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Cellular cholesterol content reflects a balance of lipid influx by lipoprotein receptors and endogenous synthesis and efflux to cholesterol acceptor particles. The beneficial effect of high density lipoprotein (HDL) in protecting against the development of cardiovascular disease is thought to be mediated predominately through its induction of cellular cholesterol efflux and "reverse cholesterol transport" from peripheral tissues to the liver. We tested the hypothesis that HDL could inhibit cellular lipid accumulation by modulating expression of peroxisome proliferator-activated receptor-gamma (PPARgamma)-responsive genes. To this end, we evaluated expression of two PPARgamma-responsive genes, CD36, a receptor for oxidized low density lipoprotein, and aP2, a fatty acid-binding protein. HDL decreased expression of macrophage CD36 and aP2 in a dose-dependent manner. HDL also decreased aP2 expression in fibroblasts, reduced accumulation of lipid, and slowed differentiation of fibroblasts into adipocytes. HDL stimulated mitogen-activated protein (MAP) kinase activity, and inhibition of CD36 expression was blocked by co-incubation with a MAP kinase inhibitor. HDL increased expression of PPARgamma mRNA and protein, induced translocation of PPARgamma from the cytoplasm to the nucleus, and increased PPARgamma phosphorylation. Our data demonstrate that despite induction and translocation of PPARgamma in response to HDL, MAP kinase-mediated phosphorylation of PPARgamma inhibited expression of PPARgamma-responsive genes and suggest mechanisms by which HDL may inhibit cellular lipid accumulation.

Scavenger receptor class B type I (SR-BI) has recently been identified as a high density lipoprotein (HDL) receptor that mediates bidirectional flux of cholesterol across the plasma membrane. We have previously demonstrated that oxidized low density lipoprotein (OxLDL) will increase expression of another class B scavenger receptor, CD36 (Han, J., Hajjar, D. P., Febbraio, M., and Nicholson, A. C. (1997) J. Biol. Chem. 272, 21654-21659). In studies reported herein, we evaluated the effects of OxLDL on expression of SR-BI in macrophages to determine how exposure to this modified lipoprotein could alter SR-BI expression and cellular lipid flux. OxLDL decreased SR-BI expression in a dose- and time-dependent manner. Incubation with OxLDL had no effect on the membrane distribution of SB-BI, and it decreased expression of both cytosolic and membrane protein. Consistent with its effect on SR-BI protein expression, OxLDL decreased SR-BI mRNA in a dose-dependent manner. The ability of OxLDL to decrease SR-BI expression was dependent on the degree of LDL oxidation. OxLDL decreased both [(14)C]cholesteryl oleate/HDL uptake and efflux of [(14)C]cholesterol to HDL in a time-dependent manner. Incubation of macrophages with 7-ketocholesterol, but not free cholesterol, also inhibited expression of SR-BI. Finally, we demonstrate that the effect of OxLDL on SR-BI is dependent on the differentiation state of the monocyte/macrophage. These results imply that in addition to its effect in inducing foam cell formation in macrophages through increased uptake of oxidized lipids, OxLDL may also enhance foam cell formation by altering SR-BI-mediated lipid flux across the cell membrane.

