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Macrophage CD36 binds and internalizes oxidized low density lipoprotein (oxLDL) to facilitate foam cell formation. CD36 expression is activated by peroxisome proliferator-activated receptor γ (PPARγ). Tamoxifen, an anti-breast cancer medicine, has demonstrated pleiotropic functions including cardioprotection with unfully elucidated mechanisms. In this study, we determined that treatment of ApoE-deficient mice with tamoxifen reduced atherosclerosis, which was associated with decreased CD36 and PPARγ expression in lesion areas. At the cellular level, we observed that tamoxifen inhibited CD36 protein expression in human THP-1 monocytes, THP-1/PMA macrophages, and human blood monocyte-derived macrophages. Associated with decreased CD36 protein expression, tamoxifen reduced cellular oxLDL accumulation in a CD36-dependent manner. At the transcriptional level, tamoxifen decreased CD36 mRNA expression, promoter activity, and the binding of the PPARγ response element in CD36 promoter to PPARγ protein. Tamoxifen blocked ligand-induced PPARγ nuclear translocation and CD36 expression, but it increased PPARγ phosphorylation, which was due to that tamoxifen-activated ERK1/2. Furthermore, deficiency of PPARγ expression in macrophages abolished the inhibitory effect of tamoxifen on CD36 expression or cellular oxLDL accumulation both in vitro and in vivo Taken together, our study demonstrates that tamoxifen inhibits CD36 expression and cellular oxLDL accumulation by inactivating the PPARγ signaling pathway, and the inhibition of macrophage CD36 expression can be attributed to the anti-atherogenic properties of tamoxifen., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Previously, we have demonstrated that 15(S)-hydroxyeicosatetranoic acid (15(S)-HETE) induces CD36 expression involving STAT1. Many studies have shown that peroxisome proliferator-activated receptor (PPAR)-γ mediates CD36 expression. Therefore, we asked the question whether these transcriptional factors interact with each other in the regulation of CD36 expression by 15(S)-HETE. Here, we show that STAT1 interacts with PPARγ in the induction of CD36 expression and foam cell formation by 15(S)-HETE. In addition, using molecular biological approaches such as EMSA, supershift EMSA, ChIP, re-ChIP, and promoter-reporter gene assays, we demonstrate that the STAT1 and PPARγ complex binds to the STAT-binding site at -107 nucleotides in the CD36 promoter and enhances its activity. Furthermore, the interaction of STAT1 with PPARγ depends on STAT1 acetylation, which is mediated by p300. In addition, our findings show that reactive oxygen species-dependent Syk and Pyk2 stimulation is required for p300 tyrosine phosphorylation and activation. Together, these results demonstrate that an interaction between STAT1, p300, and peroxisome proliferator-activated receptor-γ is required for 15(S)-HETE-induced CD36 expression, oxidized low density lipoprotein uptake, and foam cell formation, critical events underlying the pathogenesis of atherosclerosis., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Oxidized low-density lipoprotein (ox-LDL) up-regulates CD36, a scavenger receptor responsible for macrophage uptake of ox-LDL without limitation. However, the precise underlying mechanism is not completely understood. Our previous study has demonstrated that ox-LDL induces endoplasmic reticulum (ER) stress in macrophages. The goal of this study was to explore the exact relationship between ER stress and macrophage-derived foam cell formation and whether ER stress would be involved in ox-LDL-induced CD36 up-regulation. Our results showed that ox-LDL-induced lipid accumulation in macrophages was promoted synergistically by ER stress inducer tunicamycin (TM), while attenuated by ER stress inhibitor 4-phenylbutyric acid (PBA). Ox-LDL caused CD36 up-regulation with concomitant activation of ER stress as assessed by phosphorylation of inositol-requiring kinase/endonuclease-1 (IRE-1) and protein kinase-like ER kinase (PERK), up-regulation of X-box-binding protein 1 (XBP1) and glucose-regulated protein 78 (GRP 78), and nuclear translocation of activating transcription factor 6 (ATF6). TM not only up-regulated CD36 alone but also synergized with ox-LDL to increase CD36 expression. Alleviation of ER stress with PBA and siRNA against ATF6, IRE1, and GRP78 mitigated ox-LDL-induced CD36 protein up-regulation. Moreover, administration of apoE(-/-) mice with PBA suppressed the up-regulation of CD36, phospho-IRE1, and GRP78 in macrophage-dense atherosclerotic lesions and in peritoneal macrophages. Additionally, CD36 silencing attenuated ox-LDL-induced nuclear translocation of ATF6, phosphorylation of IRE1 and up-regulation of XBP1 and GRP78. These data indicate that CD36-mediated ox-LDL uptake in macrophages triggers ER stress response, which, in turn, plays a critical role in CD36 up-regulation, enhancing the foam cell formation by uptaking more ox-LDL.

IL-13 is a potent stimulator of alternative monocyte/macrophage activation. During alternative activation, the expression of several proteins is induced including 15-lipoxygenase (15-LO), a lipid-peroxidating enzyme and the scavenger receptor CD36. We previously reported that α(M)β(2) integrin activation or clustering suppresses the expression of both 15-LO and CD36. In this study we focused on exploring the molecular mechanisms that down-regulate CD36 expression and CD36-mediated foam cell formation in IL-13-stimulated monocytes/macrophages after α(M)β(2) activation. Our studies reveal that α(M)β(2) integrin activation inhibits the IL-13 activation of several critical pathways that are required for macrophage alternative activation; namely, blocking Jak2 and Tyk2 phosphorylation, which bind to the cytoplasmic tails of the IL-4Rα/IL-13Rα1 complex. This leads to the inhibition of tyrosine phosphorylation of Stats (Stat1, Stat3, and Stat6) and prevents the formation of a signaling complex (containing p38MAPK, PKCδ, and Stat3) that are critical for the expression of both 15-LO and CD36. Jak2-mediated Hck activation is also inhibited, thereby preventing Stats serine phosphorylation, which is essential for downstream Stat-dependent gene transcription. Moreover, inhibition of Jak2, Tyk2, or their downstream target 15-LO with antisense oligonucleotides profoundly inhibits IL-13-induced CD36 expression and CD36-dependent foam cell formation, whereas13(S) Hydroperoxyoctadecadienoic acid (HPODE), a 15-LO product and peroxisome proliferator-activated receptor-γ ligand, completely restores CD36 expression in monocytes treated with 15-LO antisense. α(M)β(2) integrin activation controls CD36 expression and foam cell formation in alternatively activated monocyte/macrophages by blocking Tyk2/Jak2 phosphorylation via a 15-LO-dependent pathway. The discovery of this mechanism helps our understanding of the potential role of alternatively activated macrophages in atherogenesis and highlights the impact of integrin α(M)β(2) on this process.

