Toru Tanaka

YB-1 Binds to GluR2 mRNA and CaM1 mRNA in the Brain and Regulates their Translational Levels in an Activity-Dependent Manner

The translational regulator YB-1 binds to mRNAs. In the brain, YB-1 is prominently expressed from the prenatal stage until the first week after birth, being associated with polysomes and distributed in neuronal dendrites, but its expression declines to a much lower level thereafter. It is therefore of interest to identify the mRNAs whose translation is controlled by YB-1 in the postnatal growing brain. In this study we found that YB-1 interacted with the mRNAs for glutamate receptor subunit 2 (GluR2) and calmodulin1 (CaM1) in both brain and NG108-15 cells. Overexpression or knockdown of YB-1 altered the levels of these proteins significantly in cultured cells without any change in their mRNA levels. When the cells were treated with neurotransmitters, translation of these proteins was induced within a short time, and a change in the amount of YB-1 on its target mRNAs was observed in the heavy-sedimenting polysome fractions on a sucrose gradient. Depletion of YB-1 expression by siRNA abrogated the translational activation. Furthermore, in the brain of kainic acid-treated mice, the distribution of YB-1 was shifted to much heavier fractions associated with polysomes within 30xa0min to 1xa0h after the treatment, and the distribution returned to lighter fractions within the following 2xa0h. The protein levels of GluR2 and CaM1 were also increased transiently when the distribution of YB-1 on the gradient changed. These results suggest that in the brain of growing mice, YB-1 binds to GluR2 and CaM1 mRNAs and regulates their translation in an activity-dependent manner.

Local Somatodendritic Translation and Hyperphosphorylation of Tau Protein Triggered by AMPA and NMDA Receptor Stimulation

Tau is a major component of the neurofibrillary tangles (NFT) that represent a pathological hallmark of Alzheimers disease (AD). Although generally considered an axonal protein, Tau is found in the somato-dendritic compartment of degenerating neurons and this redistribution is thought to be a trigger of neurodegeneration in AD. Here, we show the presence of tau mRNA in a dendritic ribonucleoprotein (RNP) complex that includes Ca2+-calmodulin dependent protein kinase (CaMK)IIα mRNA and that is translated locally in response to glutamate stimulation. Further, we show that Tau mRNA is a component of mRNP granules that contain RNA-binding proteins, and that it interacts with Myosin Va, a postsynaptic motor protein; these findings suggest that tau mRNA is transported into dendritic spines. We also report that tau mRNA localized in the somato-dendritic component of primary hippocampal cells and that a sub-toxic concentration of glutamate enhances local translation and hyperphosphorylation of tau, effects that are blocked by the gluatamatergic antagonists MK801 and NBQX. These data thus demonstrate that alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and N-methyl-d-aspartate (NMDA) stimulation redistributes tau to the somato-dendritic region of neurons where it may trigger neurodegeneration.

Indirubin derivatives alter DNA binding activity of the transcription factor NF-Y and inhibit MDR1 gene promoter

Indirubin derivatives exert antitumor activity. However, their effects on the expression of multidrug resistance gene 1 (MDR1) have not been investigated. Here we found three derivatives that inhibit the MDR1 gene promoter. To investigate the effects of indirubins on the DNA binding of NF-Y, a major MDR1 gene transcription factor that recognizes an inverted CCAAT element in the promoter, gel mobility shift assay was performed using the element as a probe with nuclear extracts from NG108-15, MCF7, HepG2, C2C12, and SK-N-SH cells. Among 17 compounds, 5-methoxyindirubin inhibited the DNA binding of NF-Y significantly, whereas indirubin-3-oxime and 7-methoxyindirubin 3-oxime increased the binding considerably. After evaluating a suitable concentration of each compound for transcription analysis using living tumor cells, we performed a reporter gene assay using a reporter DNA plasmid containing EGFP cDNA fused to the MDR1 gene promoter region. Indirubin-3-oxime exerted a significant inhibitory effect on the MDR1 promoter activity in MCF7 and HepG2 cells, and 5-methoxyindirubin inhibited the activity only in MCF7 cells; 7-methoxyindirubin 3-oxime suppressed the activity in all of the cell lines. We further confirmed that the compounds reduced endogenous MDR1 transcription without any inhibitory effect on NF-Y expression. Moreover, each compound increased the doxorubicin sensitivity of MCF7 cells. These results indicate that each indirubin derivative acts on the DNA binding of NF-Y and represses the MDR1 gene promoter with tumor cell-type specificity.

