(Received for publication, June 9, 1997)
From the Department of Pathology and Division of
Hematology and Oncology/Department of Medicine, Cornell University
Medical College, New York, New York 10021
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.
Oxidation of low density lipoproteins is a critical early event in the pathogenesis of atherosclerosis, and oxidized low density lipoprotein (OxLDL)1 is the proximal source of lipid that accumulates within cells of the atherosclerotic lesion (1, 2). Receptors involved in binding and internalizing modified LDL (low density lipoprotein) particles, termed "scavenger receptors", are thought to play a significant role in atherosclerotic foam cell development (2-5). The first macrophage scavenger receptor identified, isolated, and cloned (type A scavenger receptor) was identified as a receptor for AcLDL (6-9). However, since acetylation of LDL does not occur under physiological conditions, the natural ligand for this receptor was unclear until it was demonstrated that OxLDL partially competes for the binding of acetylated LDL to macrophages (10).
CD36 has now been classified as the defining member of a second class of scavenger receptors, type "B", which is distinct from the type A (I/II) receptors. It was identified using an expression-cloning strategy to isolate murine macrophage receptors that recognized OxLDL but not AcLDL (11). CD36 cDNA-transfected cells bind and internalize OxLDL, and binding of OxLDL to human macrophages is blocked by 50% by antibodies to CD36 (12, 13). CD36 is an 88-kDa transmembrane glycoprotein expressed on monocyte/macrophages (14), platelets (15), certain microvascular endothelium (16), erythroid precursors (17), adipocytes (18), and breast and retinal pigment epithelium (19). CD36 belongs to a small gene family that is highly conserved and includes at least 3 members: CD36, LIMP2 (a lysosomal membrane protein with >60% sequence homology to CD36 (20)), and CLA-1 (21). Hamster CLA-1 homologue (SR-B1) also binds OxLDL (22) and HDL (23). Both SR-BI and CD36 cDNA-transfected cells can bind anionic phospholipid liposomes (24) and CD36 on adipocytes functions to bind free fatty acids (18).
The aim of this study was to determine the impact of native and
modified lipids on the expression of CD36. Although macrophage expression of CD36 can be modulated by cytokines (25) and by adhesion
to tumor necrosis factor--activated endothelial cells (26), the
effect of lipid on CD36 expression is unknown. Modulation of scavenger
receptors by lipids may potentially impact on the accumulation of
cholesteryl esters in macrophages during atherosclerosis.
J774A.1 cells (ATCC, Rockville, MD), a murine macrophage cell line, were cultured in T25 flasks with RPMI 1640 medium containing 10% fetal calf serum, 50 µg/ml each of penicillin and streptomycin, and 2 mM glutamine. Experiments were performed when cells were about 90% confluent.
Isolation of LDL and Preparation of AcLDL and OxLDLLDL (1.019-1.063 g/ml) was isolated from normal human plasma by sequential ultracentrifugation, dialyzed with phosphate-buffered saline (PBS) containing 0.3 mM EDTA, sterilized by filtration through a 0.22-µm filter (Millipore), and stored under nitrogen at 4 °C. Protein content was determined by the method of Lowry et al. (27). AcLDL was prepared by the method of Goldstein et al. (28). LDL was oxidized by dialysis against PBS with 5 µM CuSO4 for 10 h at 37 °C. The purity and charge of both native and AcLDL were evaluated by examining electrophoretic mobility in agarose gel. The degree of oxidation of LDL and OxLDL was determined by measuring the amount of thiobarbituric acid reactive substances (29). LDL had thiobarbituric acid reactive values of <1 nmol/mg. Oxidized LDL had thiobarbituric acid reactive values of >10 and <30 nmol/mg. All lipoproteins were used for experiments within 3 weeks after preparation.
Isolation of Total RNA and Northern BlottingCells were
lysed in RNAzolTM B (Tel-Test, Inc., TX). Chloroform was
extracted, and total cellular RNA was precipitated in isopropanol. Total RNA (20 µg) from each sample was loaded on 1%
formaldehyde-agarose gels. After electrophoresis, RNA was transferred
to a Zeta-probe® GT genomic tested blotting membrane (Bio-Rad). The
blot was UV cross-linked and prehybridized with HybrisolTM
I (Oncor, Inc., Gaithersburg, MD) before the addition of
32P random prime labeling probe for CD36. The probe is a
NsiI-BglII digest (base pairs 193-805) of murine
CD36. The original murine CD36 cDNA was obtained from Dr. Gerda
Endemann (11). The cDNA probe for the murine LDL receptor was
prepared by reverse transcription-polymerase chain reaction with
primers generated from published sequences (30). The sequences of 5-
and 3
-oligonucleotides used were GACTGCAAGGACATGAGCGA (781-801) and
CGGTTGGTGAAGAGCAGATA (1201-1221), respectively. Membranes were
hybridized overnight, washed, and exposed to x-ray film (X-Omat AR,
Eastman Kodak Co.). Autoradiograms were quantified by densitometric
scanning using a UMAX (Santa Clara, CA) UC630 flatbed scanner attached
to a Macintosh IIci computer running NIH Image software (Bethesda, MD).
