Mineyoshi Aoyama

LMO3 Interacts with Neuronal Transcription Factor, HEN2, and Acts as an Oncogene in Neuroblastoma

LIM-only proteins (LMO), which consist of LMO1, LMO2, LMO3, and LMO4, are involved in cell fate determination and differentiation during embryonic development. Accumulating evidence suggests that LMO1 and LMO2 act as oncogenic proteins in T-cell acute lymphoblastic leukemia, whereas LMO4 has recently been implicated in the genesis of breast cancer. However, little is known about the role of LMO3 in either tumorigenesis or development. In the present study, we have identified LMO3 and HEN2, which encodes a neuronal basic helix-loop-helix protein, as genes whose expression levels were higher in unfavorable neuroblastomas compared with those of favorable tumors. Immunoprecipitation and immunostaining experiments showed that LMO3 was associated with HEN2 in mammalian cell nucleus. Human neuroblastoma SH-SY5Y cells stably overexpressing LMO3 showed a marked increase in cell growth, a promotion of colony formation in soft agar medium, and a rapid tumor growth in nude mice compared with the control transfectants. More importantly, the increased expression of LMO3 and HEN2 was significantly associated with a poor prognosis in 87 primary neuroblastomas. These results suggest that the deregulated expression of neuronal-specific LMO3 and HEN2 contributes to the genesis and progression of human neuroblastoma in a lineage-specific manner.

Human neuroblastomas with unfavorable biologies express high levels of brain-derived neurotrophic factor mRNA and a variety of its variants

The expression of human brain-derived neurotrophic factor (BDNF) was investigated in 16 primary human neuroblastomas with favorable biologies, 15 with unfavorable biologies, and in human neuroblastoma cell lines. We demonstrated higher expressions of human BDNF mRNA in neuroblastomas with unfavorable biologies and with N-myc amplification than in those with favorable biologies. For the first time we revealed the composition of splice variants of human BDNF mRNA and analyzed their expression in neuroblastomas by reverse transcription polymerase chain reaction (RT-PCR). Interestingly, human BDNF mRNA consisted of at least six isoforms, four isoforms resembling those of rat BDNF mRNA, a human-specific isoform and a new isoform. The expression of four isoforms were more prominent in tumors with unfavorable biologies than in those with favorable biologies (P<0.05). As previously we had reported, over 80% of the primary tumors expressed either the full-length form of BDNF receptor, TRKB, or a truncated form of TRKB lacking the tyrosine kinase domain. The full-length TRKB was predominantly detected in tumors with unfavorable biologies, and the truncated one in those with favorable biologies. These results suggest that an autocrine and/or paracrine mechanism involving BDNF may stimulate signal transduction via TRKB receptors rich in neuroblastomas with unfavorable biologies, resulting in an aberrant survival of tumor cells.

Tumor necrosis factor p55 and p75 receptors are involved in chemical-induced apoptosis of dentate granule neurons

Localized tumor necrosis factor‐α (TNFα) elevation has diverse effects in brain injury often attributed to signaling via TNFp55 or TNFp75 receptors. Both dentate granule cells and CA pyramidal cells express TNF receptors (TNFR) at low levels in a punctate pattern. Using a model to induce selective death of dentate granule cells (trimethyltin; 2 mg/kg, i.p.), neuronal apoptosis [terminal deoxynucleotidyl transferase‐mediated dUTP‐biotin in situ end labeling, active caspase 3 (AC3)] was accompanied by amoeboid microglia and elevated TNFα mRNA levels. TNFp55R (55 kDa type‐1 TNFR) and TNFp75R (75 kDa type‐2 TNFR) immunoreactivity in AC3+ neurons displayed a pattern suggestive of receptor internalization and a temporal sequence of expression of TNFp55R followed by TNFp75R associated with the progression of apoptosis. A distinct ramified microglia response occurred around CA1 neurons and healthy dentate neurons that displayed an increase in the normal punctate pattern of TNFRs. Neuronal damage was decreased with i.c.v. injection of TNFα antibody and in TNFp55R−/−p75R−/− mice that showed higher constitutive mRNA levels for interleukin (IL‐1α), macrophage inflammatory protein 1‐α (MIP‐1α), TNFα, transforming growth factor β1, Fas, and TNFRSF6‐assoicated via death domain (FADD). TNFp75R−/− mice showed exacerbated injury and elevated mRNA levels for IL‐1α, MIP‐1α, and TNFα. In TNFp55R−/− mice, constitutive mRNA levels for TNFα, IL‐6, caspase 8, FADD, and Fas‐associated phosphatase were higher; IL‐1α, MIP‐1α, and transforming growth factor β1 lower. The mice displayed exacerbated neuronal death, delayed microglia response, increased FADD and TNFp75R mRNA levels, and co‐expression of TNFp75R in AC3+ neurons. The data demonstrate TNFR‐mediated apoptotic death of dentate granule neurons utilizing both TNFRs and suggest a TNFp75R‐mediated apoptosis in the absence of normal TNFp55R activity.

