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Glia maturation factor overexpression in neuroblastoma cells activates glycogen synthase kinase-3beta and caspase-3
Asgar Zaheer1, Scott Knight, Ashna Zaheer
1Veterans Affair Medical Center, Iowa City, IA, USA. asgar-zaheer@uiowa.edu
Abstract:
In the present study we report that a replication-defective adenovirus construct of GMF cDNA (GMF-V) induced overexpression of GMF protein in neuroblastoma (N18) cells caused cytotoxicity and loss of cell viability. A significant increase in activation of GSK-3beta occurred after infection with GMF-V when compared with mock and lacZ controls. Overexpression of GMF also increased caspase-3 activity, an early marker of apoptosis. Depletion of GMF gene by introducing GMF-specific siRNA (GsiRNA) completely blocked both activation of GSK-3beta and caspase-3 activation whereas a control scrambled siRNA (CsiRNA) had no effect. A cell-permeable peptide inhibitor of GSK-3beta, and lithium completely prevented GMF-dependent activation of caspase-3. These results demonstrate that GSK-3 mediates activation of the death domain caspase by GMF overexpression. We also show that the phosphorylation of GSK-3-dependent site of Tau was a consequence of GMF-overexpression in N18 cells. Taken together our results imply that GMF is involved in the signaling leading to the activation of GSK-3beta and caspase-3 in N18 cells and strongly suggest its involvement in neurodegeneration since GSK-3beta is known to hyperphosphorylate tau which is associated with the neurotoxicity of neurofibrillary tangles in Alzheimer's disease.
Insights
Glial maturation factor (GMF) overexpression in neuroblastoma cells induces cytotoxicity via GSK-3beta and caspase-3 activation. Inhibiting GSK-3beta prevents GMF-induced apoptosis, suggesting GMF’s role in neurodegeneration.
Area of Science:
- Neuroscience
- Cell Biology
- Molecular Biology
Background:
- Glial maturation factor (GMF) is implicated in neuronal development and function.
- Dysregulation of GMF may contribute to neurodegenerative processes.
- Glycogen synthase kinase 3 beta (GSK-3beta) and caspase-3 are key players in apoptosis and neurodegeneration.
Purpose of the Study:
- To investigate the role of GMF overexpression in neuroblastoma cell death.
- To elucidate the signaling pathways, specifically involving GSK-3beta and caspase-3, activated by GMF.
- To explore the potential link between GMF, GSK-3beta, and tau phosphorylation in neurotoxicity.
Main Methods:
- Overexpression of GMF using a replication-defective adenovirus construct (GMF-V) in N18 neuroblastoma cells.
- Assessment of cell viability, cytotoxicity, and apoptosis markers (caspase-3 activity).
- Analysis of GSK-3beta activation using GMF-specific siRNA (GsiRNA) and a scrambled siRNA (CsiRNA).
- Pharmacological inhibition of GSK-3beta using a cell-permeable peptide inhibitor and lithium.
- Evaluation of tau phosphorylation at GSK-3beta-dependent sites.
Main Results:
- GMF-V induced significant cytotoxicity and loss of viability in N18 cells.
- GMF overexpression led to increased activation of GSK-3beta and caspase-3.
- GMF-specific siRNA (GsiRNA) blocked GSK-3beta and caspase-3 activation, while CsiRNA had no effect.
- GSK-3beta inhibition prevented GMF-induced caspase-3 activation.
- GMF overexpression resulted in phosphorylation of tau at GSK-3beta-dependent sites.
Conclusions:
- GMF overexpression activates GSK-3beta and caspase-3, leading to apoptosis in neuroblastoma cells.
- GSK-3beta acts as a mediator in the GMF-induced activation of death-associated caspases.
- GMF signaling contributes to tau hyperphosphorylation, a hallmark of neurodegenerative diseases like Alzheimer's.
- These findings suggest GMF's involvement in neurodegenerative pathways mediated by GSK-3beta and tau.
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