Sarcolemmal CD36 facilitates myocardial fatty acid (FA) uptake, which is markedly reduced in CD36-deficient rodents and humans. CD36 also mediates signal transduction events involving a number of cellular pathways. In taste cells and macrophages, CD36 signaling was recently shown to regulate store-responsive Ca(2+) flux and activation of Ca(2+)-dependent phospholipases A(2) that cycle polyunsaturated FA into phospholipids. It is unknown whether CD36 deficiency influences myocardial Ca(2+) handling and phospholipid metabolism, which could compromise the heart, typically during stresses. Myocardial function was examined in fed or fasted (18-22 h) CD36(-/-) and WT mice. Echocardiography and telemetry identified conduction anomalies that were associated with the incidence of sudden death in fasted CD36(-/-) mice. No anomalies or death occurred in WT mice during fasting. Optical imaging of perfused hearts from fasted CD36(-/-) mice documented prolongation of Ca(2+) transients. Consistent with this, knockdown of CD36 in cardiomyocytes delayed clearance of cytosolic Ca(2+). Hearts of CD36(-/-) mice (fed or fasted) had 3-fold higher SERCA2a and 40% lower phospholamban levels. Phospholamban phosphorylation by protein kinase A (PKA) was enhanced after fasting reflecting increased PKA activity and cAMP levels in CD36(-/-) hearts. Abnormal Ca(2+) homeostasis in the CD36(-/-) myocardium associated with increased lysophospholipid content and a higher proportion of 22:6 FA in phospholipids suggests altered phospholipase A(2) activity and changes in membrane dynamics. The data support the role of CD36 in coordinating Ca(2+) homeostasis and lipid metabolism and the importance of this role during myocardial adaptation to fasting. Potential relevance of the findings to CD36-deficient humans would need to be determined.

A pathological hallmark of Alzheimer disease (AD) is deposition of amyloid β (Aβ) in the brain. Aβ binds to microglia via a receptor complex that includes CD36 leading to production of proinflammatory cytokines and neurotoxic reactive oxygen species and subsequent neurodegeneration. Interruption of Aβ binding to CD36 is a potential therapeutic strategy for AD. To identify pharmacologic inhibitors of Aβ binding to CD36, we developed a 384-well plate assay for binding of fluorescently labeled Aβ to Chinese hamster ovary cells stably expressing human CD36 (CHO-CD36) and screened an Food and Drug Administration-approved compound library. The assay was optimized based on the cells' tolerance to dimethyl sulfoxide, Aβ concentration, time required for Aβ binding, reproducibility, and signal-to-background ratio. Using this assay, we identified four compounds as potential inhibitors of Aβ binding to CD36. These compounds were ursolic acid, ellipticine, zoxazolamine, and homomoschatoline. Of these compounds, only ursolic acid, a naturally occurring pentacyclic triterpenoid, successfully inhibited binding of Aβ to CHO-CD36 cells in a dose-dependent manner. The ursolic acid effect reached a plateau at ~20 μm, with a maximal inhibition of 64%. Ursolic acid also blocked binding of Aβ to microglial cells and subsequent ROS production. Our data indicate that cell-based high-content screening of small molecule libraries for their ability to block binding of Aβ to its receptors is a useful tool to identify novel inhibitors of receptors involved in AD pathogenesis. Our data also suggest that ursolic acid is a potential therapeutic agent for AD via its ability to block Aβ-CD36 interactions.

Increasing evidence suggests that adenosine monophosphate-activated protein kinase (AMPK) exerts protective effects for cardiovascular diseases apart from the regulation of energy homeostasis. However, the role of AMPK and its underlying mechanism on macrophage foam cell formation are poorly understood. In this study, we sought to investigate the potential effects of AMPK in modulating cholesterol deposition by using murine macrophage-derived foam cells. Incubation with 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR) markedly attenuated the cholesterol ester accumulation in oxidized low density lipoprotein-loaded macrophages. Notably, AICAR treatment significantly increased ATP-binding cassette transporters G1 (Abcg1) mRNA and protein levels without affecting mRNA and protein expression of ABCA1, scavenger receptors, including scavenger receptor-A, CD36, and scavenger receptor-BI (SR-BI), and cholesterol synthesis-related genes. The up-regulation of Abcg1 by AICAR was independent of the liver X receptor/retinoid X receptor pathway but dependent on ERK activation. AICAR elevates Abcg1 expression through a post-transcriptional mechanism that stabilizes the mRNA. Using a heterologous system with luciferase as a reporter, we further identify the Abcg1 mRNA 3'-UTR responsible for the regulatory effect of AICAR. Prevention of ABCG1 expression by small interfering RNA abolished the AICAR-mediated attenuation on foam cell formation. Furthermore, increased ABCG1 expression and reduced lipid accumulation were demonstrated in AICAR-treated macrophages isolated from apolipoprotein E-deficient mice (apoE(-/-) mice). AICAR treatment also inhibited atherosclerotic plaque formation in apoE(-/-) mice. Our findings elucidate a precise mechanism involved in the prevention of atherogenesis by AMPK.

