Studying the Subcellular Localization and DNA-Binding Activity of FoxO Transcription Factors, Downstream Effectors of PI3K/Akt

Essafi, Abdelkader; Gomes, Ana; Pomeranz, Karen M.; Zwolińska, Aleksandra; Varshochi, Rana; McGovern, Ursula; Lam, Eric W.‐F.

doi:10.1007/978-1-60327-115-8_13

Cited by 12 publications

(14 citation statements)

References 14 publications

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“…Subcellular Fractionation-Membrane and cytosolic fractions were obtained from wild type or transfected astrocytes seeded on 6-well plates (1.25 ϫ 10 5 /well) as described (26). The quality of the fractionations was determined by assaying for the presence of the cytosolic protein Cu,Zn-SOD and the membrane protein R-RAS.…”

Section: Methodsmentioning

confidence: 99%

Astrocyte Resilience to Oxidative Stress Induced by Insulin-like Growth Factor I (IGF-I) Involves Preserved AKT (Protein Kinase B) Activity

Dávila

Fernández

Torres‐Alemán

2016

Journal of Biological Chemistry

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Disruption of insulin-like growth factor I (IGF-I) signaling is a key step in the development of cancer or neurodegeneration. For example, interference of the prosurvival IGF-I/AKT/ FOXO3 pathway by redox activation of the stress kinases p38 and JNK is instrumental in neuronal death by oxidative stress. However, in astrocytes, IGF-I retains its protective action against oxidative stress. The molecular mechanisms underlying this cell-specific protection remain obscure but may be relevant to unveil new ways to combat IGF-I/insulin resistance. Here, we describe that, in astrocytes exposed to oxidative stress by hydrogen peroxide (H 2 O 2 ), p38 activation did not inhibit AKT (protein kinase B) activation by IGF-I, which is in contrast to our previous observations in neurons. Rather, stimulation of AKT by IGF-I was significantly higher and more sustained in astrocytes than in neurons either under normal or oxidative conditions. This may be explained by phosphorylation of the phosphatase PTEN at the plasma membrane in response to IGF-I, inducing its cytosolic translocation and preserving in this way AKT activity. Stimulation of AKT by IGF-I, mimicked also by a constitutively active AKT mutant, reduced oxidative stress levels and cell death in H 2 O 2 -exposed astrocytes, boosting their neuroprotective action in co-cultured neurons. These results indicate that armoring of AKT activation by IGF-I is crucial to preserve its cytoprotective effect in astrocytes and may form part of the brain defense mechanism against oxidative stress injury. IGF-I2 is a pleiotropic growth factor with important prosurvival effects in neurons (1). One of the main downstream targets of IGF-I is the Ser/Thr kinase AKT (2), which mediates cell survival and proliferation (3). Upon its activation, the IGF-I receptor recruits and phosphorylates IRS docking proteins (4), which allows translocation of phosphatidylinositol 3-kinase (PI3K) to the plasma membrane where it catalyzes the formation of the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ) (5). AKT is recruited to the membrane by interaction with these messengers so that it can be fully activated by PDK1 and mTORC2 kinases (3,6). This pathway is switched off (to prevent uncontrolled proliferation) through activation of the lipid and protein phosphatase PTEN, which catalyzes PIP 3 dephosphorylation (4).Cell metabolism generates potentially harmful reactive oxygen species (ROS), which at moderate levels can act as second messengers (7). However, chronic and/or abrupt increases of ROS (uncontainable by detoxification through cellular defenses) generates oxidative stress, a pathological cellular condition that can interfere with redox-sensitive signaling pathways (7). Neurons are particularly vulnerable to oxidative stress because of their low ROS detoxifying capacity (8).We previously described that oxidative stress interferes with the IGF-I/PI3K/AKT pathway by redox activation of p38 kinase to induce neuronal death (9). IGF-I signaling impairment by oxidative stress ha...

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Section: Methodsmentioning

confidence: 99%

Astrocyte Resilience to Oxidative Stress Induced by Insulin-like Growth Factor I (IGF-I) Involves Preserved AKT (Protein Kinase B) Activity

Dávila

Fernández

Torres‐Alemán

2016

Journal of Biological Chemistry

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“…Nuclear and cytosolic fractions were obtained from total neuronal lysates as described. 39 The quality of the fractionation was determined by assaying for the presence of the nuclear protein Neu-N.…”