Lactic acid increases aquaporin 4 expression on the cell membrane of cultured rat astrocytes

The water channel protein aquaporin (AQP) may play roles in the homeostasis of water content in the brain and brain edema. One possible mechanism of brain edema is glial swelling due to lactic acidosis associated with ischemia. Here, we investigated the effect of lactic acid on the expression and cellular distribution of AQP 4 in cultured rat astrocytes. After 24h of incubation, the AQP4 expression level increased maximally with 35mM lactic acid. The AQP4 expression levels also increased with hydrochloric acid or acetic acid. In contrast, with sodium lactate, the AQP4 levels did not increase. The increase in AQP4 expression level occurred without a significant increase in AQP4 mRNA expression level by lactic acid. Under the conditions of de novo protein synthesis inhibition with cycloheximide, lactic acid increased the AQP4 expression level. Furthermore, lactic acid increased the AQP4 expression level on the cell surface of the astrocytes, as determined by a cell surface biotinylation assay and immunocytochemical examination. The increase in AQP4 expression level on the cell membrane of astrocytes induced by lactic acid may be a new regulation mechanism of AQP4 in the brain.

Endogenous erythropoietin from astrocyte protects the oligodendrocyte precursor cell against hypoxic and reoxygenation injury

The hypoxia‐responsive cytokine erythropoietin (EPO) provides neuroprotective effects in the damaged brain during ischemic events and neurodegenerative diseases. The purpose of the present study is to evaluate the EPO/EPO receptor (EPOR) endogenous system between astrocyte and oligodendrocyte precursor cell (OPC) under hypoxia. We report here elevated EPO mRNA levels and protein release in cultured astrocytes following hypoxic stimulation by quantitative RT‐PCR and ELISA. Furthermore, the EPOR gene expressions were detected in cultured OPCs as in astrocytes and microglias by quantitative RT‐PCR. Cell staining revealed the EPOR expression in OPC. To evaluate the protective effect of endogenous EPO from astrocyte to OPCs, EPO/EPOR signaling was blocked by EPO siRNA or EPOR siRNA gene silencing in in vitro study. The suppression of endogenous EPO production in astrocytes by EPO siRNA decreased the protection to OPCs against hypoxic stress. Furthermore, OPC with EPOR siRNA had less cell survival after hypoxic/reoxygenation injury. This suggested that EPO/EPOR signaling from astrocyte to OPC could prevent OPC damage under hypoxic/reoxygenation condition. Our present finding of an interaction between astrocytes and OPCs may lead to a new therapeutic approach to OPCs for use against cellular stress and injury.

Interleukin-1β induces the expression of lipocortin 1 mRNA in cultured rat cortical astrocytes

Lipocortin 1 (LC1) has been shown to increase in neuronal damage and act as a neuroprotectant and a neurotrophic factor. IL-1beta acts as a mediator of inflammation and has been reported as a potent inducer of various neurotrophic factors including nerve growth factor and fibroblast growth factor. In this study, we investigated the relationship between LC1 and IL-1beta in cultured rat astrocytes. Time-and dose-dependent experiments of IL-1beta on rat cortical astrocytes in culture revealed that the expression of LC1 mRNA was significantly augmented by IL-1beta at 8 h, 10 ng/ml. In addition, IL-1beta evoked an extracellular secretion of LC1 without its cytotoxic effects. The effect of IL-1beta was completely abolished when we treated cells with inhibitor of mitogen-activated protein kinases (MAPKs) (PD98059) (25 microM), phospholipase A(2) inhibitor mepacrine (30 microM) and protein synthesis inhibitor cycloheximide (CHX) (10 microg/ml). This suggests that induction of LC1 by IL-1beta is through a MAPKs and phospholipaseA(2) pathway and requires protein synthesis. These results indicate that IL-1beta released in the central nervous system (CNS) injury can stimulate the transcription of the LC1 gene. Subsequent synthesis and release of LC1 may provide trophic support to neurons and modulate the action of IL-1beta in brain damage.

Novel Functions of Small Conductance Ca2+-activated K+ Channel in Enhanced Cell Proliferation by ATP in Brain Endothelial Cells

Brain capillary endothelial cells (BCECs) form the blood-brain barrier (BBB), which is essential for maintaining homeostasis of the brain. Net cellular turnover, which results from the balance between cell death and proliferation, is important in maintaining BBB homeostasis. Here we report a novel mechanism that underlies ATP-induced cell proliferation in t-BBEC 117, a cell line derived from bovine brain endothelial cells. Application of 0.1-30 μm ATP to t-BBEC 117 concentration-dependently increased intracellular Ca2+ concentration ([Ca2+]i) in two phases: an initial transient phase and a later and smaller sustained one. These two phases of [Ca2+]i rise were mainly due to Ca2+ release and sustained Ca2+ influx, respectively. The pretreatment with apamin, a selective blocker of small conductance Ca2+-activated K+ channels (SK), significantly reduced both the [Ca2+]i increase and K+ current induced by ATP. Transcripts corresponding to P2Yx, SK2, and transient receptor potential channels were detected in t-BBEC 117. Knock down of SK2 protein, which was the predominant Ca2+-activated K+ channel expressed in t-BBEC 117, by siRNA significantly reduced both the sustained phase of the [Ca2+]i rise and the K+ current induced by ATP. Cell proliferation was increased significantly by the presence of the stable ATP analogue ATPγS. This effect was blunted by UCL1684, a synthesized SK blocker. In conclusion, in brain endothelial cells ATP-induced [Ca2+]i rise activates SK2 current, and the subsequent membrane hyperpolarization enhances Ca2+ entry presumably through transient receptor potential channels. This positive feedback mechanism can account for the augmented cell proliferation by ATP.