Roles of YB-1 under arsenite-induced stress: Translational activation of HSP70 mRNA and control of the number of stress granules

BACKGROUNDnWhen cells become stressed, they form stress granules (SGs) and show an increase of the molecular chaperone HSP70. The translational regulator YB-1 is a component of SGs, but it is unclear whether it contributes to the translational induction of HSP70 mRNA. Here we examined the roles of YB-1 in SG assembly and translational regulation of HSP70 mRNA under arsenite-induced stress.nnnMETHODnUsing arsenite-treated NG108-15 cells, we examined whether YB-1 was included in SGs with GluR2 mRNA, a target of YB-1, and investigated the interaction of YB-1 with HSP70 mRNA and its effect on translation of the mRNA. We also investigated the distribution of these mRNAs to SGs or polysomes, and evaluated the role of YB-1 in SG assembly.nnnRESULTSnArsenite treatment reduced the translation level of GluR2 mRNA; concomitantly, YB-1-bound HSP70 mRNA was increased and its translation was induced. Sucrose gradient analysis revealed that the distribution of GluR2 mRNA was shifted from heavy-sedimenting to much lighter fractions, and also to SG-containing non-polysomal fractions. Conversely, HSP70 mRNA was shifted from the non-polysomal to polysome fractions. YB-1 depletion abrogated the arsenite-responsive activation of HSP70 synthesis, but SGs harboring both mRNAs were still assembled. The number of SGs was increased by YB-1 depletion and decreased by its overexpression.nnnCONCLUSIONnIn arsenite-treated cells, YB-1 mediates the translational activation of HSP70 mRNA and also controls the number of SGs through inhibition of their assembly.nnnGENERAL SIGNIFICANCEnUnder stress conditions, YB-1 exerts simultaneous but opposing actions on the regulation of translation via SGs and polysomes.

Mechanism of YB-1-mediated translational induction of GluR2 mRNA in response to neural activity through nAChR

BACKGROUNDnWe have reported previously that YB-1 induces translation of GluR2 mRNA in response to neural activity, and that HSP60 affects the association of YB-1 with polysomes. Here we examined the mechanism of YB-1-mediated translational activation of GluR2 mRNA through the nAChR.nnnMETHODSnExpression of nAChRs in NG108-15 cells was verified. Translation of GluR2 mRNA and YB-1/HSP60 interaction were examined in nicotine-treated NG108-15 cells. Effects of inhibition of α7-nAChR and the PI3K/Akt pathway were investigated. The ratios of YB-1 to GluR2 mRNA and to HSP60 were explored in polysomal and non-polysomal fractions, respectively, and the role of HSP60 in cytoplasmic retention of YB-1 was evaluated.nnnRESULTSnNicotine treatment transiently induced translation of GluR2 mRNA and Akt phosphorylation with a concomitant increase of YB-1/HSP60 interaction. Both α-bungarotoxin and LY294002 abolished the effects of nicotine. On a sucrose gradient, nicotine treatment shifted the distribution of YB-1 to much heavier-sedimenting polysome fractions. In these fractions, the ratio of YB-1 to its binding GluR2 mRNA was decreased, and ribosome association with the YB-1-bound GluR2 mRNA was increased. HSP60 was distributed only in the non-polysomal fractions as its binding to YB-1 increased. In HSP60-depleted cells, nicotine treatment induced nuclear localization of YB-1.nnnCONCLUSIONnYB-1 is released from GluR2 mRNA during α7-nAChR-mediated neurotransmission, causing the PI3K/Akt pathway to recruit ribosomes into the translational machinery, and HSP60 is involved in cytoplasmic retention of polysome-free YB-1.nnnGENERAL SIGNIFICANCEnActivation of the PI3K/Akt pathway through the α7-nAChR and YB-1/HSP60 interaction are important for YB-1-mediated translational activation of GluR2 mRNA.

