Scavenger Receptor Gene Research Articles

Stable scavenger receptor gene transfer leads to increased susceptibility to apoptosis in rabbit aortic smooth muscle cells

Stable scavenger receptor gene transfer leads to increased susceptibility to apoptosis in rabbit aortic smooth muscle cells

Secreted macrophage scavenger receptor gene transfer in LDL receptor knock-out mice reduces atherosclerotic lesion area

Secreted macrophage scavenger receptor gene transfer in LDL receptor knock-out mice reduces atherosclerotic lesion area

Analysis of interferon-gamma-dependent and -independent pathways of macrophage activation.

Macrophages are a cellular cornerstone of the innate immune response. The outcome of macrophage activity during development of an immune response to microbes results from macrophage activation by both organism-derived and host-derived factors. In order to more fully understand the spectrum of responses expressed by macrophages when encountering these distinct stimuli, we investigated the similarities and differences between interferon-gamma receptor (IFN-gammaR)-dependent macrophage activation and stimulation of macrophages through the Type A1 scavenger receptor (SR). We observed distinct patterns of macrophage activation depending on the nature of the ligand. IFN-gamma and the SR ligand lipotechoic acid (LTA) induced largely non-overlapping sets of genes. The use of two additional SR ligands, maleylated bovine serum albumin and the polydeoxynucleotide poly dI:dC, revealed differences within SR activation-induced gene expression. We also observed that priming with IFN-gamma resulted in an enhanced response to subsequent SR-mediated activation. These results suggest that full potentiation of macrophage activity during development of an antimicrobial immune response is achieved by activation of these cells through multiple receptors.

Scavenger receptors and modified lipoproteins: fatal attractions?

Lipoproteins modified by oxidation, glycation, alkylation, and nitration are generated by oxidative stress during inflammation, diabetes, and inadequate supply of dietary antioxidants. A family of genes, the scavenger receptors, recognizes and internalizes modified lipoproteins, making them susceptible to degradation. Clearance of modified lipoproteins by scavenger receptors occurs mainly in macrophages, dendritic cells, and Kupffer cells of the liver. However, scavenger receptor expression also occurs in other cells, such as endothelial cells, aortic smooth muscle cells, neuronal cells, and keratinocytes. Thus, the local clearance of oxidized low-density lipoprotein and the resolution of inflammatory processes may rely in part on the expression of scavenger receptors in "nonprofessional" phagocytes. Uptake of oxidized low-density lipoprotein, without an efficient machinery to degrade them and uncontrolled expression of scavenger receptors, may lead to cellular deregulation, apoptosis, and formation of foam cells. Diseases accompanied by oxidation of lipoproteins, such as atherosclerosis, Alzheimer disease, glomerulosclerosis, ataxia with vitamin E deficiency, and possibly age-dependent lipofuscin deposition, may share a common pathogenetic feature. This review will focus on foam cell formation, mainly within the atherosclerotic lesion, and the possible involvement of aberrant regulation of the scavenger receptor genes. To date, the regulatory mechanisms at the basis of scavenger receptor gene expression and their roles in atherosclerosis and other diseases are not well established. Knowledge on this subject could lead to a better understanding of the pathogenesis, prevention, and therapy of these diseases.

Scavenger receptor regulation and atherosclerosis

Atherosclerosis and its complications, such as coronary heart disease, heart infarction and stroke, are the leading causes of death in the developed world. High blood pressure, diabetes, smoking and a diet high in cholesterol and lipids clearly increase the likelihood of premature atherosclerosis, albeit other factors, such as the individual genetic makeup, may play an additional role. During atherosclerosis, uncontrolled cholesterol and lipid accumulation in macrophages and smooth muscle cells leads to foam cell formation and to the progression of the atherosclerotic plaque. This review will focus on foam cell formation within the atherosclerotic lesion, the involvement of the scavenger receptor genes in this process, and the possibility to interfere with scavenger receptor function to reduce the progression of atherosclerosis. To date, the regulatory mechanisms for the expression of scavenger receptor genes and their role in atherosclerosis are not well characterized. Knowledge on this subject could lead to a better understanding of the process, prevention and therapy of this disease.

