Shiu Hwa Yeh

A Role for the PI-3 Kinase Signaling Pathway in Fear Conditioning and Synaptic Plasticity in the Amygdala

Western blot analysis of neuronal tissues taken from fear-conditioned rats showed a selective activation of phosphatidylinositol 3-kinase (PI-3 kinase) in the amygdala. PI-3 kinase was also activated in response to long-term potentiation (LTP)-inducing tetanic stimulation. PI-3 kinase inhibitors blocked tetanus-induced LTP as well as PI-3 kinase activation. In parallel, these inhibitors interfered with long-term fear memory while leaving short-term memory intact. Tetanus and forskolin-induced activation of mitogen-activated protein kinase (MAPK) was blocked by PI-3 kinase inhibitors, which also inhibited cAMP response element binding protein (CREB) phosphorylation. These results provide novel evidence of a requirement of PI-3 kinase activation in the amygdala for synaptic plasticity and memory consolidation, and this activation may occur at a point upstream of MAPK activation.

Phosphorylation by c-Jun NH2-terminal Kinase 1 Regulates the Stability of Transcription Factor Sp1 during Mitosis

The transcription factor Sp1 is ubiquitously expressed in different cells and thereby regulates the expression of genes involved in many cellular processes. This study reveals that Sp1 was phosphorylated during the mitotic stage in three epithelial tumor cell lines and one glioma cell line. By using different kinase inhibitors, we found that during mitosis in HeLa cells, the c-Jun NH(2)-terminal kinase (JNK) 1 was activated that was then required for the phosphorylation of Sp1. In addition, blockade of the Sp1 phosphorylation via inhibition JNK1 activity in mitosis resulted in the ubiquitination and degradation of Sp1. JNK1 phosphorylated Sp1 at Thr278/739. The Sp1 mutated at Thr278/739 was unstable during mitosis, possessing less transcriptional activity for the 12(S)-lipoxygenase expression and exhibiting a decreased cell growth rate compared with wild-type Sp1 in HeLa cells. In N-methyl-N-nitrosourea-induced mammary tumors, JNK1 activation provided a potential relevance with the accumulation of Sp1. Together, our results indicate that JNK1 activation is necessary to phosphorylate Sp1 and to shield Sp1 from the ubiquitin-dependent degradation pathway during mitosis in tumor cell lines.

Selective Inhibition of Early—but Not Late—Expressed HIF-1α Is Neuroprotective in Rats after Focal Ischemic Brain Damage

The expression of hypoxia‐inducible factor‐1‐alpha (HIF‐1α) is upregulated in ischemic stroke, but its function is still unclear. In the present study, biphasic expression of HIF‐1α was observed during 1–12u2003h and after 48u2003h in neurons exposed to ischemic stress in vitro and in vivo. Treating neurons with 2‐methoxyestradiol (2ME2) 0.5u2003h after ischemic stress or pre‐silencing HIF‐1α with small interfering RNA (siRNA) decreased brain injury, brain edema and number of apoptotic cell, and downregulates Nip‐like protein X (Nix) expression. Conversely, applying 2ME2 to neurons 8u2003h after ischemic stress or silencing the HIF‐1α with siRNA 12u2003h after oxygen–glucose deprivation (OGD) increased neuron damage and decreased vascular endothelial growth factor (VEGF) expression. Taken together, these results demonstrate that HIF‐1α induced by ischemia in early and late times leads cellular apoptosis and survival, respectively, and provides a new insight into the divergent roles of HIF‐1α expression in neurons after ischemic stroke.

Heat Shock Protein 90 Is Important for Sp1 Stability during Mitosis

Our previous study has revealed that heat shock protein (Hsp) 90 can interact with Sp1 to regulate the transcriptional activity of 12(S)-lipoxygenase. Herein, we further found that the interaction between Hsp90 and Sp1 occurred during mitosis. By geldanamycin (GA) treatment and knockdown of Hsp90, we found that this interaction during mitosis was involved in the maintenance of Sp1 stability, and that the phospho-c-Jun N-terminal kinase (JNK)-1 level also decreased. As the JNK-1 was knocked down by the shRNA of JNK-1, Sp1 was degraded through a ubiquitin-dependent proteasome pathway. In addition, for mutation of the JNK-1 phosphorylated residues of Sp1, namely, Sp1(T278/739A) and Sp1(T278/739D), the effect of GA on Sp1 stability was reversed. Finally, based on the involvement of Hsp90 in Sp1 stability, the transcriptional activities of p21(WAF1/CIP1) and 12(S)-lipoxygenase under GA treatment were observed to have decreased. Taken together, Hsp90 is important for maintaining Sp1 stability during mitosis by the JNK-1-mediated phosphorylation of Sp1 to enable division into daughter cells and to regulate the expression of related genes in the interphase.