FAT/CD36 is a membrane scavenger receptor that facilitates long chain fatty acid uptake by muscle. Acute increases in membrane CD36 and fatty acid uptake have been reported in response to insulin and contraction. In this study we have explored protein ubiquitination as one potential mechanism for the regulation of CD36 level. CD36 expressed in Chinese hamster ovary (CHO) or HEK 293 cells was found to be polyubiquitinated via a process involving both lysines 48 and 63 of ubiquitin. Using CHO cells expressing the insulin receptor (CHO/hIR) and CD36, it is shown that addition of insulin (100 nm, 10 and 30 min) significantly reduced CD36 ubiquitination. In contrast, ubiquitination was strongly enhanced by fatty acids (200 microm palmitate or oleate, 2 h). Similarly, endogenous CD36 in C2C12 myotubes was ubiquitinated, and this was enhanced by oleic acid treatment, which also reduced total CD36 protein in cell lysates. Insulin reduced CD36 ubiquitination, increased CD36 protein, and inhibited the opposite effects of fatty acids on both parameters. These changes were paralleled by changes in fatty acid uptake, which could be blocked by the CD36 inhibitor sulfosuccinimidyl oleate. Mutation of the two lysine residues in the carboxyl-terminal tail of CD36 markedly attenuated ubiquitination of the protein expressed in CHO cells and was associated with increased CD36 level and enhanced oleate uptake and incorporation into triglycerides. In conclusion, fatty acids and insulin induce opposite alterations in CD36 ubiquitination, modulating CD36 level and fatty acid uptake. Altered CD36 turnover may contribute to abnormal fatty acid uptake in the insulin-resistant muscle.

Epidemiological studies suggest that the consumption of flavonoid-rich diets decreases the risk of cardiovascular diseases. However, the target sites of flavonoids underlying the protective mechanism in vivo are not known. Quercetin represents antioxidative/anti-inflammatory flavonoids widely distributed in the human diet. In this study, we raised a novel monoclonal antibody 14A2 targeting the quercetin-3-glucuronide (Q3GA), a major antioxidative quercetin metabolite in human plasma, and found that the activated macrophage might be a potential target of dietary flavonoids in the aorta. Immunohistochemical studies with monoclonal antibody 14A2 demonstrated that the positive staining specifically accumulates in human atherosclerotic lesions, but not in the normal aorta, and that the intense staining was primarily associated with the macrophage-derived foam cells. In vitro experiments with murine macrophage cell lines showed that the Q3GA was significantly taken up and deconjugated into the much more active aglycone, a part of which was further converted to the methylated form, in the activated macrophages. In addition, the mRNA expression of the class A scavenger receptor and CD36, which play an important role for the formation of foam cells, was suppressed by the treatment of Q3GA. These results suggest that injured/inflamed arteries with activated macrophages are the potential targets of the metabolites of dietary quercetin. Our data provide a new insight into the bioavailability of dietary flavonoids and the mechanism for the prevention of cardiovascular diseases.

Fatty acid translocase (FAT/CD36) plays an important role in facilitating long chain fatty acid transport. FAT/CD36 gene deletion protects mice from high fat diet-induced obesity. In this study we have investigated the regulatory mechanism of FAT/CD36 expression at the transcription level. FAT/CD36 expression was activated during 3T3-L1 adipocyte differentiation, and FAT/CD36 protein levels were positively correlated with CCAAT/enhancer-binding protein alpha (C/EBPalpha) and peroxisome proliferator-activated receptor gamma. However, a negative correlation was detected between FAT/CD36 and C/EBPbeta. Overexpression of C/EBPalpha or C/EBPbeta increased FAT/CD36 mRNA and protein levels in several types of cells. Restoration of C/EBPalpha or C/EBPbeta expression in C/EBPalpha- or C/EBPbeta-deficient mouse embryonic fibroblasts increased FAT/CD36 expression. However, in mouse embryonic fibroblasts C/EBPalpha was a more potent activator of FAT/CD36 expression than was C/EBPbeta. Expression of C/EBPalpha robustly increased FAT/CD36 proximal promoter-directed luciferase expression in human embryonic kidney 293 cells. A C/EBP-responsive element was identified in the FAT/CD36 promoter by using 5' and specific site mutations. The binding of C/EBPalpha in the FAT/CD36 promoter was detected by chromatin immunoprecipitation in 3T3-L1 adipocytes. These results demonstrated that C/EBPalpha regulates FAT/CD36 gene expression at the transcriptional level.

Oxidative stress is implicated in the pathogenesis of ischemia/reperfusion injury. Recently, we demonstrated that activation of CD36, a class B scavenger receptor, mediates free radical production and tissue injury in cerebral ischemia (1). Oxidized low density lipoproteins (oxLDL) are among the ligands that bind to CD36 and are elevated in acute cerebral infarction. SS31 is a cell-permeable antioxidant peptide that reduces intracellular free radicals and inhibits LDL oxidation/lipid peroxidation (2). The current study was designed to investigate whether treatment with SS31 normalizes ischemia-induced redox changes and attenuates CD36-mediated tissue injury. C57BL/6 mice were subjected to transient middle cerebral artery occlusion (MCAO). Redox status and infarct volume were measured in animals treated with either saline or SS31. Oxidative stress induced by ischemia/reperfusion profoundly depleted glutathione (GSH) concentrations in the ipsilateral cortex and striatum. Treating mice with SS31 immediately after reperfusion significantly attenuated ischemia-induced GSH depletion in the cortex and reduced infarct size. By contrast, the protective effect of SS31 was absent in CD36 knock-out mice, indicating that SS31 is acting through inhibition of CD36. Treating C57BL/6 mice with SS31 reduced CD36 expression in postischemic brain and mouse peritoneal macrophages (MPM). Further in vitro studies revealed that SS31 attenuated oxLDL-induced CD36 expression and foam cell formation in MPM. These in vivo and in vitro studies indicate that the down-regulation of CD36 by novel class antioxidant peptides may be a useful strategy to treat ischemic stroke victims.