A Null Mutation in Murine CD36 Reveals an Important Role in Fatty Acid and Lipoprotein Metabolism

A null mutation in the scavenger receptor gene CD36 was created in mice by targeted homologous recombination. These mice produced no detectable CD36 protein, were viable, and bred normally. A significant decrease in binding and uptake of oxidized low density lipoprotein was observed in peritoneal macrophages of null mice as compared with those from control mice. CD36 null animals had a significant increase in fasting levels of cholesterol, nonesterified free fatty acids, and triacylglycerol. The increase in cholesterol was mainly within the high density lipoprotein fraction, while the increase in triacylglycerol was within the very low density lipoprotein fraction. Null animals had lower fasting serum glucose levels when compared with wild type controls. Uptake of 3H-labeled oleate was significantly reduced in adipocytes from null mice. However, the decrease was limited to the low ratios of fatty acid:bovine serum albumin, suggesting that CD36 was necessary for the high affinity component of the uptake process. The data provide evidence for a functional role for CD36 in lipoprotein/fatty acid metabolism that was previously underappreciated.

Transcriptional activation of scavenger receptor expression in human smooth muscle cells requires AP-1/c-Jun and C/EBPbeta: both AP-1 binding and JNK activation are induced by phorbol esters and oxidative stress.

Reactive oxygen species generated by treatment of smooth muscle cells (SMCs) with either phorbol 12-myristate 13-acetate or with the combination of H2O2 and vanadate strongly induce expression of the class A scavenger receptor (SR-A) gene. In the current studies, cis-acting elements in the proximal 245 bp of the SR-A promoter were shown to direct luciferase reporter expression in response to oxidative stress in both SMCs and macrophages. A composite activating protein-1 (AP-1)/ets binding element located between -67 and -50 bp relative to the transcriptional start site is critical for macrophage SR-A activity. Mutation of either the AP-1 or the ets component of this site also prevented promoter activity in SMCs. Mutation of a second site located between -44 and -21 bp, which we have identified as a CCAAT/enhancer binding protein (C/EBP) element, reduced the inducible activity of the promoter in SMCs by 50%, suggesting that combinatorial interactions between these sites are necessary for optimal gene induction. Interactions between SMC nuclear extracts and the SR-A promoter were analyzed by electrophoretic mobility shift assay. c-Jun/AP-1 binding activity, specific for the -67- to -50-bp site, was induced in SMCs by the same conditions that increased SR-A expression. Moreover, phorbol 12-myristate 13-acetate, H2O2, or the combination of H2O2 and sodium orthovanadate (vanadate) activated c-Jun-activating kinase. The binding activity within SMC extracts specific for the C/EBP site was shown to be C/EBPbeta in SMCs. Taken together, these findings demonstrate that reactive oxygen species regulate the interactions between c-Jun/AP-1 and C/EBPbeta in the SR-A promoter. Furthermore, induction of oxidative stress in THP-1 cells, with a combination of 10 micromol/L vanadate and 100 micromol/L H2O2, induced macrophage differentiation, adhesion, and SR activity. These data suggest that vascular oxidative stress may contribute to the induction of SR-A expression and thereby promote the uptake of oxidatively modified low density lipoprotein by both macrophage and SMCs to produce foam cells in atherosclerotic lesions.

Differential utilization of Ras signaling pathways by macrophage colony-stimulating factor (CSF) and granulocyte-macrophage CSF receptors during macrophage differentiation.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF) independently stimulate the proliferation and differentiation of macrophages from bone marrow progenitor cells. Although the GM-CSF and M-CSF receptors are unrelated, both couple to Ras-dependent signal transduction pathways, suggesting that these pathways might account for common actions of GM-CSF and M-CSF on the expression of macrophage-specific genes. To test this hypothesis, we have investigated the mechanisms by which GM-CSF and M-CSF regulate the expression of the macrophage scavenger receptor A (SR-A) gene. We demonstrate that induction of the SR-A gene by M-CSF is dependent on AP-1 and cooperating Ets domain transcription factors that bind to sites in an M-CSF-dependent enhancer located 4.1 to 4.5 kb upstream of the transcriptional start site. In contrast, regulation by GM-CSF requires a separate enhancer located 4.5 to 4.8 kb upstream of the transcriptional start site that confers both immediate-early and sustained transcriptional responses. Results of a combination of DNA binding experiments and functional assays suggest that immediate transcriptional responses are mediated by DNA binding proteins that are constitutively bound to the GM-CSF enhancer and are activated by Ras. At 12 to 24 h after GM-CSF treatment, the GM-CSF enhancer becomes further occupied by additional DNA binding proteins that may contribute to sustained transcriptional responses. In concert, these studies indicate that GM-CSF and M-CSF differentially utilize Ras-dependent signal transduction pathways to regulate scavenger receptor gene expression, consistent with the distinct functional properties of M-CSF- and GM-CSF-derived macrophages.