Translational and transcriptional control of Sp1 against ischaemia through a hydrogen peroxide-activated internal ribosomal entry site pathway

The exact mechanism underlying increases in Sp1 and the physiological consequences thereafter remains unknown. In rat primary cortical neurons, oxygen-glucose deprivation (OGD) causes an increase in H2O2 as well as Sp1 in early ischaemia but apparently does not change mRNA level or Sp1 stability. We hereby identified a longer 5′-UTR in Sp1 mRNA that contains an internal ribosome entry site (IRES) that regulates rapid and efficient translation of existing mRNAs. By using polysomal fragmentation and bicistronic luciferase assays, we found that H2O2 activates IRES-dependent translation. Thus, H2O2 or tempol, a superoxide dismutase-mimetic, increases Sp1 levels in OGD-treated neurons. Further, early-expressed Sp1 binds to Sp1 promoter to cause a late rise in Sp1 in a feed-forward manner. Short hairpin RNA against Sp1 exacerbates OGD-induced apoptosis in primary neurons. While Sp1 levels increase in the cortex in a rat model of stroke, inhibition of Sp1 binding leads to enhanced apoptosis and cortical injury. These results demonstrate that neurons can use H2O2 as a signalling molecule to quickly induce Sp1 translation through an IRES-dependent translation pathway that, in cooperation with a late rise in Sp1 via feed-forward transcriptional activation, protects neurons against ischaemic damage.

A novel RING finger protein, Znf179, modulates cell cycle exit and neuronal differentiation of P19 embryonal carcinoma cells

Znf179 is a member of the RING finger protein family. During embryogenesis, Znf179 is expressed in a restricted manner in the brain, suggesting a potential role in nervous system development. In this report, we show that the expression of Znf179 is upregulated during P19 cell neuronal differentiation. Inhibition of Znf179 expression by RNA interference significantly attenuated neuronal differentiation of P19 cells and a primary culture of cerebellar granule cells. Using a microarray approach and subsequent functional annotation analysis, we identified differentially expressed genes in Znf179-knockdown cells and found that several genes are involved in development, cellular growth, and cell cycle control. Flow cytometric analyses revealed that the population of G0/G1 cells decreased in Znf179-knockdown cells. In agreement with the flow cytometric data, the number of BrdU-incorporated cells significantly increased in Znf179-knockdown cells. Moreover, in Znf179-knockdown cells, p35, a neuronal-specific Cdk5 activator that is known to activate Cdk5 and may affect the cell cycle, and p27, a cell cycle inhibitor, also decreased. Collectively, these results show that induction of the Znf179 gene may be associated with p35 expression and p27 protein accumulation, which lead to cell cycle arrest in the G0/G1 phase, and is critical for neuronal differentiation of P19 cells.

N‐acetylcysteine prevents β‐amyloid toxicity by a stimulatory effect on p35/cyclin‐dependent kinase 5 activity in cultured cortical neurons

