Erythropoietin involves the phosphatidylinositol 3-kinase pathway, 14-3-3 protein and FOXO3a nuclear trafficking to preserve endothelial cell integrity.
OBJECTIVE
Clinical indications for erythropoietin (EPO) in the vascular system reach far beyond the treatment of anemia, but the development of EPO as a non-toxic agent rests heavily upon the cellular pathways controlled by EPO that require elucidation.


METHODS
We modulated gene activity and examined cellular trafficking of critical pathways during oxidative stress that may work in concert with EPO to protect primary cerebral endothelial cells (ECs) during oxidative stress, namely protein kinase B (Akt1), 14-3-3 protein, the Forkhead transcription factor FOXO3a.


RESULTS
Here, we show that preservation of ECs by EPO during oxygen-glucose deprivation (OGD) required the initial activation of the phosphatidylinositol 3-kinase (PI-3K) pathway through Akt1, since specific pharmacological blockade of Akt1 activity or gene silencing of Akt1 prevented EC protection by EPO. EPO subsequently involved a series of anti-apoptotic pathways to activate STAT3, STAT5, and ERK 1/2. Furthermore, EPO maintained the inhibitory phosphorylation and integrity of the 'pro-apoptotic' transcription factor FOXO3a, promoted the binding of FOXO3a to 14-3-3 protein and regulated the intracellular trafficking of FOXO3a. Additionally, gene silencing of FOXO3a during OGD significantly increased EC survival, but did not synergistically improve cytoprotection by EPO, illustrating that EPO relied upon the blockade of the FOXO3a pathway.


CONCLUSIONS
Our work defines a novel cytoprotective pathway in ECs that involves PI-3 K, STAT3, STAT5, ERK 1/2, 14-3-3 protein and FOXO3a, which can be targeted for the development of EPO as a clinically effective and safe agent in the vascular system.
Chong
Z Z
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Division of Cellular and Molecular Cerebral Ischemia, Center for Molecular Medicine and Genetics, Institute of Environmental Health Sciences, Wayne State University School of Medicine, Detroit, MI 48201, USA.
Maiese
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D048948:14-3-3 Proteins / Q000378:metabolism; D000818:Animals; D015687:Cell Hypoxia; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism; D004921:Erythropoietin / Q000494:pharmacology*; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors / Q000378:metabolism; D005947:Glucose / Q000172:deficiency; D008297:Male; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D050796:STAT3 Transcription Factor / Q000378:metabolism; D050799:STAT5 Transcription Factor / Q000378:metabolism
D048948:14-3-3 Proteins; D004921:Erythropoietin; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors; D005947:Glucose; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D050796:STAT3 Transcription Factor; D050799:STAT5 Transcription Factor; C499515:FOXO3 protein, rat; C494088:Stat3 protein, rat; C494920:Akt1 protein, rat