Association of 14-3-3γ and Phosphorylated Bad Attenuates Injury in Ischemic Astrocytes

Chen, Xiao Qian; Fung, Yin-Wan Wendy; Yu, Albert Cheung Hoi

doi:10.1038/sj.jcbfm.9600032

Cited by 44 publications

(63 citation statements)

References 30 publications

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“…A Western blot analysis showed that the expression level of 14-3-3γ protein decreased (28.8%) in N2a/ Ngb-siRNA cells compared with N2a/vec cells ( Figure 3C). We further confirmed the protective role of 14-3-3γ from oxidative stress by transfecting 14-3-3γ plasmids [24] into N2a/ Ngb-siRNA cells. A WST-8 assay demonstrated that overexpression of 14-3-3γ in N2a/Ngb-siRNA cells significantly enhanced cell viability following treatment with 150 µmol/L of H 2 O 2 compared with vector controls ( Figure 3D).…”

Section: Resultssupporting

confidence: 56%

“…14-3-3 proteins exist predominantly in the brain and mediate essential apoptotic signaling pathways [28,29] . We have previously reported that 14-3-3γ is up-regulated and protects astrocytes from ischemia-induced apoptosis [24,25] . We compared the expression of 14-3-3γ transcripts ( Figure 3B) and proteins ( Figure 3C) in N2a/NgbsiRNA and N2a/vec cells.…”

Section: Resultsmentioning

confidence: 99%

“…14-3-3γ protein mediates essential apoptotic signaling pathways by binding to proapoptotic proteins such as Bad, Bax, ASK-1, and p53 [28] . We previously demonstrated that 14-3-3γ is up-regulated in and protects astrocytes from ischemic injury [24,25] . In ischemic neurons, Ngb is up-regulated and exerts a protective role [11] .…”

Section: Discussionmentioning

confidence: 99%

“…The total RNA (2 µg) was then used to perform RT using M-MLV transcriptase (Promega, USA), oligo (dT 15 ) primer (Promega, USA) and a total volume of 25 µL. Semi-quantitative PCR, 28 cycles of 94 °C, 45 s; 60 °C, 45 s; 72 °C, 45 s plus a final extension at 72°C for 10 min, was performed as previously reported, using β-actin as an internal control [23,24] . The primers used in PCR were Ngb: 5'-ctctggaacatggcactgtc-3' and 5'-gcactggctcgtctcttact-3'; β-actin: 5'-cagccttccttcttgggtat-3' and 5'-gctcagtaacagtccgccta-3'; and 14-3-3γ: 5'-gttggtctggctcttcatcat-3' and 5'-aggtgcagagtagacttgggtg-3'.…”

Section: Rt-pcrmentioning

confidence: 99%

“…Cell extracts were prepared and Western blot analysis was performed, as previously described [24,25] . Briefly, 10−50 µg of total protein was resolved on 12%−15% reducing SDS-PAGE gels and electrophoretically transferred to nitrocellulose membranes.…”

Section: Western Blot Analysismentioning

confidence: 99%

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Silencing neuroglobin enhances neuronal vulnerability to oxidative injury by down-regulating 14-3-3γ

Zhou

Lai

et al. 2009

Acta Pharmacol Sin

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Aim: To explore the protective role and mechanism of endogenous neuroglobin (Ngb) in neuronal cells under oxidative stress. Methods: A stable N2a neuroblastoma cell line expressing the Ngb-siRNA plasmid (N2a/Ngb-siRNA) was established by neomycin screening. Reverse transcription (RT)-PCR and Western blot analysis were used to detect Ngb gene and protein levels. Hydrogen peroxide was used to induce oxidative stress in N2a cells. Cytotoxicity and cell viability were measured by lactate dehydrogenase (LDH) and WST-8 assays. Hoechst staining demonstrated that the quantity of apoptotic cells among N2a/Ngb-siRNA cells following hydrogen peroxide treatment significantly increased compared with controls. In N2a/Ngb-siRNA cells, the expression level of activated caspase-3 significantly increased, whereas the expression of 14-3-3γ decreased compared with that of N2a/vec cells. Transfection of 14-3-3γ plasmids significantly enhanced the viability of N2a/Ngb-siRNA cells following hydrogen peroxide treatment compared with vector controls. Conclusion: Ngb contributes to neuronal defensive machinery against oxidative injuries by regulating 14-3-3γ expression.

