydrolase); and by NAADP (nicotinic acid adenine dinucleotide phosphate) and TPC2 (two pore channel 2). We also discuss the Ca(2+) influx mediated by SOCs (store-operated Ca(2+) channels), TRPCs (transient receptor potential cation channels) and LTCC (L-type Ca(2+) channels) in the pluripotent ESCs as well as in neural differentiation of ESCs. Moreover, we describe the integration of Ca(2+) signaling in the other signaling pathways that are known to regulate the fate of ESCs.
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Señalización del Calcio , Calcio/metabolismo , Diferenciación Celular , Autorrenovación de las Células , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Neuronas/citología , Neuronas/metabolismo , Animales , HumanosRESUMEN
Nitric oxide (NO) is known to be involved in modulating production of styrylpyrone polyphenols in the basidiomycete Inonotus obliquus. However, it remains unknown how NO orchestrates fungal styrylpyrone biosynthesis. Here, we show that a transient NO burst correlated with an enhanced expression of phenylalanine ammonia lyase (PAL), 4-coumarate CoA ligase (4CL), and styrylpyrone synthase (SPS), the key enzymes involved in styrylpyrone biosynthesis, and subsequently an increased production of styrylpyrone polyphenols. In parallel, the NO burst also resulted in S-nitrosylation of PAL, 4CL, and SPS, which compromised their enzymatic activities mediating a post-translational feedback mechanism that keeps NO-dependent transcriptional activation in check. Moreover, dysfunction of thioredoxin reductase (TrxR) further increased the formation of S-nitrosylated proteins, implicating the significance of the Trx system in maintaining a low level of protein-nitrosothiols. Three thioredoxin-like proteins (TrxLs) from I. obliquus show in vitro denitrosylation potential toward S-nitrosylated proteins via trans-denitrosylation or mixed disulfide intermediates. Thus, S-nitrosylation triggered by the NO burst limits over production of fungal styrylpyrone polyphenols, and denitrosylation by TrxLs that act in concert with TrxR play a key role in maintaining redox balance and orchestrating catalytic activities of the enzymes engaged in styrylpyrone synthetic metabolism.
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Basidiomycota/metabolismo , Redes y Vías Metabólicas , Óxido Nítrico/metabolismo , Pironas/metabolismo , Estirenos/metabolismo , Coenzima A Ligasas , Retroalimentación Fisiológica , Proteínas Fúngicas/metabolismo , Regulación Enzimológica de la Expresión Génica , Hidroximetilglutaril-CoA Sintasa/metabolismo , Fenilanina Amoníaco-Liasa , Polifenoles/metabolismoRESUMEN
CD38 is a multifunctional membrane enzyme and the main mammalian ADP-ribosyl cyclase, which catalyzes the synthesis and hydrolysis of cADPR, a potent endogenous Ca(2+) mobilizing messenger. Here, we explored the role of CD38 in the neural differentiation of mouse embryonic stem cells (ESCs). We found that the expression of CD38 was decreased during the differentiation of mouse ESCs initiated by adherent monoculture. Perturbing the CD38/cADPR signaling by either CD38 knockdown or treatment of cADPR antagonists inhibited the neural commitment of mouse ESCs, whereas overexpression of CD38 promoted it. Moreover, CD38 knockdown dampened reactive oxygen species (ROS) production during neural differentiation of ESCs by inhibiting NADPH oxidase activity, while CD38 overexpression enhanced it. Similarly, application of hydrogen peroxide mitigated the inhibitory effects of CD38 knockdown on neural differentiation of ESCs. Taken together, our data indicate that the CD38 signaling pathway is required for neural differentiation of mouse ESCs by modulating ROS production.