Section: Discussionmentioning

confidence: 99%

Two-step activation of FOXO3 by AMPK generates a coherent feed-forward loop determining excitotoxic cell fate

et al. 2012

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Cerebral ischemia and excitotoxic injury induce transient or permanent bioenergetic failure, and may result in neuronal apoptosis or necrosis. We have previously shown that ATP depletion and activation of AMP-activated protein kinase (AMPK) during excitotoxic injury induces neuronal apoptosis by transcription of the pro-apoptotic BH3-only protein, Bim. AMPK, however, also exerts pro-survival functions in neurons. The molecular switches that determine these differential outcomes are not well understood. Using an approach combining biochemistry, single-cell imaging and computational modeling, we here demonstrate that excitotoxic injury activated the bim promoter in a FOXO3-dependent manner. The activation of AMPK reduced AKT activation, and led to dephosphorylation and nuclear translocation of FOXO3. Subsequent mutation studies indicated that bim gene activation during excitotoxic injury required direct FOXO3 phosphorylation by AMPK in the nucleus as a second activation step. Inhibition of this phosphorylation prevented Bim expression and protected neurons against excitotoxic and oxygen/glucose deprivation-induced injury. Systems analysis and computational modeling revealed that these two activation steps defined a coherent feed-forward loop; a network motif capable of filtering any effects of short-term AMPK activation on bim gene induction. This may prevent unwanted AMPK-mediated Bim expression and apoptosis during transient or physiological bioenergetic stress. Excitotoxicity has been implicated in the pathogenesis of cerebral ischemia and several neurodegenerative disorders, 1-3 and results from abnormal levels of the excitatory neurotransmitter glutamate (GLUT) in the synaptic cleft. [4][5][6]

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“…ChIP was performed as previously described [26]. Briefly, 5 × 10 6 cells per ChIP assay were cross-linked with 1% formaldehyde for 10 min at 37°C and then quenched with 125 mM glycine.…”

Section: Methodsmentioning

confidence: 99%

Estrogen receptor α-coupled Bmi1 regulation pathway in breast cancer and its clinical implications

Wang

Liu

et al. 2014

BMC Cancer

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BackgroundBmi1 has been identified as an important regulator in breast cancer, but its relationship with other signaling molecules such as ERα and HER2 is undetermined.MethodsThe expression of Bmi1 and its correlation with ERα, PR, Ki-67, HER2, p16INK4a, cyclin D1 and pRB was evaluated by immunohistochemistry in a collection of 92 cases of breast cancer and statistically analyzed. Stimulation of Bmi1 expression by ERα or 17β-estradiol (E2) was analyzed in cell lines including MCF-7, MDA-MB-231, ERα-restored MDA-MB-231 and ERα-knockdown MCF-7 cells. Luciferase reporter and chromatin immunoprecipitation assays were also performed.ResultsImmunostaining revealed strong correlation of Bmi1 and ERα expression status in breast cancer. Expression of Bmi1 was stimulated by 17β-estradiol in ERα-positive MCF-7 cells but not in ERα-negative MDA-MB-231 cells, while the expression of Bmi1 did not alter expression of ERα. As expected, stimulation of Bmi1 expression could also be achieved in ERα-restored MDA-MB-231 cells, and at the same time depletion of ERα decreased expression of Bmi1. The proximal promoter region of Bmi1 was transcriptionally activated with co-transfection of ERα in luciferase assays, and the interaction of the Bmi1 promoter with ERα was confirmed by chromatin immunoprecipitation. Moreover, in breast cancer tissues activation of the ERα-coupled Bmi1 pathway generally correlated with high levels of cyclin D1, while loss of its activity resulted in aberrant expression of p16INK4a and a high Ki-67 index, which implied a more aggressive phenotype of breast cancer.ConclusionsExpression of Bmi1 is influenced by ERα, and the activity of the ERα-coupled Bmi1 signature impacts p16INK4a and cyclin D1 status and thus correlates with the tumor molecular subtype and biologic behavior. This demonstrates the important role which is played by ERα-coupled Bmi1 in human breast cancer.

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Studying the Subcellular Localization and DNA-Binding Activity of FoxO Transcription Factors, Downstream Effectors of PI3K/Akt

Cited by 12 publications

References 14 publications

Astrocyte Resilience to Oxidative Stress Induced by Insulin-like Growth Factor I (IGF-I) Involves Preserved AKT (Protein Kinase B) Activity

Astrocyte Resilience to Oxidative Stress Induced by Insulin-like Growth Factor I (IGF-I) Involves Preserved AKT (Protein Kinase B) Activity

Two-step activation of FOXO3 by AMPK generates a coherent feed-forward loop determining excitotoxic cell fate

Estrogen receptor α-coupled Bmi1 regulation pathway in breast cancer and its clinical implications

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