Subventricular Zone-Derived Oligodendrogenesis in Injured Neonatal White Matter in Mice Enhanced by a Nonerythropoietic Erythropoietin Derivative†‡§

Perinatal hypoxia‐ischemia (HI) frequently causes white‐matter injury, leading to severe neurological deficits and mortality, and only limited therapeutic options exist. The white matter of animal models and human patients with HI‐induced brain injury contains increased numbers of oligodendrocyte progenitor cells (OPCs). However, the origin and fates of these OPCs and their potential to repair injured white matter remain unclear. Here, using cell‐type‐ and region‐specific genetic labeling methods in a mouse HI model, we characterized the Olig2‐expressing OPCs. We found that after HI, Olig2+ cells increased in the posterior part of the subventricular zone (pSVZ) and migrated into the injured white matter. However, their oligodendrocytic differentiation efficiency was severely compromised compared with the OPCs in normal tissue, indicating the need for an intervention to promote their differentiation. Erythropoietin (EPO) treatment is a promising candidate, but it has detrimental effects that preclude its clinical use for brain injury. We found that long‐term postinjury treatment with a nonerythropoietic derivative of EPO, asialo‐erythropoietin, promoted the maturation of pSVZ‐derived OPCs and the recovery of neurological function, without affecting hematopoiesis. These results demonstrate the limitation and potential of endogenous OPCs in the pSVZ as a therapeutic target for treating neonatal white‐matter injury. STEM Cells2012;30:2234–2247

Role of ES cell-expressed Ras (ERas) in tumorigenicity of gastric cancer.

ERas, a unique member of the Ras family, was initially found only in embryonic stem (ES) cells, where it plays a crucial role in the transformation of transplanted ES cells to teratomas. ERas is involved in ES cell survival, and unlike other Ras family members, is constitutively active without any mutations. The aim of this study was to investigate the expression and role of ERas in human gastric cancer. To test whether ERas played a significant role in human cancer cells, we examined its expression and function in gastric cancer. ERas was expressed in gastric cancer cell lines at different levels. Induction of ERas expression activated the phosphatidylinositol 3 kinase (PI3K)/Akt axis and then enhanced anchorage-independent growth and ERas knockdown by siRNA suppressed cell invasion. Immunohistochemical analyses revealed that ERas was expressed in 38.7% (55/142) of human gastric carcinoma tissues, and its expression was significantly associated with metastasis to the liver (P < 0.0001) and lymph nodes (P < 0.05). ERas up-regulated transcription regulatory factors including ZFHX1A, ZFHX1B, and TCF3, which repress E-cadherin. These data suggest that ERas is activated in a significant population of gastric cancer, where it may play a crucial role in gastric cancer cell survival and metastases to liver via down-regulation of E-cadherin.

The type 1 interleukin 1 receptor is not required for the death of murine hippocampal dentate granule cells and microglia activation

Alterations in inflammatory process, neuronal death, and glia response have been observed under manipulation of interleukin-1 (IL-1) and subsequent signaling through the type 1 IL-1 receptor (IL-1R1). To investigate the influence of IL-1R1 activation in the pathophysiology of a chemical-induced injury to the murine hippocampus, we examined the level and pattern of neuronal death and neuroinflammation in male weanling mice exposed to trimethyltin hydroxide (2.0 mg TMT/kg, i.p.). Dentate granule cell death occurred at 6 h post-TMT as detected by active caspase 3 immunostaining and presence of lectin positive microglia. The severity of neuronal death and microglia response increased by 12-24 h with elevations in mRNA levels for TNFalpha and IL-1alpha. In IL-1R1 null (IL-1R1-/-) mice, the pattern and severity of neuronal death at 24 or 72 h post-TMT was similar as compared to wildtype (WT) mice. In both groups, mRNA levels for TNFalpha and MIP-1alpha were elevated, no significant change was seen in either IL-1alpha or IL-1beta, and the early activation of microglia, including their ability to progress to a phagocytic phenotype, was maintained. Compared to WT mice, IL-1R1-/- mice displayed a limited glial fibrillary acidic protein (GFAP) astrocytic response, as well as a preferential induction in mRNA levels of Fas signaling components. Cumulatively, these results indicate that IL-1R1 activation is not necessary for TMT-induced death of dentate granule neurons or local activation of microglia; however, IL-1R1 signaling is involved in mediating the structural response of astrocytes to injury and may regulate apoptotic mechanisms via Fas signaling components.