Translational level of acetylcholine receptor α mRNA in mouse skeletal muscle is regulated by YB-1 in response to neural activity

Y-box-binding protein 1 (YB-1) binds to mRNAs and affects translation. In this study, we focused on skeletal muscle, in which YB-1 expression is restricted to the early postnatal period, and found that YB-1 binds to acetylcholine receptor α-subunit (AChR α) mRNA. Although transcription of the AChR α gene is known to be regulated by myogenic transcription factors, translational control of the mRNA in response to neuromuscular transmission has not been examined. In undifferentiated C2C12 myoblasts, expression of AChR α remained at a low level. However, translation of the mRNA was increased by knockdown of YB-1. Continued overexpression of YB-1 prevented the cells from differentiating. In myotubes, which show clustering of AChRs, translation of the mRNA was induced within 3h after treatment with nicotine. The effect of nicotine was inhibited by α-bungarotoxin, and in the presence of cycloheximide the level of AChR α was reduced, even after nicotine treatment. Sucrose gradient analysis revealed that in nicotine-treated myotubes, YB-1-containing polysomes were shifted to the heavier-sedimenting fractions, and showed an apparent decrease in the amount of YB-1 bound to AChR α mRNA. These results suggest that in skeletal muscle cells, neural activity reduces the molar ratio of YB-1 relative to its binding AChR α mRNA, leading to an increase of ribosome binding to the mRNA, and thus activating translation. Furthermore, in postnatal growing mice, as has already been shown, the level of AChR α mRNA declined during the early period with maturation of neuromuscular synapses, but the translation level was found to increase transiently at postnatal day 10, when the level of YB-1 was markedly reduced. It is suggested that although the level of AChR α mRNA is reduced, the translation can be induced by alteration of the ratio of YB-1 protein to the mRNA.

Endothelial Robo4 regulates IL-6 production by endothelial cells and monocytes via a crosstalk mechanism in inflammation

Roundabout4 (Robo4) is an endothelial cell-specific receptor that stabilizes vasculature in pathological angiogenesis. Previous studies have shown that Robo4 is a potential therapeutic target for inflammatory diseases, but its precise roles in inflammation remain unclear. To investigate physiological Robo4 functions in inflammation, we performed a loss-of-function study inxa0vitro and inxa0vivo using lipopolysaccharide (LPS)-induced endotoxemia models. Subcutaneous injection of LPS into Robo4-knockout mice reduced circulating IL-6 levels. siRNA-mediated Robo4 knockdown suppressed IL-6 production induced by LPS, IL-1β, and TNFα, in human umbilical vein endothelial cells (HUVECs). Coculture experiments with HUVECs and a monocytic cell line, U937xa0cells, demonstrated that Robo4 knockdown suppresses IL-6 production by both endothelial cells and U937xa0cells. Further coculture experiments demonstrated that Robo4 knockdown inhibited a novel IL-6 amplification mechanism mediated by crosstalk between endothelial cells and U937xa0cells via direct interactions and two mediators, GM-CSF and IL-1β. Taken together, we demonstrated novel Robo4 functions in inflammation, i.e., it promotes IL-6 production by endothelial cells and immune cells via crosstalk.

Four nucleocytoplasmic-shuttling proteins and p53 interact specifically with the YB-NLS and are involved in anticancer reagent-induced nuclear localization of YB-1.