We recently identified a novel family of oxidized choline glycerophospholipid (oxPC) molecular species enriched in atheroma that serve as endogenous ligands for the scavenger receptor CD36 (oxPC(CD36)), facilitating macrophage cholesterol accumulation and foam cell formation (Podrez, E. A., Poliakov, E., Shen, Z., et al. (2002) J. Biol. Chem. 277, 38517-38523). A high affinity CD36 recognition motif was defined within oxPC(CD36), an oxidatively truncated sn-2 acyl group with a terminal gamma-hydroxy (or oxo)-alpha,beta-unsaturated carbonyl. The fate of these species once formed in vivo is unknown. Here we show that a subset of oxPC(CD36), a phosphatidylcholine molecular species possessing sn-2 esterified fatty acyl hydroxyalkenal groups, can undergo a slow intramolecular cyclization and dehydration reaction to form novel oxPC species possessing a sn-2 acyl group that incorporates a terminal furyl moiety (oxPC-furan). Using high performance liquid chromatography with on-line tandem mass spectrometry in combination with unambiguous organic synthesis, we confirm that oxPC-furans, ultimately derived from phospholipids with sn-2 esterified docosahexaenoic, arachidonic, or linoleic acids, are formed during exposure of model membranes and isolated lipoproteins to physiological oxidant systems. In vivo generation of oxPC-furans at sites of enhanced oxidant stress is also demonstrated, such as within brain tissues following cerebral ischemia. Cell binding studies reveal that in contrast to their oxPC(CD36) precursors, oxPC-furans lack CD36 binding activity. Taken together, the present studies identify oxPC-furans as a novel family of oxidized phospholipids that are formed in vivo from phospholipid hydroxyalkenals but that lack CD36 binding activity.

Toll-like receptors (TLRs) are receptors of the innate immune system responsible for recognizing pathogen-associated molecular patterns. TLR2 seems to be the most promiscuous TLR receptor able to recognize the most diverse set of pathogen-associated patterns. Its promiscuity has been attributed to its unique ability to heterodimerize with TLRs 1 and 6 and, most recently, to its association with CD36 in response to diacylated lipoproteins. Thus, it seems that TLR2 forms receptor clusters in response to different microbial ligands. In this study we investigated TLR2 cell surface heterotypic interactions in response to different ligands as well as internalization and intracellular trafficking. Our data show that TLR2 forms heterodimers with TLR1 and TLR6 and that these heterodimer pre-exist and are not induced by the ligand. Upon stimulation by the specific ligand, these heterodimers are recruited within lipid rafts. In contrast, heterotypic associations of TLR2/6 with CD36 are not preformed and are ligand-induced. All TLR2 receptor clusters accumulate in lipid rafts and are targeted to the Golgi apparatus. This localization and targeting is ligand-specific. Activation occurs at the cell surface, and the observed trafficking is independent of signaling.

Microglial interaction with extracellular beta-amyloid fibrils (fAbeta) is mediated through an ensemble of cell surface receptors, including the B-class scavenger receptor CD36, the alpha(6)beta(1)-integrin, and the integrin-associated protein/CD47. The binding of fAbeta to this receptor complex has been shown to drive a tyrosine kinase-based signaling cascade leading to production of reactive oxygen species and stimulation of phagocytic activity; however, little is known about the intracellular signaling cascades governing the microglial response to fAbeta. This study reports a direct mechanistic link between the fAbeta cell surface receptor complex and downstream signaling events responsible for NADPH oxidase activation and phagosome formation. The Vav guanine nucleotide exchange factor is tyrosine-phosphorylated in response to fAbeta peptides as a result of the engagement of the microglia fAbeta cell surface receptor complex. Co-immunoprecipitation studies demonstrate an Abeta-dependent association between Vav and both Lyn and Syk kinases. The downstream target of Vav, the small GTPase Rac1, is GTP-loaded in an Abeta-dependent manner. Rac1 is both an essential component of the NADPH oxidase and a critical regulator of microglial phagocytosis. The direct role of Vav in fAbeta-stimulated intracellular signaling cascades was established using primary microglia obtained from Vav(-/-) mice. Stimulation of Vav(-/-) microglia with fAbeta failed to generate NADPH oxidase-derived reactive oxygen species and displayed a dramatically attenuated phagocytic response. These findings directly link Vav phosphorylation to the Abeta-receptor complex and demonstrate that Vav activity is required for fAbeta-stimulated intracellular signaling events upstream of reactive oxygen species production and phagosome formation.

Atherosclerosis development is accelerated severalfold in patients with Type 2 diabetes. In the initial stages of disease, monocytes transmigrate into the subendothelial space and differentiate into foam cells. Scavenger receptors and ATP binding cassette (ABC) Transporters play an important role in foam cell formation as they regulate the influx and efflux of oxidized lipids. Here, we show that peritoneal macrophages isolated from Type 2 diabetic db/db mice have decreased expression of the ABC transporter ABCG1 and increased expression of the scavenger receptor CD36. We found a 2-fold increase in accumulation of esterified cholesterol in diabetic db/db macrophages compared with wild-type control macrophages. Diabetic db/db macrophages also had impaired cholesterol efflux to high density lipoprotein but not to lipid-free apo A-I, suggesting that the increased esterified cholesterol in diabetic db/db macrophages was due to a selective loss of ABCG1-mediated efflux to high density lipoprotein. Additionally, we were able to confirm down-regulation of ABCG1 using C57BL/6J peritoneal macrophages cultured in elevated glucose in vitro (25 mM glucose for 7 days), suggesting that ABCG1 expression in diabetic macrophages is regulated by chronic exposure to elevated glucose. Diabetic KK(ay) mice were also studied and were found to have decreased ABCG1 expression without an increase in CD36. These observations demonstrate that ABCG1 plays a major role in macrophage cholesterol efflux and that decreased ABCG1 function can facilitate foam cell formation in Type 2 diabetic mice.