The same blot was used to rehybridize with 32P-labeled
probe for glyceraldehyde phosphate dehydrogenase (GAPDH) to verify the
amount of total RNA loaded.
Cells in a T25 flask were
released by a rubber scraper, pelleted by sedimentation for 5 min at
500 × g, and washed twice with PBS. After lysis with
lysis buffer (20 mM Tris, pH 7.5, 137 mM NaCl,
2 mM EDTA, 1% Triton X-100, 25 mM
-glycerophosphate, 2 mM sodium pyrophosphate, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml of aprotinin,
and 100 mM NaVO4), the lysate was
microcentrifuged for 10 min at 4 °C, and the supernatant was
transferred to a new test tube. Protein (100 µg) from each sample was
loaded and separated on a 15% SDS-polyacrylamide gel and transferred
to a nitrocellulose membrane. The membrane was blocked with a solution
of 0.1% Tween 20/PBS (PBS-T) containing 1% BSA for 2 h, and then
incubated with rabbit polyclonal anti-FAT (the rat homologue of CD36)
antibody (31) at 2 µg/ml in PBS-T/BSA for 1 h at room
temperature followed by washing 3 times for 10 min with PBS-T buffer.
The blot was reblocked with PBS-T/BSA for an additional hour before
adding horseradish peroxidase conjugated goat anti-rabbit IgG (1:400 dilution) for another hour at room temperature. After washing 3 times
for 10 min with PBS-T the membrane was incubated for 1 min in a mixture
of equal volumes of Western blot chemiluminescence reagents 1 and 2 (Renaissance®, NEN Life Science Products). The membrane was then
exposed to film for 2 min before development.
Unilamellar phospholipid liposomes were prepared by extrusion through polycarbonate membranes (24). Phosphatidylcholine liposomes contained a 2:1 molar ratio of phosphatidylcholine to cholesterol whereas phosphatidylcholine/phosphatidylinositol liposomes had a 1:1:1 ratio of phosphatidylinositol, phosphatidylcholine, and cholesterol. Liposomes were used within 1 week of preparation. Apoprotein B100 was obtained from Sigma.
The
effect of native LDL (LDL) and modified LDL (AcLDL and OxLDL) on CD36
mRNA expression was evaluated in a murine macrophage cell line,
J774 cells. The cells were cultured overnight in serum-free medium and
then treated for 12 h with LDL, AcLDL, or OxLDL (each at 12.5, 25, 50, and 100 µg/ml protein). Changes in cell morphology and viability
were not observed. As shown in Fig. 1,
native LDL significantly increased (8-fold) CD36 mRNA expression at
an LDL concentration of 25 µg/ml. AcLDL induced CD36 mRNA
expression by 5-fold with peak expression induced at a concentration of
50 µg/ml. OxLDL increased CD36 mRNA expression (maximal induction of 6-fold) at a concentration of 25 µg/ml.
To study the kinetics of induction of CD36 mRNA expression by
lipoproteins, we carried out the time course study as shown in Fig.
2. Maximum induction was observed about
2 h after treatment with AcLDL and 4 h with LDL and OxLDL.
Increased expression of CD36 mRNA persisted through 24 h with
each treatment group.
Induction of CD36 Protein
To investigate if the induction of
CD36 mRNA expression by lipoproteins was associated with an
increase of CD36 protein, macrophages were treated with either LDL,
AcLDL, or OxLDL (50 µg/ml protein) for 6 h. As seen in Fig.
3, CD36 protein is detected at two major bands of approximately 53 kDa and 88 kDa, which represent the non-glycosylated form and glycosylated form of the protein. CD36 protein was increased significantly by LDL, AcLDL, and OxLDL by 2-, 3-, and 4-fold, respectively. Only OxLDL significantly increased the
glycosylated (88 kDa) form of the protein.