CD36, the macrophage type B scavenger receptor, binds and internalizes oxidized low density lipoprotein, a key event in the development of macrophage foam cells within atherosclerotic lesions. Expression of CD36 in monocyte/macrophages is dependent on differentiation status and exposure to soluble mediators. In this study, we investigated the effect of transforming growth factor-beta1 (TGF-beta1) and TGF-beta2 on the expression of CD36 in macrophages. Treatment of phorbol ester-differentiated THP-1 macrophages with TGF-beta1 or TGF-beta2 significantly decreased expression of CD36 mRNA and surface protein. TGF-beta1/TGF-beta2 also inhibited CD36 mRNA expression induced by oxidized low density lipoprotein and 15-deoxyDelta(12,14) prostaglandin J(2), a peroxisome proliferator-activated receptor (PPAR)-gamma ligand, suggesting that the TGF-beta1/TGF-beta2 down-regulated CD36 expression by inactivating PPAR-gamma-mediated signaling. TGF-beta1/TGF-beta2 increased phosphorylation of both mitogen-activated protein (MAP) kinase and PPAR-gamma, whereas MAP kinase inhibitors reversed suppression of CD36 and inhibited PPAR-gamma phosphorylation induced by TGF-beta1/TGF-beta2. Finally, MAP kinase inhibitors alone increased expression of CD36 mRNA and surface protein but had no effect on PPAR-gamma protein levels. Our data demonstrate for the first time that TGF-beta1 and TGF-beta2 decrease expression of CD36 by a mechanism involving phosphorylation of MAP kinase, subsequent MAP kinase phosphorylation of PPAR-gamma, and a decrease in CD36 gene transcription by phosphorylated PPAR-gamma.

CD36 is a multifunctional cell-surface receptor that binds adhesion molecules such as thrombospondin-1 and collagen and modified lipids and/or lipoproteins. It participates in cellular uptake of photoreceptor outer segments and scavenging of apoptotic cells and oxidized low density lipoprotein (Ox-LDL). Recognition and internalization of Ox-LDL by mononuclear phagocytes may play an important role in the development of atherosclerotic lesions. We have utilized a series of recombinant bacterial glutathione S-transferase/CD36 fusion proteins that span nearly all of the CD36 molecule to characterize the structural domain on CD36 that recognizes Ox-LDL. We found that the Ox-LDL-binding domain is different from the thrombospondin-1-binding domain located at amino acids 93-120. A fusion protein containing the region extending from amino acids 5 to 143 formed specific, saturable, and reversible complexes with Ox-LDL. As with intact CD36, binding was blocked by excess unlabeled Ox-LDL and antibodies to CD36. The stoichiometry and affinity of the fusion protein for Ox-LDL were similar to those of the intact protein. We also demonstrated that this fusion protein competitively inhibited binding of Ox-LDL to purified platelet CD36 and to CD36 expressed on peripheral blood monocytes and CD36 cDNA-transfected melanoma cells. The use of smaller peptides and fusion proteins including those spanning amino acids 28-93 and 5-93 has further narrowed the binding site to a region from amino acids 28 to 93, although participation of a sequence in the noncontiguous region 120-155 cannot be excluded. This study, for the first time, demonstrates unique regions of the scavenger receptor CD36 that bind the Ox-LDL ligand. Our structural analysis of the receptor provides information as to potential control of the trafficking of modified lipoproteins into the blood vessel wall.

The uptake of oxidized low density lipoprotein (OxLDL) by macrophages is a key event implicated in the initiation and development of atherosclerotic lesions. Two macrophage surface receptors, CD36 (a class B scavenger receptor) and the macrophage scavenger receptor (a class A scavenger receptor), have been identified as the major receptors that bind and internalize OxLDL. Expression of CD36 in monocyte/macrophages in tissue culture is dependent both on the differentiation state as well as exposure to soluble mediators (cytokines and growth factors). The regulatory mechanisms of this receptor in vivo are undetermined as is the role of lipoproteins themselves in modulating CD36 expression. We studied the effect of lipoproteins, native LDL and modified LDL (acetylated LDL (AcLDL) and OxLDL) on the expression of CD36 in J774 cells, a murine macrophage cell line. Exposure to lipoproteins resulted in a marked induction of CD36 mRNA expression (4-8-fold). Time course studies showed that maximum induction was observed 2 h after treatment with AcLDL and at 4 h with LDL and OxLDL. Increased expression of CD36 mRNA persisted for 24 h with each treatment group. Induction of CD36 mRNA expression was paralleled by an increase in CD36 protein as determined by Western blot with the greatest induction by OxLDL (4-fold). In the presence of actinomycin D, treatment of macrophages with LDL, AcLDL, or OxLDL did not affect CD36 mRNA stability, implying that CD36 mRNA was transcriptionally regulated by lipoproteins. To determine the mechanism(s) by which lipoproteins increased expression of CD36 we evaluated the effects of lipoprotein components on CD36 mRNA expression. ApoB 100 increased CD36 mRNA expression significantly, whereas phospholipid/cholesterol liposomes had less effect. Incubation of macrophages with bovine serum albumin or HDL reduced expression of CD36 mRNA in a dose-dependent manner. Finally, to evaluate the in vivo relevance of the induction of CD36 mRNA expression by lipoproteins, peritoneal macrophages were isolated from mice following intraperitoneal injection of lipoproteins. Macrophage expression of CD36 mRNA was significantly increased by LDL, AcLDL, or OxLDL in relation to mice infused with phosphate-buffered saline, with OxLDL causing the greatest induction (8-fold). This is the first demonstration that exposure to free and esterified lipids augments functional expression of the class B scavenger receptor, CD36. These data imply that lipoproteins can further contribute to foam cell development in atherosclerosis by up-regulating a major OxLDL receptor.