Vitamin E deficiency increases expression of the CD36 scavenger receptor, suggesting specific molecular mechanisms and signaling pathways modulated by alpha-tocopherol. We show here that alpha-tocopherol down-regulated CD36 expression (mRNA and protein) in oxidized low density lipoprotein (oxLDL)-stimulated THP-1 monocytes, but not in unstimulated cells. Furthermore, alpha-tocopherol treatment of monocytes led to reduction of fluorescent oxLDL-3,3'-dioctadecyloxacarbocyanine perchlorate binding and uptake. Protein kinase C (PKC) appears not to be involved because neither activation of PKC by phorbol 12-myristate 13-acetate nor inhibition by PKC412 was affected by alpha-tocopherol. However, alpha-tocopherol could partially prevent CD36 induction after stimulation with a specific agonist of peroxisome proliferator-activated receptor-gamma (PPARgamma; troglitazone), indicating that this pathway is susceptible to alpha-tocopherol action. Phosphorylation of protein kinase B (PKB) at Ser473 was increased by oxLDL, and alpha-tocopherol could prevent this event. Expression of PKB stimulated the CD36 promoter as well as a PPARgamma element-driven reporter gene, whereas an inactive PKB mutant had no effect. Moreover, coexpression of PPARgamma and PKB led to additive induction of CD36 expression. Altogether, our results support the existence of PKB/PPARgamma signaling pathways that mediate CD36 expression in response to oxLDL. The activation of CD36 expression by PKB suggests that both lipid biosynthesis and fatty acid uptake are stimulated by PKB.

Accumulating evidence indicates that secretory phospholipase A2 (sPLA2) enzymes promote atherogenic processes. We have previously showed the presence of Group V sPLA2 (GV sPLA2) in human and mouse atherosclerotic lesions, its hydrolysis of low density lipoprotein (LDL) particles, and the ability of GV sPLA2-modified LDL (GV-LDL) to induce macrophage foam cell formation in vitro. The goal of this study was to investigate the mechanisms involved in macrophage uptake of GV-LDL. Peritoneal macrophages from C57BL/6 mice (wild type (WT)), C57BL/6 mice deficient in LDL receptor (LDLR-/-), or SR-A and CD36 (DKO) were treated with control LDL, GV-LDL, oxidized LDL (ox-LDL) or LDL aggregated by vortexing (vx-LDL). As expected, ox-LDL induced significantly more cholesterol ester accumulation in WT and LDLR-/- compared with DKO macrophages. In contrast, there was no difference in the accumulation of GV-LDL or vx-LDL in the three cell types. 125I-ox-LDL exhibited high affinity, saturable binding to WT cells that was significantly reduced in DKO cells. Vx-LDL and GV-LDL showed low affinity, non-saturable binding that was similar for both cell types, and significantly higher compared with control LDL. GV-LDL degradation in WT and DKO cells was similar. Analyses by confocal microscopy indicated a distinct intracellular distribution of Alexa-568-labeled GV-LDL and Alexa-488-labeled ox-LDL. Uptake of GV-LDL (but not ox-LDL or vx-LDL) was significantly reduced in cells preincubated with heparin or NaClO3, suggesting a role for proteoglycans in GV-LDL uptake. Our data point to a physiological modification of LDL that has the potential to promote macrophage foam cell formation independent of scavenger receptors.

Fatty acid-binding proteins are cytosolic fatty acid chaperones, and the adipocyte isoform, aP2, plays an important role in obesity and glucose metabolism. Recently, this protein has been detected in macrophages where it strongly contributes to the development of atherosclerosis. Here, we investigated the role of aP2 in macrophage biology and the molecular mechanisms underlying its actions. We demonstrate that aP2-deficient macrophages display defects in cholesterol accumulation and alterations in pro-inflammatory responsiveness. Deficiency of aP2 alters the lipid composition in macrophages and enhances peroxisome proliferator-activated receptor gamma activity, leading to elevated CD36 expression and enhanced uptake of modified low density lipoprotein. The increased peroxisome proliferator-activated receptor gamma activity in aP2-deficient macrophages is also accompanied by a significant stimulation of the liver X receptor alpha-ATP-binding cassette transporter A1-mediated cholesterol efflux pathway. In parallel, aP2-deficient macrophages display reduced IkappaB kinase and NF-kappaB activity, resulting in suppression of inflammatory function including reduced cyclooxygenase-2 and inducible nitric-oxide synthase expression and impaired production of inflammatory cytokines. Our results demonstrate that aP2 regulates two central molecular pathways to coordinate macrophage cholesterol trafficking and inflammatory activity.

Serum amyloid A protein (SAA) is an acute-phase reactant, known to mediate pro-inflammatory cellular responses. This study reports that CLA-1 (CD36 and LIMPII Analogous-1; human orthologue of the Scavenger Receptor Class B Type I (SR-BI)) mediates SAA uptake and downstream SAA signaling. Flow cytometry experiments revealed more than a 5-fold increase of Alexa-488 SAA uptake in HeLa cells stably transfected with CLA-1. Alexa 488-HDL uptake directly correlated with SAA uptake when determined in several CLA-1 stably transfected HeLa cell clones expressing various levels of CLA-1. SAA directly binds to CLA-1 as determined by cross-linking and colocalization of anti-CLA-1 antibody with SAA. SAA was co-internalized with transferrin to the endocytic recycling compartment pointing to a potential site of SAA metabolism. Alexa-488 SAA uptake in the CLA-1-overexpressing HeLa cells, as well as in THP-1 monocyte cell line, can be efficiently blocked by unlabeled SAA, high density lipoprotein, and other CLA-1 ligands. At the same time, markedly enhanced levels of phosphorylation of the mitogen-activated protein kinases (MAPKs), ERK1/2, and p38, were observed in cells stably transfected with CLA-1 cells following SAA stimulation when compared with mock transfected cells. The levels of the SAA-induced interleukin-8 (IL-8) secretion by CLA-1-overexpressing cells also significantly exceeded (5- to 10-fold) those detected for control cells. Synthetic amphipathic peptides possessing a structural alpha-helical motif inhibited SAA-induced activation of both MAPKs and IL-8 secretion in THP-1 cells. The results of this study demonstrate for the first time that CLA-1 functions as an endocytic SAA receptor and is involved in SAA-mediated cell signaling events associated with the immune-related and inflammatory effects of SAA.