Lipoproteins Do Not Alter the Half-life of CD36 mRNA
Because alterations in steady-state mRNA can reflect
changes in either transcription rates or mRNA stability, we studied
the effect of lipoproteins on the half-life of CD36 mRNA. In the
absence of actinomycin D, CD36 mRNA steady-state levels were
increased in response to LDL, AcLDL, or OxLDL (Figs. 1 and
4). In the presence of actinomycin D,
CD36 mRNA decreased with time (Fig. 4). The half-life of CD36
mRNA was estimated to be approximately 5 h based on the
densitometric scanning. Lipoproteins did not increase CD36 mRNA
expression in the presence of actinomycin D, and the decrease in the
CD36 message, measured as the ratio of CD36 mRNA to GAPDH mRNA,
was similar to cells treated with actinomycin D alone. These data
demonstrate that lipoproteins do not alter CD36 mRNA stability and
imply that regulation of CD36 by lipoproteins is at the level of
transcription.
Specificity of Effect
To rule out the possibility of
nonspecific membrane effects arising from the loading of macrophages
with sterols as a cause of CD36 induction, we evaluated the effect of
lipoproteins on the expression of another lipoprotein receptor, the LDL
receptor. Native, acetylated, and oxidized LDL all down-regulate this
receptor (Fig. 5), as would be expected,
because of its sterol response element (32).
Evaluation of Lipoprotein Components in CD36 Induction
To
determine if CD36 induction resulted from uptake of component lipids of
the lipoproteins or binding of apoB 100, we evaluated the effects of
the components of LDL on the expression of CD36 mRNA. Macrophages
were treated with apoB 100 (1 µg/ml) or unilamellar phospholipid/cholesterol liposomes (150 µg/ml) or a combination of
both for 20 h. As seen in Fig. 6,
apoB either alone or in combination with liposomes induced CD36
mRNA expression remarkably (>4-fold). Phospholipid/cholesterol
liposomes alone had a less significant effect (~2-fold). Incubation
with 25-hydroxycholesterol or cholesterol caused a similar increase of
CD36 expression to that by liposomes; however, results were difficult
to interpret because of the inhibitory effects of ethanol in which they
were dissolved (data not shown).
Effect of Cholesterol Acceptors on CD36 mRNA
Since
addition of lipoproteins to macrophages induced CD36 mRNA, we next
evaluated the effect of cholesterol acceptors on CD36 expression. Both
HDL and BSA can act as cholesterol acceptors and facilitate the efflux
of cholesterol from cultured cells (33). We demonstrate in Fig.
7 that incubation of macrophages with BSA (0.5-5%) causes a dose-dependent reduction in the
expression of CD36 mRNA. HDL at a concentration of 25-50 µg/ml
had a similar effect (data not shown). To exclude the possibility that
the inhibitory effects of BSA or HDL on expression of CD36 mRNA
might be due to a contaminating endotoxin (LPS), two experiments were
performed. First, experiments were performed with certified endotoxin
free (<0.1 ng/mg) BSA with similar results. Second, macrophages were treated with a broad range concentration of LPS (0.1-20 µg/ml). LPS
had no effect on CD36 mRNA expression.
Induction of CD36 mRNA by Murine Peritoneal Macrophages in Vivo
To investigate if the induction of CD36 in vitro
could be mimicked in an in vivo system and in primary
(i.e. non-cell line) cells, mice were injected
intraperitoneally with lipoproteins. Four mice were injected with
either 1 ml of PBS (control) or lipoproteins (LDL, AcLDL, or OxLDL, 200 µg/ml) in PBS. Peritoneal macrophages were collected by lavage,
centrifuged, and plated in Petri dishes for 30 min at 37 °C. After
removal of non-adherent cells, RNA was isolated from adherent
macrophages. Whereas LDL and AcLDL treatment modestly increased CD36
expression by 2- and 3-fold, respectively, OxLDL increased CD36
expression by 8-fold relative to macrophages isolated from control
(PBS-treated) mice (Fig. 8).
Scavenger receptors are a family of cell surface receptors expressed by macrophages that are believed to mediate the binding and uptake of modified lipids in atherosclerotic lesions. Therefore, regulation of scavenger receptor expression is thought to be a critical determinant of lipid accumulation at these sites. Cytokines produced by cells comprising the atheroma (macrophages, endothelial cells, smooth muscle cells, and lymphocytes) modulate macrophage scavenger receptor expression in vitro and are thought to participate in modulating expression in vivo. Although the role of lipid accumulation on expression of class A scavenger receptors has been evaluated in one study (34), their effect on the expression on CD36, a class B scavenger receptor, has not been addressed.
The LDL receptor, which contains a sterol regulatory element in the 5
region of the gene, is down-regulated by high intracellular cholesterol
levels and for this reason is not likely to be involved in the foam
cell development (35). However, scavenger receptors, which do not
contain sterol regulatory elements, are constitutively expressed in the
presence of high intracellular cholesterol/cholesteryl ester levels.