The binding and assembly of the coagulation proteases on the endothelial cell surface are important steps not only in the generation of thrombin and thrombogenesis, but also in vascular cell signaling. Effector cell protease receptor (EPR-1) was identified as a novel leukocyte cell surface receptor recognizing the coagulation serine protease Factor Xa but not the precursor Factor X. We now demonstrate that EPR-1 is expressed on vascular endothelial cells and smooth muscle cells. Northern blots of endothelial and smooth muscle cells demonstrated three abundant mRNA bands of 3.0, 1.8, and 1.3 kDa. 125I-Labeled Factor Xa bound to endothelial cells in a dose-dependent saturable manner, and the binding was inhibited by antibody to EPR-1. No specific binding was observed with a recombinant mutant Factor X in which the activation site was substituted by Arg196 --> Gln to prevent the proteolytic conversion to Xa. EPR-1 was identified immunohistochemically on microvascular endothelial and smooth muscle cells. Functionally, exposure of smooth muscle cells or endothelial cells to Factor Xa induced a 3-fold and a 2-fold increase in [3H]thymidine uptake, respectively. However, receptor occupancy alone is insufficient for mitogenic signaling because the active site of the enzyme is required for mitogenesis. Thus, EPR-1 represents a site of specific protease-receptor complex assembly, which during local initiation of the coagulation cascade could mediate cellular signaling and responses of the vessel wall.

Regulation of expression of the scavenger receptor is thought to play a critical role in the accumulation of lipid by macrophages in atherosclerosis. Tumor necrosis factor-alpha (TNF-alpha) has been shown to suppress macrophage scavenger receptor function (van Lenten, B.J., and Fogelman, A.M. (1992) J. Immunol. 148, 112-6). However, the mechanism by which it does so is unknown. We evaluated the mechanism by which TNF-alpha inhibited macrophage scavenger receptor surface expression and binding of acetylated low density lipoprotein (aLDL). Binding of aLDL to phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 macrophages was suppressed by TNF-alpha in a dose-dependent manner. Inhibition of aLDL binding was paralleled by a reduction of macrophage scavenger receptor protein as detected by the Western blot. TNF-alpha partially decreased macrophage scavenger receptor mRNA steady state levels in PMA-differentiated THP-1 macrophages, a result that was confirmed by reverse transcription-polymerase chain reaction. PMA increased the luciferase activity driven by the macrophage scavenger receptor promoter in the transfected cells, whereas TNF-alpha partially reduced luciferase activity. However, macrophage scavenger receptor mRNA half-life was dramatically reduced in cells treated with TNF-alpha relative to untreated cells. Reduction in macrophage scavenger receptor message in response to TNF-alpha was dependent on new protein synthesis because it was blocked by cycloheximide. These results indicate that TNF-alpha regulates macrophage scavenger receptor expression in PMA-differentiated THP-1 macrophages by transcriptional and post-transcriptional mechanisms but principally by destabilization of macrophage scavenger receptor mRNA.