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ADP-Ribosil Ciclasa 1/biosíntesis , Diferenciación Celular/fisiología , Glicoproteínas de Membrana/biosíntesis , Células Madre Embrionarias de Ratones/metabolismo , Neuronas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , ADP-Ribosil Ciclasa 1/genética , Animales , Células Cultivadas , Técnicas de Silenciamiento del Gen , Glicoproteínas de Membrana/genética , RatonesRESUMEN
Autophagy is a catabolic lysosomal degradation process essential for cellular homeostasis and cell survival. Dysfunctional autophagy has been associated with a wide range of human diseases, e.g., cancer and neurodegenerative diseases. A large number of small molecules that modulate autophagy have been widely used to dissect this process and some of them, e.g., chloroquine (CQ), might be ultimately applied to treat a variety of autophagy-associated human diseases. Here we found that vacuolin-1 potently and reversibly inhibited the fusion between autophagosomes and lysosomes in mammalian cells, thereby inducing the accumulation of autophagosomes. Interestingly, vacuolin-1 was less toxic but at least 10-fold more potent in inhibiting autophagy compared with CQ. Vacuolin-1 treatment also blocked the fusion between endosomes and lysosomes, resulting in a defect in general endosomal-lysosomal degradation. Treatment of cells with vacuolin-1 alkalinized lysosomal pH and decreased lysosomal Ca(2+) content. Besides marginally inhibiting vacuolar ATPase activity, vacuolin-1 treatment markedly activated RAB5A GTPase activity. Expression of a dominant negative mutant of RAB5A or RAB5A knockdown significantly inhibited vacuolin-1-induced autophagosome-lysosome fusion blockage, whereas expression of a constitutive active form of RAB5A suppressed autophagosome-lysosome fusion. These data suggest that vacuolin-1 activates RAB5A to block autophagosome-lysosome fusion. Vacuolin-1 and its analogs present a novel class of drug that can potently and reversibly modulate autophagy.
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Autofagia , Compuestos Heterocíclicos de 4 o más Anillos/química , Lisosomas/metabolismo , Fagosomas/metabolismo , Proteínas de Unión al GTP rab5/metabolismo , Adenosina Trifosfatasas/metabolismo , Calcio/metabolismo , Proliferación Celular , Supervivencia Celular , Cloroquina/química , Endosomas/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Células Hep G2 , Homeostasis , Humanos , Concentración de Iones de Hidrógeno , Concentración 50 Inhibidora , Lentivirus/genética , Metabolismo , Microscopía Electrónica de Transmisión , MutaciónRESUMEN
Store-operated Ca(2+) entry (SOCE) is an important Ca(2+) influx pathway in non-excitable cells. STIM1, an ER Ca(2+) sensor, and Orai1, a plasma membrane Ca(2+) selective channel, are the two essential components of the Ca(2+) release activated channel (CRAC) responsible for SOCE activity. Here we explored the role of STIM1 and Orai1 in neural differentiation of mouse embryonic stem (ES) cells. We found that STIM1 and Orai1 were expressed and functionally active in ES cells, and expressions of STIM1 and Orai1 were dynamically regulated during neural differentiation of mouse ES cells. STIM1 knockdown inhibited the differentiation of mouse ES cells into neural progenitors, neurons, and astrocytes. In addition, STIM1 knockdown caused severe cell death and markedly suppressed the proliferation of neural progenitors. Surprisingly, Orai1 knockdown had little effect on neural differentiation of mouse ES cells, but the neurons derived from Orai1 knockdown ES cells, like those from STIM1 knockdown cells, had defective SOCE. Taken together, our data indicate that STIM1 is involved in both early neural differentiation of ES cells and survival of early differentiated ES cells independent of Orai1-mediated SOCE.