Expression of the class A (type I/II) scavenger receptor was shown to
be increased in macrophages and macrophage-derived foam cells relative
to freshly isolated monocytes, but expression of the receptor in foam
cells was equivalent to differentiated macrophages (34). In addition,
expression of the class A scavenger receptor is increased in monocytes
from hyperlipidemic patients (36).
To the best of our knowledge, this is the first demonstration of increased macrophage CD36 expression by exposure to lipoproteins. These findings are important since they demonstrate for the first time that a macrophage scavenger receptor can be up-regulated by both native and modified lipoproteins. CD36 expression is increased in murine heart tissue in mice fed a high fat diet (37). Immunohistochemical evaluation of heart tissue demonstrated that CD36 expression was limited to microvascular endothelial cells. In monocytes, CD36 expression is increased by M-CSF, phorbol 12-myristate 13-acetate, interleukin-4 (25, 38), and adhesion to tumor necrosis factor-activated endothelium (26) and is reduced by treatment with LPS (25). For this reason, it is unlikely that our results can be explained by LPS (endotoxin) contamination of our lipoproteins.
Lipoproteins (particularly minimally modified LDL and oxidized LDL) can modulate signal transduction and gene expression in macrophages and other cells (39). LDL stimulates phosphoinositide turnover and elevates cytosolic calcium levels in vascular smooth muscle cells (40). Minimally modified LDL induces the expression of monocyte adhesion molecules (41), tissue factor (42), MCP-1 (monocyte chemoattractant protein-1) (43), and M-CSF (44) in endothelial cells. Induction of MCP-1 and M-CSF is cyclic AMP dependent (44). Oxidized LDL induces expression of interleukin-8 in monocytes (45). Some of the effects of modified LDL can be mimicked by its constituent lipids. For example, the mitogenic effects of oxidized LDL can be mimicked by lysophosphatidylcholine (46), and oxidized phospholipid appears to be the lipid constituent in oxidized LDL that induces endothelial cell expression of MCP-1 (47) and adhesion molecules (48). The effects of oxidized LDL on gene expression are gene- and stimulus-dependent (49).
The mechanism(s) by which lipoproteins induce expression of CD36 remain unclear. Although apoB alone induced CD36 expression, it was not induced to the same degree by the intact lipoprotein. The slight induction of CD36 by phospholipid/cholesterol liposomes does not rule out cholesterol enrichment or delivery in mediating CD36 expression since the degree of uptake and alterations of cholesterol content were not verified. However, the dose dependent reduction of CD36 mRNA expression in response to BSA is consistent with the possibility that alterations in cellular cholesterol levels are modulating CD36 expression. Cholesterol has been shown to increase expression of macrophage apolipoprotein E (50) and sterol carrier protein 2 (51). Cholesterol inhibits the expression of HMG-CoA reductase and the LDL receptor, which contains sterol response elements (35). Alternatively, delivery of other bioactive lipids present in both native and modified lipoproteins may modulate expression of CD36. In support of this hypothesis, it has been shown that fatty acids can modulate CD36 expression. N-3 fatty acids (eicosapentaenoic acid and docosahexaenoic acid) inhibited CD36 expression in human monocytic cells while N-6 fatty acids (arachidonic acid and linoleic acid) tended to increase expression of CD36 (52).
Our results demonstrate that increased CD36 steady-state levels are not
the result of alterations in message stability (Fig. 4) and therefore
most likely involve increased transcription of CD36. Whether this is
directly through a lipoprotein effect on the CD36 promoter or
indirectly through the induction of other mediators or transcriptional
factors is unknown. In this regard, it has been shown that oxidized
lipoproteins can influence gene expression by causing oxidative stress
and activating the transcription factor NF-B (53). The promoter of
the CD36 gene has been isolated and partially sequenced (54, 55), but
the role of NF-
B in the transcription of CD36 has not been
determined.
The relative role of CD36 and the type A scavenger receptors in terms of their contribution to lipid accumulation in the atherosclerotic lesion is undetermined. The use of knockout mice that lack the expression of these receptors may shed some light on the in vivo function of these scavenger receptors. A "natural knockout" gives some indication that CD36 is likely to be an important receptor for oxidized LDL in vivo. A genetic polymorphism in the CD36 gene has been identified in an Asian population (56) and shown to result in deficient expression of CD36 (NAKa- phenotype). Monocyte-derived macrophages isolated from these patients bound 40% less OxLDL and accumulated 40% less cholesterol ester than cells derived from normal controls (57), further implicating CD36 as a physiological OxLDL receptor.
In summary, we have shown for the first time that both native LDL and modified LDL (AcLDL and OxLDL) can up-regulate expression of the macrophage class B scavenger receptor, CD36. Our results imply that exposure to, or cellular accumulation of, free and esterified lipids may augment the expression of CD36 and further contribute to foam cell development in atherosclerosis.