ABSTRACT
ATF4 is a pro-oncogenic transcription factor whose translation is activated by eIF2 phosphorylation through delayed re-initiation involving two uORFs in the mRNA leader. However, in yeast, the effect of eIF2 phosphorylation can be mimicked by eIF5 overexpression, which turns eIF5 into translational inhibitor, thereby promoting translation of GCN4, the yeast ATF4 equivalent. Furthermore, regulatory protein termed eIF5-mimic protein (5MP) can bind eIF2 and inhibit general translation. Here, we show that 5MP1 overexpression in human cells leads to strong formation of 5MP1:eIF2 complex, nearly comparable to that of eIF5:eIF2 complex produced by eIF5 overexpression. Overexpression of eIF5, 5MP1 and 5MP2, the second human paralog, promotes ATF4 expression in certain types of human cells including fibrosarcoma. 5MP overexpression also induces ATF4 expression in Drosophila The knockdown of 5MP1 in fibrosarcoma attenuates ATF4 expression and its tumor formation on nude mice. Since 5MP2 is overproduced in salivary mucoepidermoid carcinoma, we propose that overexpression of eIF5 and 5MP induces translation of ATF4 and potentially other genes with uORFs in their mRNA leaders through delayed re-initiation, thereby enhancing the survival of normal and cancer cells under stress conditions.
Subject(s)
Activating Transcription Factor 4/metabolism , DNA-Binding Proteins/metabolism , Eukaryotic Initiation Factor-2/metabolism , Eukaryotic Initiation Factor-5/metabolism , Peptide Chain Initiation, Translational , Animals , Carcinogenesis/pathology , Cell Line, Tumor , Drosophila melanogaster/metabolism , Eukaryotic Initiation Factor-3 , Fibrosarcoma/pathology , Gene Knockdown Techniques , HEK293 Cells , HeLa Cells , Humans , Male , Mass Spectrometry , Mice, NudeABSTRACT
Multiple pathologic conditions, including hemorrhage, tumor angiogenesis, and ischemia-reperfusion events, will result in hypoxia and subsequent reperfusion. Previous studies have analyzed the lipid changes within whole tissues and indicated that ischemia-reperfusion altered tissue and cellular phospholipids. Using an in vitro cell culture model of hypoxia and reoxygenation, we examined the endothelial lipid changes. We hypothesized that phospholipid scramblase 1, a protein that regulates bilayer asymmetry, is involved in altering the phospholipids of endothelial cells during hypoxia, a component of ischemia, leading to ß2-glycoprotein I and IgM binding and subsequent lipid-mediated, inflammatory responses. We have completed the first comprehensive study of steady-state phospholipid scramblase 1 mRNA levels, protein expression, and activity under conditions of hypoxia and reoxygenation. Phospholipid scramblase 1 regulates phosphatidylserine exposure in response to oxygen stress, leading to ß2-glycoprotein I and IgM binding and lipid-mediated, inflammatory responses.
Subject(s)
Endothelium, Vascular/metabolism , Phospholipid Transfer Proteins/metabolism , Reperfusion Injury/metabolism , Vasculitis/metabolism , beta 2-Glycoprotein I/metabolism , Animals , Cell Hypoxia , Cell Line , Endothelium, Vascular/pathology , Immunoglobulin M/genetics , Immunoglobulin M/metabolism , Inflammation/genetics , Inflammation/metabolism , Inflammation/pathology , Mice , Phospholipid Transfer Proteins/genetics , Reperfusion Injury/genetics , Reperfusion Injury/pathology , Vasculitis/genetics , Vasculitis/pathology , beta 2-Glycoprotein I/geneticsABSTRACT
Ischemia, lack of blood flow, and reperfusion, return of blood flow, are a common phenomenon affecting millions of Americans each year. Roughly 30,000 Americans per year experience intestinal ischemia-reperfusion (IR), which is associated with a high mortality rate. Previous studies of the intestine established a role for neutrophils, eicosanoids, the complement system and naturally occurring antibodies in IR-induced pathology. Furthermore, data indicate involvement of a lipid or lipid-like moiety in mediating IR-induced damage. It has been proposed that antibodies recognize exposure of neo-antigens, triggering action of the complement cascade. While it is evident that the pathophysiology of IR-induced injury is complex and multi-factorial, we focus this review on the involvement of eicosanoids, phospholipids and neo-antigens in the early pathogenesis. Lipid changes occurring in response to IR, neo-antigens exposed and the role of a phospholipid transporter, phospholipid scramblase 1 will be discussed.
Subject(s)
Intestinal Mucosa/metabolism , Intestines/pathology , Membrane Lipids/metabolism , Reperfusion Injury/metabolism , Animals , Antibodies/immunology , Antigens/immunology , Humans , Hypoxia/metabolism , Intestines/blood supply , Intestines/immunology , Phospholipid Transfer Proteins/metabolism , Reperfusion Injury/immunology , Signal TransductionABSTRACT
The mortality rate due to intestinal ischemia/reperfusion (IR) remains at 60-80%. As toll-like receptor (TLR) 4 has been shown to be critical for IR injury in several organs, including the intestine, and TLR9 is necessary for IR-induced damage of the liver, we investigated the hypothesis that TLR9 is involved in intestinal IR-induced damage. Wildtype (C57Bl/6) and TLR9(-/-) mice were subjected to intestinal IR or Sham treatment. Several markers of damage and inflammation were assessed, including mucosal injury, eicosanoid production, cytokine secretion and complement deposition. Although IR-induced injury was not altered, PGE(2) production was decreased in TLR9(-/-) mice. Attenuated PGE(2) production was not due to differences in percentage of lipids or COX-2 transcription. The data indicate that TLR9 is not required for IR-induced injury or inflammation of the intestine.
