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.
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 β2-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 β2-glycoprotein I and IgM binding and lipid-mediated, inflammatory responses.
Ischemia, lack of blood flow, and reperfusion, return of blood flow, is 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 exposure of neo-antigens are recognized by antibodies, 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.
Ischemia, lack of blood flow, and reperfusion, return of blood flow, is 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 70-80% mortality rate. Previous studies of the intestine established a role for innate immune cells and naturally occurring antibodies (Ab) in IR-induced pathology. Furthermore, administration of two monoclonal Ab that recognize phospholipids (PL) or the PL binding protein β2-glycoprotein I (β2-GPI) restores tissue damage in IR-resistant Rag-1-/- mice to wildtype levels. These data indicate involvement of a lipid or lipid-like moiety in mediating IR-induced damage. We hypothesized that PL scramblase 1 (PLSCR1), a protein regulating bilayer asymmetry, is involved in altering the PL of endothelial cells during hypoxia leading to β2-GPI binding and subsequent inflammation. To test our hypothesis, an endothelial cell line, MS1, was subjected to hypoxia (1% O2) followed by reoxygenation as an in vitro model of IR. PLSCR1 transcription, protein expression and activity were assessed. Mass spectrometry detected PL alterations and β2-GPI binding was confirmed by immunohistochemistry. Inflammatory markers including prostaglandins were also evaluated. Our data demonstrate hypoxia and reoxygenation-induced PLSCR1 activity in PL scrambling of endothelial cells. The altered PL arrangement enhances β2-GPI binding and leads to intestinal IR pathology.
Ischemia/reperfusion (IR)-induced injury results in severe tissue damage and causes up to 30,000 human deaths per year in the US. In a mouse model of intestinal IR, C57Bl/6 mice experience significant intestinal damage; however Rag-1-/- mice, which are antibody deficient, do not sustain significant damage. Complement activation is also critical as indicated by the lack of damage in complement receptor 2 deficient (CR2-/-) mice. Additionally, when Rag-1-/- or CR2-/- mice are pretreated with C57Bl/6 antibodies or specific anti-phospholipid monoclonal antibodies, damage is restored to C57Bl/6 levels. We hypothesized that antibodies recognize a specific lipid antigen produced during IR. After subjecting C57Bl/6, CR2-/- and Rag-1-/- mice to either Sham or intestinal ischemia followed by reperfusion, we determined jejunal prostaglandin E2 (PGE2) production and lipid profile. Mass spectrometry analysis of extracted lipids indicated that lysolipids and arachidonic acid (AA) levels increased by 15 min post-ischemia in C57Bl/6 mice. Differences in lysolipids or AA will be detected between C57Bl/6, CR2-/- and Rag-1-/- mice. C57Bl/6 mice produced significantly more Cox-2 mRNA and PGE2 than either CR2-/- or Rag-1-/- mice. Pretreating Rag-1-/- mice with antibodies purified from C57Bl/6 mice restored IR-induced PGE2 production to C57Bl/6 levels. We conclude that antibodies do not influence lipid metabolism, but are required for activation of Cox-2 and subsequent production of PGE2.
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