Herpesvirus infection of arterial smooth muscle cells has been shown to cause cholesteryl ester (CE) accumulation. However, the effects of human herpes simplex virus type 1 (HSV-1) infection on cholesterol binding and internalization, intracellular metabolism, and efflux have not been evaluated. In addition, the effects of viral infection on signal transduction pathways that impact upon cholesterol metabolism have not been studied. We show in studies reported herein that HSV-1 infection of arterial smooth muscle cells enhances low density lipoprotein (LDL) binding and uptake which parallels an increase in LDL receptor steady state mRNA levels and transcription of the LDL receptor gene. HSV-2 also increases CE synthesis and 3-hydroxy- 3-methylglutaryl-CoA reductase activity but concomitantly reduces CE hydrolysis and cholesterol efflux. Interestingly, this viral infection was associated with a time-dependent decrease in protein kinase A activity and an increase in viral-induced protein kinase (VPK) activity commensurate with the accumulation of esterified cholesterol. The relationship between increased VPK activity and alterations in CE accumulation in virally infected cells was explored using an HSV-1 VPK- mutant in which the portion of the HSV-1 genome encoding VPK had been deleted. Cholesteryl ester accumulation was significantly increased (> 50-fold) in HSV-1-infected cells compared to uninfected cells. However, the HSV-1 VPK- mutant had no significant effect on CE accumulation. The relationship between VPK activity and these alterations in cholesterol metabolism was further supported by the observation that staurosporine and calphostin C (protein kinase inhibitors) reduced protein kinase activity in HSV-1-infected cells. These results suggest several potential mechanisms by which alterations in kinase activities in response to HSV-1 infection of vascular cells may alter cholesterol trafficking processes that eventually lead to CE accumulation.

Basic fibroblast growth factor (bFGF) has been implicated in the regulation of cell proliferation and cholesterol metabolism. In studies reported herein, we show bFGF increases low density lipoprotein (LDL) binding, uptake, and degradation in arterial smooth muscle cells in a dose-dependent manner. This increase was paralleled by an increase in LDL receptor mRNA steady state levels. To determine if bFGF activated transcription of the LDL receptor gene, we transiently transfected smooth muscle cells with a gene construct consisting of the 5'-upstream promoter region of the DNA from the human LDL receptor gene ligated to a plasmid containing the luciferase gene. We found that bFGF and a protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate, significantly induced luciferase activity driven by the LDL receptor promoter, whereas 25-hydroxycholesterol reduced the luciferase activity in bFGF-stimulated cells. These findings show that bFGF and PKC are inducing LDL receptor gene transcription. We also evaluated potential signal transduction pathways induced by bFGF to establish the mechanism(s) leading to the activation of the LDL receptor gene. Activation of the activity of FGF receptor tyrosine kinase in smooth muscle cells by ligand binding resulted in tyrosine phosphorylation of one of the FGF receptors and a 90-kDa-protein as well as increased tyrosine phosphorylation of phospholipase C-gamma. Parallel observations were made in that increased PKC and protein kinase A activities occurred with bFGF as compared with control cells. Inhibitors of receptor tyrosine kinase and other protein kinases significantly reduced transcription and surface expression of LDL receptor. Finally, several key enzymes that are central to the regulation of LDL-cholesteryl ester metabolism were also studied in bFGF-stimulated cells. An increase in acyl-CoA:cholesterol acyltransferase activity and cholesterol esterification was observed with bFGF stimulation, but there was no effect on the lysosomal or cytoplasmic cholesteryl ester hydrolase activities. Our findings suggest potential signal transduction pathways activated by bFGF which play a role in regulating transcription and surface expression of the LDL receptor.