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Section: Resultssupporting

confidence: 56%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Rt-pcrmentioning

confidence: 99%

Section: Western Blot Analysismentioning

confidence: 99%

See 3 more Smart Citations

Silencing neuroglobin enhances neuronal vulnerability to oxidative injury by down-regulating 14-3-3γ

Zhou

Lai

et al. 2009

Acta Pharmacol Sin

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Differential role of 14‐3‐3 family members in Xenopus development

Lau

Muslin

2006

Developmental Dynamics

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The 14-3-3 proteins are intracellular dimeric phosphoserine/threonine binding molecules that participate in signal transduction, checkpoint control, nutrient sensing, and cell survival pathways. Previous work established that 14-3-3 proteins are required in early Xenopus laevis development by modulating fibroblast growth factor signaling. Although this general requirement for 14-3-3 proteins in Xenopus early embryogenesis is established, there is no information about the specific role of individual 14-3-3 genes. Botanical studies previously demonstrated functional specificity among 14-3-3 genes during plant development. In this study, an antisense morpholino oligo microinjection approach was used to characterize the requirement for six specific 14-3-3 family members in Xenopus embryogenesis. Microinjection experiments followed by Western blot analysis showed that morpholinos reduced specific 14-3-3 protein levels. Embryos lacking specific 14-3-3 isoforms displayed unique phenotypic defects. In particular, reduction of 14-3-3 tau () protein, and to a lesser extent, 14-3-3 epsilon (⑀), resulted in embryos with prominent gastrulation and axial patterning defects and reduced mesodermal marker gene expression. In contrast, reduction of 14-3-3 zeta () protein caused no obvious phenotypic abnormalities. Reduction of 14-3-3 gamma (␥) protein resulted in eye defects without gastrulation abnormalities. Therefore, individual 14-3-3 genes have separable functions in vertebrate embryonic development.

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Traumatic scratch injury in astrocytes triggers calcium influx to activate the JNK/c-Jun/AP-1 pathway and switch on GFAP expression

Gao

Wang

Jiang

et al. 2013

Glia

Self Cite

116

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Astrocyte activation is a hallmark of central nervous system injuries resulting in glial scar formation (astrogliosis). The activation of astrocytes involves metabolic and morphological changes with complex underlying mechanisms, which should be defined to provide targets for astrogliosis intervention. Astrogliosis is usually accompanied by an upregulation of glial fibrillary acidic protein (GFAP). Using an in vitro scratch injury model, we scratched primary cultures of cerebral cortical astrocytes and observed an influx of calcium in the form of waves spreading away from the wound through gap junctions. Using the calcium blocker BAPTA-AM and the JNK inhibitor SP600125, we demonstrated that the calcium wave triggered the activation of JNK, which then phosphorylated the transcription factor c-Jun to facilitate the binding of AP-1 to the GFAP gene promoter to switch on GFAP upregulation. Blocking calcium mobilization with BAPTA-AM in an in vivo stab wound model reduced GFAP expression and glial scar formation, showing that the calcium signal, and the subsequent regulation of downstream signaling molecules, plays an essential role in brain injury response. Our findings demonstrated that traumatic scratch injury to astrocytes triggered a calcium influx from the extracellular compartment and activated the JNK/c-Jun/AP-1 pathway to switch on GFAP expression, identifying a previously unreported signaling cascade that is important in astrogliosis and the physiological response following brain injury.

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Association of 14-3-3γ and Phosphorylated Bad Attenuates Injury in Ischemic Astrocytes

Cited by 44 publications

References 30 publications

Silencing neuroglobin enhances neuronal vulnerability to oxidative injury by down-regulating 14-3-3γ

Silencing neuroglobin enhances neuronal vulnerability to oxidative injury by down-regulating 14-3-3γ

Differential role of 14‐3‐3 family members in Xenopus development

Traumatic scratch injury in astrocytes triggers calcium influx to activate the JNK/c-Jun/AP-1 pathway and switch on GFAP expression

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