Eukaryotic Initiation Factor-2αPhosphorylation Mediates Foam Cell Formation Induced By Apolipoprotein E-Deficient Lipoproteins

Atherosclerosis is a chronic vasculo-occlusive disease characterized by the intimal accumulation of macrophages, smooth muscle cells, and T lymphocytes, in addition to lipids and extracellular matrix components. Accumulating evidence suggests that atherosclerosis is a multifactorial diorder and several different mechanisms have been implicated in atherogenesis and lesion progression over the past two decades. However, the precise mechanisms by which atherosclerosis develops and progresses has not been fully elucidated.Endoplasmic reticulum stress (ER stress) is caused by perturbation of any of the three homeostatic functions of the ER, protein folding, functioning as an intracellular Ca2+store, and as a site for synthesis of sterols and phospholipids. Accumulation of unfolded proteins in the ER lumen results in activation of a common response to this stress situation, the unfolded protein response (UPR). The initial intent of the UPR is adaptation and restoration of the normal ER function. The adaptive mechanisms employed to combat ER stress and restore normal function include upregulation of the folding capacity of the ER and downregulation of its biosynthetic capacity. Eukaryotic initiation factor (eIF-2α) phosphorylation mediated by RNA-dependent protein kinase-like endoplasmic reticulum kinase (PERK) is one of the UPR signaling pathways that functions to attenuate global translation and reduce the biosynthetic load, thus preventing further accumulation of unfolded proteins. Increased evidence indicates that ER stress might be involved in every aspect of atherosclerosis, from the endothelial dysfunction to plaque formation, even plaque rupture. One recent study from our laboratory have demonstrated that apolipoprotein (apo) E-deficient, apoB48-containing (E-/B48) lipoproteins-induced macrophage foam cell formation is associated with a enhanced phosphorylation of macrophage PERK and eIF-2α, an increased protein level of ATF4, and a decreased global protein synthesis, indicating ER stress is involved in macrophage foam cell formation induced by E-/B48lipoproteins. Whereas, incubation of mouse peritoneal macrophages (MPMs) with eIF-2a kinase inhibitor2-aminopurine (2-AP) suppresses phosphorylation of eIF-2α, reduces intralysosomal lipoprotein accumulation, providing evidence for a potential role of eIF-2a phosphorylation in E-/B48lipoproteins-induced foam cell formation.Although2-AP has been extensively used as a specific inhibitor for eIF-2a kinases, there are scattered reports showing that this chemical compound may inhibit other kinases in some circumstances, such as mitogen-activated protein kinases. To confirm the results derived from2-AP, we inhibited phosphorylation of endogenous eIF-2a by dominant-negative mutation of eIF-2a, and examined the causal relationship between eIF-2a phosphorylation and lipid accumulation in macrophages induced by E-/B48lipoproteins. Data from this report demonstrate that overexpression of a nonphosphorylatable eIF-2a mutant in macrophages inhibited E"/B48lipoprotein-induced phosphorylation of endogenous eIF-2a, alleviated the suppressive effect of E-/B48lipoproteins on lysosomal hydrolase synthesis, and inhibited cellular lipid accumulation. More importantly, we observed that incubation of macrophages with E-/B48lipoproteins induced phosphorylation of PERK, but not other eIF-2a kinases, such as double-stranded RNA-activated protein kinase (PKR) and general control non-derepressible-2(GCN2), and that overexpression of a nonphosphorylatable PERK mutant attenuated the eIF-2a phosphorylation and reduced cholesterol ester accumulation in macrophages induced by E-/B48lipoproteins. In addition, our data also demonstrate that incubation of macrophages with E-/B48lipoproteins induces intracellular lipoprotein and cholesterol ester accumulation, which is associated with a significant decline in cholesterol efflux. Furthermore, E-/B48lipoproteins shows a lower inducibility to mRNAs encoding ABCA1and ABCG1, parallel to the protein expression of ABC A1and ABCG1.Transfection of macrophages with a nonphosphorylatable eIF-2a mutant alleviates the suppressive effect of E-/B48lipoproteins on protein expression of ABC transporters, increases macrophage cholesterol efflux, reduces intralysosomal lipoprotein accumulation, and inhibits macrophage foam cell formation. Part Ⅰ-Ⅰ Role of eIF2α-S51A mutant in macrophage foam cell formation induced by apolipoprotein E-deficient lipoproteinsObjective:Our previous work demonstrates that transformation of macrophages into foam cells by E-/B48lipoproteins is associated with an increased phosphorylation of eIF-2a, an signaling of endoplasmic reticulum stress. The objective of this study is to determine the causal role of eIF-2a phosphorylation in E-/B48lipoprotein-induced foam cell formation.Methods:Raw264.7were transfected with a nonphosphorylatable eIF-2α mutant (eIF2α-S51A) or an empty vector as control, and then incubated with E+/B48lipoproteins, E-/B48lipoproteins, or culture media alone. The E-/B48lipoprotein-induced changes in cholesterol contents were measured with enzymatic colorimetric method; the catabolism of E-/B48lipoprotein was measured with radioimmunoassay; polysome associated mRNA was isolated