Elevated plasma levels of cytokines have been demonstrated in inflammatory, malignant, and infectious diseases. These disease states are often associated with abnormal lipid metabolism and reductions in plasma cholesterol levels. To determine if inflammatory cytokines could influence hepatic lipid metabolism, we evaluated low density lipoprotein (LDL) receptor function and gene expression in cytokine stimulated HepG2 cells, a hepatoblastoma-derived cell line which shares many functional similarities with normal hepatocytes. Tumor necrosis factor-alpha (TNF) and interleukin-1 beta (IL-1) increased LDL binding to HepG2 cells in a dose-responsive manner. Other cytokines including macrophage-colony stimulating factor, granulocyte macrophage-colony stimulating factor, and gamma-interferon had no significant effects on LDL binding. Increased binding in response to TNF or IL-1 was paralleled by increased steady-state levels of LDL receptor mRNA. Evaluation of LDL receptor mRNA half-life revealed no significant change in mRNA stability between control and TNF- or IL-1-stimulated cells. A fusion gene construct consisting of 1563 base pairs of the 5'-flanking DNA of the human LDL receptor gene was coupled to a luciferase reporter gene, transfected into HepG2 cells, and promoter activity was assayed after TNF and IL-1 challenge to the cells. TNF and IL-1 increased promoter activity 200-400%, while treatment with LDL inhibited promoter activity by 70-85%. TNF or IL-1 co-incubation with LDL could not override transcriptional inhibition by LDL. Pretreatment with cycloheximide prevented induction of LDL receptor mRNA by TNF, but not by IL-1, suggesting stimulation of LDL receptor transcription by TNF requires protein synthesis. We propose that TNF and IL-1, acting via distinct signal transduction pathways, increase surface LDL receptors by increasing gene transcription. Our findings suggest that cytokine-induced hypocholesterolemia may be related to TNF and/or IL-1 stimulation of hepatic LDL receptor gene expression and function.

We have determined whether macrophage derived-foam cells, a prominent component of the atherosclerotic lesion, express more urokinase-type plasminogen activator (uPA) and whether their ability to generate plasmin stimulates the release of matrix-bound growth factors. Steady state levels of uPA mRNA and both membrane and intracellular uPA activities were significantly increased in foam cells. When cultured on cell-derived matrices containing bound 125I-basic fibroblast growth factor (bFGF), both macrophage and foam cells released intact 125I-bFGF into their media. The release of 125I-bFGF by either cell was significantly enhanced in the presence of plasminogen. However, foam cells, which expressed more membrane uPA, released more 125I-bFGF than control cells. The release of matrix-bound bFGF was independent of heparanase activity, since neither macrophage nor foam cells degraded 35SO4-labeled heparan sulfate proteoglycans. In addition, media derived from foam cells cultured on cell-derived matrices in the presence of plasminogen had increased levels of transforming growth factor (TGF) beta activity as compared to cells grown in the absence of plasminogen. In contrast, plasminogen had no effect on TGF-beta activity recovered in the media of foam cells grown on plastic. Moreover, when macrophage were cultured on matrices containing bound 125I-TGF-beta, the release of labeled TGF-beta was increased in the presence of plasminogen. This is the first demonstration that foam cells can release two important growth regulators, bFGF and TGF-beta, from the extracellular matrix, and provides a mechanism by which macrophage and foam cells can stimulate atherosclerotic lesion development.

The effects of transforming growth factor-beta (TGF-beta) on low density lipoprotein (LDL) receptor-mediated cholesterol metabolism were evaluated in vascular smooth muscle cells. TGF-beta significantly increased the binding, uptake, and degradation of 125I-LDL. This increase was paralleled by an increase in LDL receptor mRNA steady state levels and an increase in cholesterol esterification. The increase in LDL cholesterol metabolism was independent of proliferation. LDL receptor expression in response to TGF-beta was not affected by coincubation with an antibody against platelet-derived growth factor or by cyclooxygenase inhibitors in arterial smooth muscle cells, suggesting that TGF-beta's effect was not mediated through platelet-derived growth factor or prostaglandins, as demonstrated in other cell systems. However, coincubation with pertussis toxin abrogated the effect of TGF-beta on LDL receptor expression, suggesting that a pertussis toxin-sensitive G-protein may be involved in the signal transduction pathway. These results are discussed in terms of their potential effects on cellular cholesterol trafficking.