A naturally occurring isoform of the human macrophage scavenger receptor (SR-A) gene generated by alternative splicing blocks modified LDL uptake

The class A macrophage scavenger receptors (SR-A) are macrophage-specific trimeric integral membrane glycoproteins that have been implicated in many macrophage-associated physiological and pathological processes including atherosclerosis, Alzheimer's disease, and host defense. There are two forms of the receptor that have been previously cloned, and both are generated by alternative splicing of a single gene. Here we report the cloning of a third, alternatively spliced isoform of the human SR-A gene (type III hSR-A). The novel isoform is expressed in the human monocytic leukemia cell line THP-1 and also in primary human monocyte derived macrophages. When expressed in CHO-K1 cells, type III hSR-A does not internalize AcLDL despite having the domain shown to mediate this function in type I and II hSR-A. We show that type III protein has altered intracellular processing and is trapped within the endoplasmic reticulum, making it unable to perform endocytosis. Type III protein acts as a dominant negative isoform by reducing modified LDL uptake in CHO cells stably expressing either type I or type II SR-A. The demonstration that a naturally occurring splice variant of SR-A mRNA can act as a dominant negative isoform suggests a novel mechanism for regulation of scavenger receptor activity in macrophages.—Gough, P. J., D. R. Greaves, and S. Gordon. A naturally occurring isoform of the human macrophage scavenger receptor (SR-A) gene generated by alternative splicing blocks modified LDL uptake.

4.P.301 Retrovirus-mediated scavenger receptor gene transfer leads to foam cell formation in eucaryotic cell lines

4.P.301 Retrovirus-mediated scavenger receptor gene transfer leads to foam cell formation in eucaryotic cell lines

4.P.302 Novel elements located at −504 to −399 bp of the promoter region regulated the expression of the human macrophage scavenger receptor gene in murine macrophages

4.P.302 Novel elements located at −504 to −399 bp of the promoter region regulated the expression of the human macrophage scavenger receptor gene in murine macrophages

Heritability analysis of lipids and three gene loci in twins link the macrophage scavenger receptor to HDL cholesterol concentrations.

We studied 100 healthy monozygotic and 72 dizygotic twin pairs (mean age, 34 +/- 14 years) to test for genetic influences on blood lipids and to examine relevant gene loci. Total cholesterol (TC), LDL cholesterol (LDL-C), HDL cholesterol (HDL-C), and triglyceride (TG) levels were determined after a 12-hour fast. Zygosity was determined with the use of microsatellite markers. Heritability estimates were conducted by using the lisrel 8 program; a sib-pair analysis was conducted by using the sibpal program. Linear regression analyses were carried out between identical-by-descent status and squared within-pair differences of TC, LDL-C, HDL-C, and TG values. Heritability estimates of the lipid serum concentrations ranged from .58 to .66. A significant linkage relationship was found for HDL-C (P = .008) and TGs (P = .05) with D8S261 on chromosome 8p. However, no linkage was found between any of the lipid variables and the lipoprotein lipase gene locus (LPL GZ14/15 and D8S282). Because D8S261 is located approximately halfway between the LPL and macrophage scavenger receptor genes, we examined the nearby markers D8S549 and D8S1731. Linkage was found for HDL-C and D8S549 (P = .001) and for HDL-C and D8S1731 (P = .04). On the other hand, we found no linkage between the LDL receptor gene locus and LDL-C serum concentrations nor between the LPL gene locus and the various other lipid fractions. Our data suggest a significant influence of the macrophage scavenger receptor gene locus on HDL-C and weak influence on TG levels. We suggest that inherited variability in the macrophage scavenger receptor gene has an influence on serum lipid concentrations.

Novel elements located at -504 to -399 bp of the promoter region regulated the expression of the human macrophage scavenger receptor gene in murine macrophages

The expressions of type I and type II macrophage scavenger receptors (MSRs) are highly specific in macrophages and related cell types. Although some reports have described the regulation of MSR gene expression and proposed some cis-elements related to cell-specific expression, the regulation of MSR remains largely unclear. This is due, in part, to an unacceptably low efficiency of transfection into monocyte/ macrophage cells. In the present study, we optimized the conditions of electroporation in murine macrophage (P388D1) cells. The efficiency of electroporation was increased 20-fold compared with previous methods. Using the optimized method, we focused on studying the regulation of the human MSR promoter in macrophages. We presently demonstrate that: a) the proximal -10 to +50 bp human MSR promoter region is necessary for the cell type-specific expression of human MSR; b) the 6.5 kbp upstream sequence suppresses the expression of human MSR; c) a promoter region extending from -504 to -399 bp produced the greatest increase in transcriptional activity; d) macrophage cell-specific transcription factors bind to the region as determined by electrophoretic mobility shift assay (EMSA) and a footprint assay; and e) mutations of the region reduced about 40-75% of the promoter activity in a transfecting assay. We concluded that novel elements located at the -504 to -399 bp region may play an important role in the regulation of the MSR gene expression in macrophages. We speculate that macrophage-specific factors binding to those elements may be responsible for the transcription regulation of the MSR gene in macrophages.