Although previous studies have indicated that the neuroprotective effect of N‐acetylcysteine (NAC) required activation of the Ras‐extracellular‐signal‐regulated kinase (ERK) pathway, the detailed mechanisms and signal cascades leading to activation ERK are not clear. In the present study, we investigated the effect of NAC on Aβ25–35‐induced neuronal death. Pretreatment of neurons with NAC 1 hr before application of Aβ prevented Aβ‐mediated cell death. NAC increased cyclin‐dependent kinase 5 (Cdk5) phosphorylation, an effect that was blocked by Cdk5 inhibitor. The neuroprotective effect of NAC was significantly attenuated by Cdk5 inhibitors or in neurons transfected with Cdk5 or p35 small interfering RNA (siRNA). Conversely, pretreatment of neurons with the calpain inhibitors calpeptin or MDL28170 enhanced the neuroprotective effect of NAC. Aβ25–35 caused a significant decrease in the level of p35, with a concomitant increase in p25, which was completely prevented by NAC. This effect of NAC was blocked by the Cdk5 inhibitors roscovitine and butyrolactone. In addition, NAC increased Cdk5/p35 kinase activity but reduced Cdk5 kinase activity. Aβ25–35 treatment decreased phosphorylated levels of ERK, which could be reversed by NAC. The effect of NAC was completely blocked by Cdk5 inhibitors. NAC reversed the Aβ25–35–induced decrease in the expression of Bcl‐2, which could be blocked by the MAPK kinase (MEK) inhibitor or Cdk5 inhibitors. These results suggest that NAC‐mediated neuroprotection against Aβ toxicity is likely mediated by the p35/Cdk5‐ERKs‐Bcl‐2 signal pathway.

Specificity protein 1-zinc finger protein 179 pathway is involved in the attenuation of oxidative stress following brain injury.

After sudden traumatic brain injuries, secondary injuries may occur during the following days or weeks, which leads to the accumulation of reactive oxygen species (ROS). Since ROS exacerbate brain damage, it is important to protect neurons against their activity. Zinc finger protein 179 (Znf179) was shown to act as a neuroprotective factor, but the regulation of gene expression under oxidative stress remains unknown. In this study, we demonstrated an increase in Znf179 protein levels in both in vitro model of hydrogen peroxide (H2O2)-induced ROS accumulation and animal models of traumatic brain injury. Additionally, we examined the sub-cellular localization of Znf179, and demonstrated that oxidative stress increases Znf179 nuclear shuttling and its interaction with specificity protein 1 (Sp1). Subsequently, the positive autoregulation of Znf179 expression, which is Sp1-dependent, was further demonstrated using luciferase reporter assay and green fluorescent protein (GFP)-Znf179-expressing cells and transgenic mice. The upregulation of Sp1 transcriptional activity induced by the treatment with nerve growth factor (NGF) led to an increase in Znf179 levels, which further protected cells against H2O2-induced damage. However, Sp1 inhibitor, mithramycin A, was shown to inhibit NGF effects, leading to a decrease in Znf179 expression and lower cellular protection. In conclusion, the results obtained in this study show that Znf179 autoregulation through Sp1-dependent mechanism plays an important role in neuroprotection, and NGF-induced Sp1 signaling may help attenuate more extensive (ROS-induced) damage following brain injury.

Suberoylanilide hydroxamic acid represses glioma stem-like cells

BackgroundGlioma stem-like cells (GSCs) are proposed to be responsible for high resistance in glioblastoma multiforme (GBM) treatment. In order to find new strategies aimed at reducing GSC stemness and improving GBM patient survival, we investigated the effects and mechanism of a histone deacetylases (HDACs) inhibitor, suberoylanilide hydroxamic acid (SAHA), since HDAC activity has been linked to cancer stem-like cell (CSC) abundance and properties.MethodsHuman GBM cell lines were plated in serum-free suspension cultures allowed for sphere forming and CSC enrichment. Subsequently, upon SAHA treatment, the stemness markers, cell proliferation, and viability of GSCs as well as cellular apoptosis and senescence were examined in order to clarify whether inhibition of GSCs occurs.ResultsWe demonstrated that SAHA attenuated cell proliferation and diminished the expression stemness-related markers (CD133 and Bmi1) in GSCs. Furthermore, at high concentrations (more than 5xa0μM), SAHA triggered apoptosis of GSCs accompanied by increases in both activation of caspase 8- and caspase 9-mediated pathways. Interestingly, we found that a lower dose of SAHA (1xa0μM and 2.5xa0μM) inhibited GSCs via cell cycle arrest and induced premature senescence through p53 up-regulation and p38 activation.ConclusionSAHA induces apoptosis and functions as a potent modulator of senescence via the p38-p53 pathway in GSCs. Our results provide a perspective on targeting GSCs via SAHA treatment, and suggest that SAHA could be used as a potent agent to overcome drug resistance in GBM patients.