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Canales de Calcio/metabolismo , Calcio/metabolismo , Células Madre Embrionarias/metabolismo , Animales , Canales de Calcio/genética , Muerte Celular/fisiología , Diferenciación Celular/fisiología , Supervivencia Celular/fisiología , Células Madre Embrionarias/citología , Técnicas de Silenciamiento del Gen , Ratones , Proteína ORAI1 , Molécula de Interacción Estromal 1RESUMEN
Extracellular signal-regulated kinases (ERKs) have been implicated to be dispensable for self-renewal of mouse embryonic stem (ES) cells, and simultaneous inhibition of both ERK signaling and glycogen synthase kinase 3 (GSK3) not only allows mouse ES cells to self-renew independent of extracellular stimuli but also enables more efficient derivation of naïve ES cells from mouse and rat strains. Interestingly, some ERKs stay active in mouse ES cells which are maintained in regular medium containing leukemia inhibitory factor (LIF) and bone morphogenetic protein (BMP). Yet, the upstream signaling for ERK activation and their roles in mouse ES cells, other than promoting or priming differentiation, have not been determined. Here we found that mouse ES cells express three forms of Raf kinases, A-Raf, B-Raf, and C-Raf. Knocking-down each single Raf member failed to affect the sustained ERK activity, neither did A-Raf and B-Raf double knockdown or B-Raf and C-Raf double knockdown change it in ES cells. Interestingly, B-Raf and C-Raf double knockdown, not A-Raf and B-Raf knockdown, inhibited the maximal ERK activation induced by LIF, concomitant with the slower growth of ES cells. On the other hand, A-Raf, B-Raf, and C-Raf triple knockdown markedly inhibited both the maximal and sustained ERK activity in ES cells. Moreover, Raf triple knockdown, similar to the treatment of U-0126, an MEK inhibitor, significantly inhibited the survival and proliferation of ES cells, thereby compromising the colony propagation of mouse ES cells. In summary, our data demonstrate that all three Raf members are required for ERK activation in mouse ES cells and are involved in growth and survival of mouse ES cells.
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Células Madre Embrionarias/citología , Proteínas Proto-Oncogénicas A-raf , Proteínas Proto-Oncogénicas B-raf , Proteínas Proto-Oncogénicas c-raf , Animales , Western Blotting , Butadienos/farmacología , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Células Cultivadas , Células Madre Embrionarias/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Citometría de Flujo , Técnicas de Silenciamiento del Gen , Ratones , Nitrilos/farmacología , Proteínas Proto-Oncogénicas A-raf/genética , Proteínas Proto-Oncogénicas A-raf/metabolismo , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas c-raf/genética , Proteínas Proto-Oncogénicas c-raf/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de SeñalRESUMEN
Autophagy is an evolutionarily conserved lysosomal degradation pathway, yet the underlying mechanisms remain poorly understood. Nicotinic acid adenine dinucleotide phosphate (NAADP), one of the most potent Ca(2+) mobilizing messengers, elicits Ca(2+) release from lysosomes via the two pore channel 2 (TPC2) in many cell types. Here we found that overexpression of TPC2 in HeLa or mouse embryonic stem cells inhibited autophagosomal-lysosomal fusion, thereby resulting in the accumulation of autophagosomes. Treatment of TPC2 expressing cells with a cell permeant-NAADP agonist, NAADP-AM, further induced autophagosome accumulation. On the other hand, TPC2 knockdown or treatment of cells with Ned-19, a NAADP antagonist, markedly decreased the accumulation of autophagosomes. TPC2-induced accumulation of autophagosomes was also markedly blocked by ATG5 knockdown. Interestingly, inhibiting mTOR activity failed to increase TPC2-induced autophagosome accumulation. Instead, we found that overexpression of TPC2 alkalinized lysosomal pH, and lysosomal re-acidification abolished TPC2-induced autophagosome accumulation. In addition, TPC2 overexpression had no effect on general endosomal-lysosomal degradation but prevented the recruitment of Rab-7 to autophagosomes. Taken together, our data demonstrate that TPC2/NAADP/Ca(2+) signaling alkalinizes lysosomal pH to specifically inhibit the later stage of basal autophagy progression.