Enhanced gene expression of scavenger receptor in peripheral blood monocytes from patients on cuprophane haemodialysis

Macrophage scavenger receptor (SR) is implicated in playing a key role in macrophage-derived foam cell formation by taking up a large amount of modified low-density lipoproteins (LDL). It has also been postulated that alpha 2-macroglobulin receptor/LDL receptor-related protein (alpha 2MG/LRP) is involved in the development of foam cells by taking up apo E-enriched chylomicrons and VLDL remnants, and lipoprotein lipase-triglyceride-rich lipoprotein complexes. Accumulation of these lipid-loaded monocyte/ macrophages in the subendothelial space is considered to be an early event of atherogenesis. Since atherogenesis is considered to be accelerated in dialysis patients, we attempted to investigate whether gene expression of SR and alpha 2MG/LRP are altered in peripheral blood monocytes from patients on haemodialysis with a cuprophane (Cu) or polymethylmethacrylate (PMMA) membranes. Peripheral blood monocytes (PBM) were prepared from patients undergoing haemodialysis with a Cu membrane (n = 9), patients undergoing haemodialysis with a PMMA membrane (n = 9), and healthy controls (n = 7). In a separate experiment we examined SR gene expression in uraemic patients (n = 12) and healthy controls (n = 9). SR and alpha 2MG/LRP mRNA were semiquantitated using reverse-transcription polymerase chain reaction (RT-PCR) assay followed by Southern blotting. SR mRNA expression in PBM from patients on chronic haemodialysis with a Cu membrane was about twofold higher than that in PBM from patients on chronic haemodialysis with a PMMA membrane or the controls (P < 0.05). alpha 2MG/LRP mRNA expression in PBM showed no difference among these, three groups. SR gene expression in monocytes from uraemic patients was not increased compared with that in the controls. PBM from patients under Cu membrane dialysis showed higher gene expression of SR than patients under PMMA membrane dialysis, uraemic patients, or healthy controls. This increased gene expression of SR in monocytes may be associated with the pathogenesis of accelerated atherosclerosis in patients on dialysis with a Cu membrane.

Scavenger receptor mRNAs in rat brain microglia are induced by kainic acid lesioning and by cytokines.

The expression and localization of two distinct mRNAs from the macrophage scavenger receptor gene family were studied in rat brain cells in vivo and in vitro. In general, brains of control male rats showed low level signals by in situ hybridization for the macrophage scavenger receptor (MSR) and murine adherent macrophage (MAMA) receptor. In contrast, the reticular thalamic nucleus had a subpopulation of intensely labeled cells. Kainic acid (KA) treatment induced MSR and MAMA mRNA levels on different schedules in brain regions that are susceptible to KA, including hippocampal areas CA1 and CA3. The combination of immunocytochemistry and in situ hybridization localized the MSR and MAMA mRNA to microglia of KA-treated rats. Northern blot hybridization detected both MSR and MAMA mRNAs in primary cultures of mixed glia that contained microglia. Both MSR and MAMA mRNA were induced by treatment of primary mixed glia with lipopolysaccharide and interferon-gamma, but not TGF beta 1. MSR, but not MAMA, mRNA levels were increased after treatment with interleukin-1 alpha. These results demonstrate the differential regulation of scavenger receptor mRNAs in microglia that is consistent with distinct roles for scavenger receptors in responses to neurodegeneration.

Nuclear integration of JAK/STAT and Ras/AP-1 signaling by CBP and p300.

We report that interferon gamma (IFN-gamma) inhibits transcription of the macrophage scavenger receptor gene by antagonizing the Ras-dependent activities of AP-1 and cooperating ets domain transcription factors, apparently as a result of competition between AP-1/ets factors and activated STAT1 for limiting amounts of CBP and p300. Consistent with this model, STAT1 alpha interacts directly with CBP in cells, and microinjection of anti-CBP and anti-p300 antibodies blocks transcriptional responses to IFN-gamma. Cells lacking STAT1 fail to inhibit AP-1/ets activity, and overexpression of CBP both potentiates IFN-gamma-dependent transcription and relieves AP-1/ets repression. Thus, CBP and p300 integrate both positive and negative effects of IFN-gamma on gene expression by serving as essential coactivators of STAT1 alpha, modulating gene-specific responses to simultaneous activation of two or more signal transduction pathways.