using sucrose gradient and the E-/B48lipoprotein-induced changes in total and polysome associated mRNAs were measured with real-time PCR; the celluar proteins changes induced by E-/B48lipoprotein were measured with Westeron Blot.Results:Incubation of empty vector-transfected macrophages with E-/B48lipoproteins induced intracellular lipoprotein and cholesterol ester accumulation, which is associated with a significant decline in lipoprotein degradation. In addition, E-/B48lipoproteins enhance macrophage eIF-2α phosphorylation and inhibit the translation efficiency of mRNAs encoding lysosomal acid lipase (LAL), cathepsin B (Cath B), with a parallel reduction in the level of these proteins. In contrast, E-/B48lipoproteins did not enhance eIF-2α phosphorylation in the eIF2α-S51A-transfected macrophages. In addition, treatment of eIF2α-S51A-transfected macrophages with E-/B48lipoproteins induces less lipid accumulation, less suppressive effect on lipoprotein degradation, and less inhibitory effect on the expression of lysosomal hydrolases, as compared with the empty vector-transfected macrophages.Conclusin:Our data using eIF2α-S51A as a tool to demonstrate that induction of eIF-2a phosphorylation is a mechanism by which E-/B48lipoproteins down-regulate lysosomal hydrolases, such as LAL and Cath B, which in turn reduces the degradation of E-/B48lipoproteins, result in intralysosomal E-/B48lipoprotein accumulation, and induce foam cell formation.Part I-II PERK is responsible for eIF-2a phosphorylation induced by E-/B48lipoproteinsObjective:There are four eIF-2a kinases that have been identified in mammalian cells. The objective of this study is to determine whether PERK is the solely kinase that is responsible for eIF-2a phosphorylation induced by E-/B48lipoproteins.Methods:Raw264.7were transfected with a nonphosphorylatable PERK mutant (PERK-K618A) or an empty vector as control, and then incubated with E+/B48lipoproteins, E-/B48lipoproteins, or culture media alone. The E-/B48lipoprotein-induced changes in cellular proteins and lipids were measured with Westeron Blot and enzymatic colorimetric method, respectively.Results:We observed that incubation of macrophages with E-/B48lipoproteins increased the phosphorylation of PERK, but not other eIF-2a kinases, such as PKR and GCN2. Overexpression of a nonphosphorylatable PERK mutant inhibited the PERK and eIF-2a phosphorylation, and alleviated cholesterol ester accumulation induced by E-/B48lipoproteins.Conclusion:These observations suggest that activation of the PERK-eIF2a signaling pathway is a mechanism by which E-/B48lipoproteins down-regulate lysosomal hydrolase synthesis, inhibit lysosomal lipoprotein degradation, and increase intracellular lipoprotein and cholesterol ester accumulation, resulting in foam cell formation. Part Ⅱ Role of eIF-2a phosphorylation in cholesterol efflux mediated by ABC transportersReverse cholesterol transport (RCT) is cholesterol trafficking from peripheral tissues to the liver, where it is excreted in the bile as unchanged cholesterol or after conversion to bile acids. Cholesterol efflux from macrophages represents the first critical step of RCT and is thought to be an important anti-atherogenic process.Both ABCA1and ABCG1are members of a large family of ATP-binding cassette transporters that have common structural motifs and use ATP as an energy source to transport a variety of substrates, including ions, lipids, and cytotoxins. Cholesterol efflux mediated by ABCA1and ABCG1plays an important role in maintaining lipid homeostasis in macrophages and suppressing the development of atherosclerosis.Objective:Both ABCA1and ABCG1have been suggested to play an important role in determining macrophage cholesterol efflux. The objective of this study is to determine the role of eIF-2a phosphorylation in cholesterol efflux mediated by ABCA1and ABCG1Methods:Raw264.7were transfected with a nonphosphorylatable eIF-2a mutant (eIF2a-S51A) or an empty vector as control, and then incubated with E+/B48lipoproteins, E-/B48lipoproteins, or culture media alone. The E-/B48lipoprotein-induced changes in cellular proteins were measured with Westeron Blot; polysome associated mRNA was isolated using sucrose gradient and the E-/B48lipoprotein-induced changes in total and polysome associated mRNAs were measured with real-time PCR; cholesterol efflux were measured with liquid scintillation.Results:Our data demonstrated that E-/B48lipoproteins decreased cholesterol efflux, and inhibited the mRNAs encoding ABCA1and ABCG1, with a parallel reduction in the level of these proteins. These changes were overcome by overexpression of a nonphosphorylatable eIF-2a mutant in macrophages.Conclusion:In the absence of apoE, apoB48-carrying lipoproteins induces eIF-2a phosphorylation, which in turn inhibits the expression of cholesterol transporters, and therefore reduces lipoprotein degradation and cholesterol efflux, leading to foam cell formation, a hallmark feature of atherosclerosis.Since we don’t observe that eIF-2α phosphorylation inhibit the translation efficiency of mRNAs encoding ABCA1and ABCG1, further research is needed to explore whether or not eIF2α phosphorylation reduce the translation efficiency of the mRNAs encoding the transcription factor, such as LXR, in turn downregulate the protein expression of ABCA1and ABCG1induced by E-/B48lipoproteins.