The self-association of proteins to form amyloid fibrils has been implicated in the pathogenesis of a number of diseases including Alzheimer's, Parkinson's, and Creutzfeldt-Jakob diseases. We recently reported that the myeloid scavenger receptor CD36 initiates a signaling cascade upon binding to fibrillar beta-amyloid that stimulates recruitment of microglia in the brain and production of inflammatory mediators. This receptor plays a key role in the pathogenesis of atherosclerosis, prompting us to evaluate whether fibrillar proteins were present in atherosclerotic lesions that could initiate signaling via CD36. We show that apolipoprotein C-II, a component of very low and high density lipoproteins, readily forms amyloid fibrils that initiate macrophage inflammatory responses including reactive oxygen production and tumor necrosis factor alpha expression. Using macrophages derived from wild type and Cd36(-/-) mice to distinguish CD36-specific events, we show that fibrillar apolipoprotein C-II activates a signaling cascade downstream of this receptor that includes Lyn and p44/42 MAPKs. Interruption of this signaling pathway through targeted deletion of Cd36 or blocking of p44/42 MAPK activation inhibits macrophage tumor necrosis factor alpha gene expression. Finally, we demonstrate that apolipoprotein C-II in human atheroma co-localizes to regions positive for markers of amyloid and macrophage accumulation. Together, these data characterize a CD36-dependent signaling cascade initiated by fibrillar amyloid species that may promote atherogenesis.

Cholesterol elimination from the body involves reverse cholesterol transport from peripheral tissues in which the elimination of high density lipoprotein (HDL) and low density lipoprotein (LDL) cholesterol by the liver and subsequent biliary excretion as free cholesterol and bile acids are important. In situations of peripheral fat and cholesterol accumulation, such as obesity, these pathways may be overloaded, contributing to increased cholesterol deposition. Leptin has an important role in obesity, suppressing food intake and increasing energy expenditure. This hormone, which is absent in genetically obese ob/ob mice, is also thought to be involved in the coordination of lipid excretion pathways, although available data are somewhat inconsistent. We therefore studied the expression of the hepatic HDL receptor, scavenger receptor class B type I (SR-BI), and the LDL receptor as well as the rate-limiting enzyme in bile acid synthesis, cholesterol 7alpha-hydroxylase (Cyp7a1), in leptin-deficient ob/ob mice and their wild-type controls. In ob/ob mice, protein levels of both LDL receptor and SR-BI were reduced, whereas LDL receptor mRNA levels were increased and those of SR-BI were reduced, regardless of challenge with a 2% cholesterol diet. In ob/ob mice, the enzymatic activity and mRNA for Cyp7a1 were reduced, and the increase in response to dietary cholesterol was blunted. Upon short-term (2 days) treatment with leptin, a dose-dependent increase was seen in the SR-BI protein and mRNA, whereas the Cyp7a1 protein and mRNA were reduced. Our findings indicate that leptin is an important regulator of hepatic SR-BI expression and, thus, HDL cholesterol levels, whereas it does not stimulate Cyp7a1 and bile acid synthesis.

We demonstrated previously that oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (ox-PAPC) and, specifically, the component lipid 1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphorylcholine increase interleukin-8 (IL-8) synthesis in aortic endothelial cells. The goal of the current studies was to characterize the receptor complex mediating the increased transcription of IL-8. We demonstrate that scavenger receptor class A, types I and II, lectin-like ox-LDL receptor-1, macrophage receptor with collagenous structure, and CD36 are not responsible for the increase in IL-8. Using dominant-negative constructs and antisense oligonucleotides, we demonstrate a role for Toll-like receptor 4 (TLR4) as the ox-PAPC receptor mediating IL-8 transcription. We demonstrate that a glycosylphosphatidylinositol (GPI)-anchored protein is also necessary because phosphatidylinositol-specific phospholipase C pretreatment inhibited the effect of ox-PAPC. CD14, a GPI-anchored protein that associates with TLR4 in mediating lipopolysaccharide action, did not appear to mediate ox-PAPC action because ox-PAPC-induced IL-8 transcription was not blocked by anti-CD14 neutralizing antibodies nor was it augmented by the addition of soluble CD14 or overexpression of membrane CD14. Instead, anti-TLR4 antibodies immunoprecipitated a 37-kDa protein that also bound ox-PAPC. A protein of this same size was found in aerolysin overlays used to detect GPI-anchored proteins. Therefore, these studies suggest that ox-PAPC may initially bind to a 37-kDa GPI-anchored protein, which interacts with TLR4 to induce IL-8 transcription.

Hepatic cholesterol(ester) uptake from serum coupled to intracellular processing and biliary excretion are important features in the removal of excess cholesterol from the body. ATP-binding cassette (ABC) transporters play an important role in hepatic cholesterol transport. The liver consists of different cell types, and ABC transporters may exert different physiological functions dependent on the individual cell type. Therefore, in the current study, using real time PCR we compared the mRNA expression of ABC transporters and genes involved in the regulation of cholesterol metabolism in liver parenchymal, endothelial, and Kupffer cells. It appears that liver parenchymal cells contain high expression levels compared with endothelial and Kupffer cells of scavenger receptor class BI ( approximately 3-fold), peroxisome proliferator-activated receptor (PPAR)alpha and PPARgamma (8-20-fold), cholesterol 7alpha-hydroxylase A1 (>100-fold), and ABCG5/G8 ( approximately 5-fold). Liver endothelial cells show a high expression of cholesterol 27-hydroxylase, liver X receptor (LXR)beta, PPARdelta, and ABCG1, suggesting a novel specific role for these genes in endothelial cells. In Kupffer cells, the expression level of LXRalpha, ABCA1, and in particular ABCG1 is high, leading to an ABCG1 mRNA expression level that is 70-fold higher than in parenchymal cells. It can be calculated that 51% of the total liver ABCG1 expression resides in Kupffer cells and 24% in endothelial cells, suggesting an intrahepatic-specific role for ABCG1 in Kupffer and endothelial cells. Because of a specific stimulation of ABCG1 in parenchymal cells by a high cholesterol diet, the contribution of parenchymal cells to the total liver increased from 25 to 60%. Our data indicate that for studies of the role of ABC transporters and their regulation in liver, their cellular localization should be taken into account, allowing proper interpretation of metabolic changes, which are directly related to their (intra)cellular expression level.