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Álcalis/metabolismo , Autofagia , Canales de Calcio/metabolismo , Lisosomas/metabolismo , Fusión de Membrana , Fagosomas/metabolismo , Animales , Autofagia/efectos de los fármacos , Calcio/farmacología , Diferenciación Celular/efectos de los fármacos , Células Madre Embrionarias/citología , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/metabolismo , Endosomas/efectos de los fármacos , Endosomas/metabolismo , Endosomas/ultraestructura , Células HeLa , Humanos , Concentración de Iones de Hidrógeno/efectos de los fármacos , Lisosomas/efectos de los fármacos , Lisosomas/ultraestructura , Fusión de Membrana/efectos de los fármacos , Ratones , NADP/análogos & derivados , NADP/metabolismo , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fagosomas/efectos de los fármacos , Fagosomas/ultraestructura , Unión Proteica/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , Proteínas de Unión al GTP rab/efectos de los fármacos , Proteínas de Unión a GTP rab7RESUMEN
Nicotinic adenine acid dinucleotide phosphate (NAADP) is one of the most potent endogenous Ca(2+) mobilizing messengers. NAADP mobilizes Ca(2+) from an acidic lysosome-related store, which can be subsequently amplified into global Ca(2+) waves by calcium-induced calcium release (CICR) from ER/SR via Ins(1,4,5)P 3 receptors or ryanodine receptors. A body of evidence indicates that 2 pore channel 2 (TPC2), a new member of the superfamily of voltage-gated ion channels containing 12 putative transmembrane segments, is the long sought after NAADP receptor. Activation of NAADP/TPC2/Ca(2+) signaling inhibits the fusion between autophagosome and lysosome by alkalizing the lysosomal pH, thereby arresting autophagic flux. In addition, TPC2 is downregulated during neural differentiation of mouse embryonic stem (ES) cells, and TPC2 downregulation actually facilitates the neural lineage entry of ES cells. Here we propose the mechanism underlying how NAADP-induced Ca(2+) release increases lysosomal pH and discuss the role of TPC2 in neural differentiation of mouse ES cells.
RESUMEN
Cyclic adenosine diphosphate ribose is an endogenous Ca(2+) mobilizer involved in diverse cellular processes. A cell membrane-permeable cyclic adenosine diphosphate ribose analogue, cyclic inosine diphosphoribose ether (cIDPRE), can induce Ca(2+) increase in intact human Jurkat T-lymphocytes. Here we synthesized a coumarin-caged analogue of cIDPRE (Co-i-cIDPRE), aiming to have a precisely temporal and spatial control of bioactive cIDPRE release inside the cell using UV uncaging. We showed that Co-i-cIDPRE accumulated inside Jurkat cells quickly and efficiently. Uncaging of Co-i-cIDPRE evoked Ca(2+) release from endoplasmic reticulum, with concomitant Ca(2+) influx in Jurkat cells. Ca(2+) release evoked by uncaged Co-i-cIDPRE was blocked by knockdown of ryanodine receptors (RyRs) 2 and 3 in Jurkat cells. The associated Ca(2+) influx, on the other hand, was abolished by double knockdown of Stim1 and TRPM2 in Jurkat cells. Furthermore, Ca(2+) release or influx evoked by uncaged Co-i-cIDPRE was recapitulated in HEK293 cells that overexpress RyRs or TRPM2, respectively, but not in wild-type cells lacking these channels. In summary, our results indicate that uncaging of Co-i-cIDPRE incites Ca(2+) release from endoplasmic reticulum via RyRs and triggers Ca(2+) influx via TRPM2.
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Membrana Celular/metabolismo , ADP-Ribosa Cíclica/análogos & derivados , Alquenos/metabolismo , Western Blotting , Calcio , Línea Celular , Cumarinas/metabolismo , ADP-Ribosa Cíclica/metabolismo , Fluorescencia , Células HEK293 , Humanos , Células Jurkat , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Molécula de Interacción Estromal 1 , Canales Catiónicos TRPM/genética , Canales Catiónicos TRPM/metabolismoRESUMEN
Intracellular pH (pHi) and Ca(2+) regulate essentially all aspects of cellular activities. Their inter-relationship has not been mechanistically explored. In this study, we used bases and acetic acid to manipulate the pHi. We found that transient pHi rise induced by both organic and inorganic bases, but not acidification induced by acid, produced elevation of cytosolic Ca(2+). The sources of the Ca(2+) increase are from the endoplasmic reticulum (ER) Ca(2+) pools as well as from Ca(2+) influx. The store-mobilization component of the Ca(2+) increase induced by the pHi rise was not sensitive to antagonists for either IP(3)-receptors or ryanodine receptors, but was due to inhibition of the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA), leading to depletion of the ER Ca(2+) store. We further showed that the physiological consequence of depletion of the ER Ca(2+) store by pHi rise is the activation of store-operated channels (SOCs) of Orai1 and Stim1, leading to increased Ca(2+) influx. Taken together, our results indicate that intracellular alkalinization inhibits SERCA activity, similar to thapsigargin, thereby resulting in Ca(2+) leak from ER pools followed by Ca(2+) influx via SOCs.