Human cytomegalovirus increases modified low density lipoprotein uptake and scavenger receptor mRNA expression in vascular smooth muscle cells.

Evidence suggests a possible role for human cytomegalovirus (HCMV) in the development of arteriosclerosis. One of the earliest events in plaque formation is the accumulation of lipid-laden foam cells, derived from macrophages and smooth muscle cells (SMCs). The lipid accumulation that occurs depends upon the uptake of oxidized LDL (Ox-LDL), a process in which the scavenger receptor (SR) has been postulated to play an important role. We therefore examined the effects of HCMV on this process. We demonstrate that HCMV infection of human SMCs increases modified LDL uptake and stimulates class A SR gene (SR-A) mRNA expression. In addition, infection of rat SMCs with HCMV, which causes immediate early gene expression (IE72/IE84), but no early or late HCMV gene products and no cytopathic effects, also increases SMC uptake of Ox-LDL and acetylated LDL, with either effect blocked by an excess of either cold Ox-LDL or acetylated-LDL, and by fucoidin, an SR competitor. Cotransfection of an IE72, but not an IE84, expression plasmid and a plasmid containing a Class A SR promoter/reporter gene construct enhances SR promoter activity. Since increased Ox-LDL uptake is believed to play an important role in arteriosclerosis, these results provide a link between HCMV infection and arteriosclerotic plaque formation.

Lovastatin inhibits gene expression of type-I scavenger receptor in THP-1 human macrophages

Lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, inhibits the synthesis of mevalonic acid and is widely used as an anti-atherosclerotic drug. The macrophage scavenger receptor (SCR), a trimeric membrane glycoprotein, is postulated to play a key role in atheroma macrophage foam cell formation. HMG-CoA reductase is involved in the control of the synthesis of glycoproteins and farnesylated proteins, including ras proteins, which are involved in the transcriptional regulation of SCR gene expression. Accordingly, we examined whether lovastatin alters the gene expression of SCRs in THP-1 cell derived human macrophages. Lovastatin (5–15 μM) caused a significant dose-related reduction in steady state levels of type-I SCR mRNA in phorbol 12-myristate 13-acetate (PMA)-treated THP-1 cells. The addition of exogenous mevalonate (1 mM) completely restored the lovastatin-induced decrease of type-I SCR mRNA levels. While the addition of the isoprenoid end-product, isopentenyl adenine (50 μM), had little effect on the type-I SCR mRNA levels in lovastatin-treated cells, the addition of isoprenoid farnesol (5 μM) largely restrored the lovastatin-induced decrease of type-I SCR mRNA levels. Actinomycin D treatment showed that degradation rates of type-I SCR mRNA did not differ between the THP-1 derived cells with and without lovastatin treatment. Nuclear run-on assays showed that lovastatin markedly decreased the transcription of SCR gene in the cells. These results suggest that lovastatin inhibits the transcription of type-I SCR gene by affecting mevalonate metabolism, possibly through the farnesyl-pyrophosphate related end-product(s) in the THP-1-derived macrophages.

Modification of macrophage function and effects on atherosclerosis

Various factors are involved in the maturation and differentiation process of monocytes to macrophages and subsequent foam cell formation in atherosclerotic lesions. Macrophage colonystimulating factor, in particular, specifically promotes growth and differentiation of the monocyte-macrophage lineage, by activating various functions in mature macrophages and by influencing the atherosclerotic process. Vascular smooth muscle cells migrate from the media to the intima of the arterial wall, where they are transformed into macrophage-like foam cells that express two characteristic macrophage genes, scavenger receptor and macrophage colony-stimulating factor receptor genes.

Scavenger receptors in innate immunity

Scavenger receptors are cell-surface proteins expressed by mammalian monocytes and macrophages and by invertebrate hemocytes, among other cell types. They exhibit distinctive ligand-binding properties, recognizing a wide range of ligands that include microbial surface constituents and intact microbes. The ligand-binding properties and expression patterns of these receptors suggest that they may function in one or more host-defense-related processes. Significant advances in scavenger receptor biology have recently been reported, including the identification of several new scavenger receptor genes.