The neural cell adhesion molecule and the voltage-sensitive sodium channel alpha-subunit are the only two molecules in mammals known to be modified by alpha-2,8-linked polysialic acid (polySia). We found a new polySia-containing glycoprotein in human milk and identified it as CD36, a member of the B class of the scavenger receptor superfamily. The polySia-containing glycan chain(s) were removed by alkaline treatment but not by peptide:N-glycanase F digestion, indicating that milk CD36 contained polySia on O-linked glycan chain(s). Polysialylation of CD36 occurs not only in human milk but also in mouse milk. However, CD36 in human platelets is not polysialylated. PolySia CD36 is secreted in milk at any lactation stage and reaches peak level at 1 month after parturition. Thus, it is suggested that polySia of milk CD36 is significant for neonatal development in terms of protection and nutrition.

Cholesterol is a key lipid in the stratum corneum, where it is critical for permeability barrier homeostasis. The epidermis is an active site of cholesterol synthesis, but inhibition of epidermal cholesterol synthesis with topically applied statins only modestly affects epidermal permeability barrier function, suggesting a possible compensatory role for extraepidermal cholesterol. Scavenger receptor class B type I (SR-BI) is a recently described cell surface receptor for high density lipoproteins (HDL) that mediates the selective uptake of cholesterol esters from circulating HDL. In the present study, we demonstrate that SR-BI is present in cultured human keratinocytes and that calcium-induced differentiation markedly decreases SR-BI levels. Additionally, the cell association of [(3)H]cholesterol-labeled HDL decreased in differentiated versus undifferentiated keratinocytes. Furthermore, the inhibition of cholesterol synthesis with simvastatin resulted in a 3-4-fold increase in both SR-BI mRNA and protein levels, whereas conversely, addition of 25-hydroxycholesterol suppressed SR-BI levels by approximately 50%. SR-BI mRNA is also expressed in murine epidermis, increasing by 50% in parallel with cholesterol requirements following acute barrier disruption. Because the increase is completely blocked by occlusion with a vapor-impermeable membrane, changes in epidermal SR-BI expression are regulated specifically by barrier requirements. Lastly, using immunofluorescence we demonstrated that SR-BI is present in human epidermis predominantly in the basal layer and increases following barrier disruption. In summary, the present study demonstrates first that SR-BI is expressed in keratinocytes and regulated by cellular cholesterol requirements, suggesting that it plays a role in keratinocyte cholesterol homeostasis. Second, the increase in SR-BI following barrier disruption suggests that SR-BI expression increases to facilitate cholesterol uptake leading to barrier restoration.

Lipoproteins originating from axon and myelin breakdown in injured peripheral nerves are believed to supply cholesterol to regenerating axons. We have used compartmented cultures of rat sympathetic neurons to investigate the utilization of lipids from lipoproteins for axon elongation. Lipids and proteins from human low density lipoproteins (LDL) and high density lipoproteins (HDL) were taken up by distal axons and transported to cell bodies, whereas cell bodies/proximal axons internalized these components from only LDL, not HDL. Consistent with these observations, the impairment of axonal growth, induced by inhibition of cholesterol synthesis, was reversed when LDL or HDL were added to distal axons or when LDL, but not HDL, were added to cell bodies. LDL receptors (LDLRs) and LR7/8B (apoER2) were present in cell bodies/proximal axons and distal axons, with LDLRs being more abundant in the former. Inhibition of cholesterol biosynthesis increased LDLR expression in cell bodies/proximal axons but not distal axons. LR11 (SorLA) was restricted to cell bodies/proximal axons and was undetectable in distal axons. Neither the LDL receptor-related protein nor the HDL receptor, SR-B1, was detected in sympathetic neurons. These studies demonstrate for the first time that lipids are taken up from lipoproteins by sympathetic neurons for use in axonal regeneration.

The oxidation of methionine residues in many proteins, including the serine proteinase inhibitor alpha1-antitrypsin (AAT), can result in functional inactivation. In this study we investigated the pro-inflammatory properties of oxidized AAT (oxAAT), specifically its ability to activate human monocytes in culture. Monocytes stimulated with oxAAT at concentrations up to 0.2 mg/ml for 24 h showed significant elevation in monocyte chemoattractant protein-1, cytokine interleukin-6, and tumor necrosis factor-alpha expression and increased NADPH oxidase activity. Monocytes activated with oxAAT showed surprising effects on lipid metabolism. Expression of low density lipoprotein (LDL) receptors increased by up to 76% compared with controls but was not accompanied by any changes in (125)I-labeled LDL binding and, paradoxically, decreased LDL uptake, degradation, and intracellular cholesterol synthesis. oxAAT also down-regulated the scavenger receptor CD36, which takes up and is up-regulated by oxidized LDL and is down-regulated by cholesterol efflux. As a by-product of oxidative events accompanying inflammation, oxAAT has multiple effects on cytokine expression, generation of reactive oxygen species, and on intracellular lipid metabolism. The up-regulation of monocyte-derived reactive oxygen by oxAAT could potentially result in self-amplification of AAT oxidation and, thereby, the other effects deriving from it. This implies that there are as yet unidentified regulatory processes that control this cycle.

