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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BFBNIB, GIS, IJS, KISLJ, NUK, PNG, UL, UM, UPUK
Close homolog of L1 (CHL1) is a cell adhesion molecule of the immunoglobulin superfamily. It promotes neuritogenesis and survival of neurons in vitro. In vivo, CHL1 promotes nervous system ...development, regeneration after trauma, and synaptic function and plasticity. We identified programmed cell death 6 (PDCD6) as a novel binding partner of the CHL1 intracellular domain (CHL1‐ICD). Co‐immunoprecipitation, pull‐down assay with CHL1‐ICD, and proximity ligation in cerebellum and pons of 3‐day‐old and 6‐month‐old mice, as well as in cultured cerebellar granule neurons and cortical astrocytes indicate an association between PDCD6 and CHL1. The Ca2+‐chelator BAPTA‐AM inhibited the association between CHL1 and PDCD6. The treatment of cerebellar granule neurons with a cell‐penetrating peptide comprising the cell surface proximal 30 N‐terminal amino acids of CHL1‐ICD inhibited the association between CHL1 and PDCD6 and PDCD6‐ and CHL1‐triggered neuronal survival. These results suggest that PDCD6 contributes to CHL1 functions in the nervous system.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
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.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Store-operated Ca2+ entry (SOCE) is an important Ca2+ influx pathway in non-excitable cells. STIM1, an ER Ca2+ sensor, and Orai1, a plasma membrane Ca2+ selective channel, are the two essential ...components of the Ca2+ 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.
•STIM1 and Orai1 were expressed and functionally active in mouse ES cells.•STIM1 knockdown inhibited the neural differentiation of mouse ES cells.•Orai1 knockdown had little effect on neural differentiation of mouse ES cells.•The neurons derived from Orai1 or STIM1 knockdown ES cells had defective SOCE.•STIM1 is required for neural differentiation of mouse ES cells independent of SOCE.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Nogo-A, a glycoprotein expressed in oligodendrocytes and central nervous system myelin, inhibits regeneration after injury. Antibodies against Nogo-A neutralize this inhibitory activity, improve ...locomotor recovery in spinal cord-injured adult mammals, and promote regrowth/sprouting/saving of damaged axons beyond the lesion site. Nogo-A is also expressed by neurons. Complete ablation of Nogo-A in all cell types expressing it has been found to lead to recovery in some studies but not in others. Neuronal ablation of Nogo-A reduces axonal regrowth after injury. In view of these findings, we hypothesized that, in addition to neutralizing Nogo-A in oligodendrocytes and myelin, Nogo-A antibodies may act directly on neuronal Nogo-A to trigger neurite outgrowth and neuronal survival. Here, we show that polyclonal and monoclonal antibodies against Nogo-A enhance neurite growth and survival of cultured cerebellar granule neurons and increase expression of the neurite outgrowth-promoting L1 cell adhesion molecule and polysialic acid. Application of inhibitors of signal transducing molecules, such as c-src, c-fyn, protein kinase A, and casein kinase II reduce antibody-triggered neurite outgrowth. These observations indicate that the recovery-promoting functions of antibodies against Nogo-A may not only be due to neutralizing Nogo-A in oligodendrocytes and myelin, but also to their interactions with Nogo-A on neurons.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
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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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
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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BFBNIB, GIS, IJS, KISLJ, NUK, PNG, UL, UM, UPUK
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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BFBNIB, GIS, IJS, KISLJ, NUK, PNG, UL, UM, UPUK
Abstract
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.
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.
●Mouse ES (mES) cells express all three Raf members, A-Raf, B-Raf, and C-Raf.●Leukemia inhibitory factor (LIF) temporally activates ERKs in mES cells.●B-Raf and C-Raf are required for LIF-induced maximal ERKs activity in mES cells.●All Raf members are required for LIF-induced sustained ERK activity in mES cells.●All Raf members are required the survival and proliferation of mES cells.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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