In cancer cells, anticancer reagents often trigger nuclear accumulation of YB-1, which participates in the progression of cancer malignancy. YB-1 has a non-canonical nuclear localization signal (YB-NLS). Here we found that four nucleocytoplasmic-shuttling RNA-binding proteins and p53 interact specifically with the YB-NLS and co-accumulate with YB-1 in the nucleus of actinomycin D-treated cells. To elucidate the roles of these YB-NLS-binding proteins, we performed a dominant-negative experiment in which a large excess of YB-NLS interacts with the YB-NLS-binding proteins, and showed inhibitory effects on actinomycin D-induced nuclear transport of endogenous YB-1 and subsequent MDR1 gene expression. Furthermore, the YB-NLS-expressing cells were also found to show increased drug sensitivity. Our results suggest that these YB-NLS-associating proteins are key factors for nuclear translocation/accumulation of YB-1 in cancer cells.

Indirubin 3′-oxime inhibits anticancer agent-induced YB-1 nuclear translocation in HepG2 human hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is a disease with poor prognosis. Nuclear accumulation of YB-1 is closely related to the malignancy of HCC. Treatment with anticancer agents often induces translocation of YB-1 from cytoplasm to nucleus and activates the expression of multidrug resistance gene 1 (MDR1). Therefore, any effective inhibitor of this phenomenon would be useful for cancer treatment. Here we examined various indirubin derivatives and found that indirubin 3-oxime inhibits actinomycin D-induced nuclear transport of YB-1 and suppresses the activation of MDR1 gene expression in the human hepatocellular carcinoma cell line HepG2. Furthermore, use of both indirubin 3-oxime and actinomycin D in combination increased the anticancer effect on HepG2 cells. Indirubin 3-oxime is a novel and efficient inhibitor of anticancer agent-induced YB-1 nuclear translocation.

Mechanism responsible for inhibitory effect of indirubin 3′-oxime on anticancer agent-induced YB-1 nuclear translocation in HepG2 human hepatocellular carcinoma cells

&NA; YB‐1 nuclear translocation/accumulation caused by anticancer agents leads to malignant transformation. Nuclear import of YB‐1 requires a nuclear localization signal (YB‐NLS). Previously, we identified five nucleocytoplasmic‐shuttling proteins as YB‐NLS binding proteins, and showed that they co‐accumulate in the nucleus with YB‐1 in response to treatment with actinomycin D. In addition, another group reported that transportin‐1 is the molecule responsible for YB‐1 nuclear translocation, binding to a region (PY‐NLS) consistent with the YB‐NLS. Recently, we found that indirubin 3′‐oxime inhibits the nuclear localization of YB‐1 in HepG2 cells and increases their sensitivity to actinomycin D. Here, we found that YB‐1 nuclear translocation is dependent on the cellular mRNA level and that indirubin 3′‐oxime inhibits the interaction between YB‐1 and transportin‐1. Interestingly, in cells showing inhibition of actinomycin D‐induced YB‐1 nuclear translocation by the compound, the YB‐NLS‐binding proteins as well as transportin‐1 and its cargos were imported to the nucleus. Furthermore, the compound inhibited nuclear localization of the GFP‐conjugated full‐length YB‐1 but not that of GFP‐conjugated YB‐NLS. These results indicate that indirubin 3′‐oxime is a specific inhibitor of anticancer agent‐induced YB‐1 nuclear translocation, interacting with YB‐1 itself in a region other than the YB‐NLS/PY‐NLS. This compound would increase the efficacy of cancer therapy. HighlightsYB‐1 nuclear translocation is dependent on the cellular mRNA level.Indirubin 3′‐oxime hinders interaction between YB‐1 and transportin‐1.Indirubin 3′‐oxime does not inhibit nuclear import of YB‐NLS‐binding proteins.Indirubin 3′‐oxime does not inhibit nuclear import of transportin‐1 and its cargos.Indirubin 3′‐oxime acts on YB‐1 specifically and inhibits its nuclear translocation.