Increasing evidence has implicated the membrane protein CD36 (FAT) in binding and transport of long chain fatty acids (FA). To determine the physiological role of CD36, we examined effects of its overexpression in muscle, a tissue that depends on FA for its energy needs and is responsible for clearing a major fraction of circulating FA. Mice with CD36 overexpression in muscle were generated using the promoter of the muscle creatine kinase gene (MCK). Transgenic (MCK-CD36) mice had a slightly lower body weight than control litter mates. This reflected a leaner body mass with less overall adipose tissue, as evidenced by magnetic resonance spectroscopy. Soleus muscles from transgenic animals exhibited a greatly enhanced ability to oxidize fatty acids in response to stimulation/contraction. This increased oxidative ability was not associated with significant alterations in histological appearance of muscle fibers. Transgenic mice had lower blood levels of triglycerides and fatty acids and a reduced triglyceride content of very low density lipoproteins. Blood cholesterol levels were slightly lower, but no significant decrease in the cholesterol content of major lipoprotein fractions was measured. Blood glucose was significantly increased, while insulin levels were similar in the fed state and higher in the fasted state. However, glucose tolerance curves, determined at 20 weeks of age, were similar in control and transgenic mice. In summary, the study documented, in vivo, the role of CD36 to facilitate cellular FA uptake. It also illustrated importance of the uptake process in muscle to overall FA metabolism and glucose utilization.

Hepatic lipase (HL) and scavenger receptor type B class I (SR-BI) have both been implicated in high density lipoprotein (HDL)-cholesteryl ester uptake in cholesterol-utilizing tissues. Inactivation of HL by gene-directed targeting in mice results in up-regulation of SR-BI expression in adrenal gland (Wang, N., Weng, W., Breslow, J. L., and Tall, A. R. (1996) J. Biol. Chem. 271, 21001-21004). The net effect on HDL-cholesteryl ester uptake is not known. We determined the impact of acute in vivo inhibition of rat adrenal HL activity by antibodies on SR-BI expression and on human and rat HDL-[3H]cholesteryl ether (CEth) uptake in the adrenal gland. Rat HDL was isolated from rats in which HL activity had been inhibited for 1 h. The rats were studied under basal conditions (not ACTH-treated) and after previous treatment with ACTH for 6 days (ACTH-treated). Intravenous injection of anti-HL resulted in 70% lowering of adrenal HL activity in both conditions which were maintained for at least 8 h. In not ACTH-treated rats, inhibition of adrenal HL increased adrenal SR-BI mRNA (5.2-fold) and mass (1. 6-fold) within 4 h. HL inhibition resulted in 41% and 14% more adrenal accumulation of human HDL-[3H]CEth during 4 and 24 h, respectively. The adrenal uptake of rat HDL-[3H]CEth increased by 68%, 4 h after the antibody injection. ACTH treatment increased total adrenal HL activity from 3.7 +/- 0.5 milliunits to 34.0 +/- 17. 2 milliunits, as well as adrenal SR-BI mRNA from 2.9 +/- 0.7 arbitrary units (A.U.) to 86.8 +/- 41.1 A.U. and SR-BI mass from 7.7 +/- 1.8 A.U. to 63.16 +/- 46.7 A.U. The human HDL-[3H]CEth uptake by adrenals was also significantly increased from 0.58 +/- 0.11% of injected dose to 7.24 +/- 1.58% of injected dose. Inhibition of adrenal HL activity did not result in further induction of SR-BI expression and did not affect human HDL-[3H]CEth uptake. These findings indicate that SR-BI expression may be influenced by changes in HL activity. HL activity is not needed for the SR-BI-mediated HDL-cholesteryl ester uptake by rat adrenal glands.

CD36 is a cell surface glycoprotein composed of a single polypeptide chain, which interacts with thrombospondin, collagens type I and IV, oxidized low density lipoprotein, fatty acids, anionic phospholipids, and erythrocytes parasitized with Plasmodium falciparum. Its expression is restricted to a few cell types, including monocyte/macrophages. In these cells, CD36 is involved in phagocytosis of apoptotic cells, and foam cell formation by uptake of oxidized low density lipoprotein. To study the molecular mechanisms that control the transcription of the CD36 gene in monocytic cells we have isolated and analyzed the CD36 promoter. Transient expression experiments of 5'-deletion fragments of the CD36 promoter coupled to luciferase demonstrated that as few as 158 base pairs upstream from the transcription initiation site were sufficient to direct the monocyte-specific transcription of the reporter gene. Within the above region, the fragment spanning nucleotides -158 to -90 was required for optimal transcription in monocytic cells. Biochemical analysis of the region -158/-90 revealed a binding site for transcription factors of the polyomavirus enhancer-binding protein 2/core-binding factor (PEBP2/CBF) family at position -103. Disruption of the PEBP2/CBF site markedly diminished the role of the PEBP2/CBF factors in the constitutive transcription of the CD36 gene. The involvement of members of the PEBP2/CBF family in chromosome translocations associated with acute myeloid leukemia, and in the transcriptional regulation of the myeloid-specific genes encoding for myeloperoxidase, elastase, and the colony-stimulating factor receptor, highlights the relevance of the regulation of the CD36 gene promoter in monocytic cells by members of the PEBP2/CBF family.

CD36 is an integral membrane glycoprotein expressed by several cell types, including endothelial cells of the microvasculature, erythrocytes, platelets, and monocytes. In the monocytic lineage, CD36 is expressed during the late stages of differentiation in the bone marrow, in circulating monocytes, and in some tissue resident macrophages, and it is thought to mediate the phagocytosis of apoptotic cells and the endocytic uptake of modified lipoproteins. Here we analyze the synthesis, processing, and intracellular transport of CD36 in U937 and THP-1, two human cell lines representing different stages of monocytic maturation. In both cell lines, phorbol 12-myristate 13-acetate induces the expression of CD36. A 74-kDa intracellular precursor is first synthesized that has the hallmarks of a resident protein of the endoplasmic reticulum. The precursor protein is later processed into a mature form of 90-105 kDa which is transported to the cell surface. The kinetics of processing differ significantly in U937 and THP-1. These differences are specific for the CD36, as two unrelated proteins (CD11b and CD45R) are processed and transported to the surface at similar rates in the two cell lines. A 33-kDa endoglycosidase H-sensitive glycoprotein specifically associates with the 74-kDa precursor. Coprecipitation of gp33 correlates with slow processing of CD36 precursor, suggesting that gp33 may play a role in regulating the intracellular transport of CD36, during monocyte maturation.