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. Author manuscript; available in PMC: 2007 Sep 1.
Published in final edited form as: Cell Metab. 2006 Sep;4(3):211–221. doi: 10.1016/j.cmet.2006.06.007

A CD36-dependent signaling cascade is necessary for macrophage foam cell formation

SO Rahaman 1,*, DJ Lennon 2,*, M Febbraio 3, EA Podrez 4, SL Hazen 5, RL Silverstein 6
PMCID: PMC1855263  NIHMSID: NIHMS12472  PMID: 16950138

Summary

Accumulation of macrophage foam cells in intima of atherosclerotic blood vessels is a critical component of atherogenesis mediated by scavenger receptor-dependent internalization of oxidized LDL. We demonstrated by co-immunoprecipitation and cytoplasmic tail pull down assays that the macrophage scavenger receptor CD36 associates with a signaling complex containing Lyn and MEKK2 (a MAP kinase pathway component). We found that MAP kinases JNK1 and JNK2 were specifically phosphorylated in macrophages exposed to oxLDL. Using cells isolated from SRA, TLR2, or CD36 null mice, and phospholipid ligands specific for either SRA or CD36, we showed that JNK activation was mediated by CD36. Both foam cell formation and activation of JNK2 in hyperlipidemic mice were dramatically diminished in the absence of CD36. Furthermore, inhibition of Src or JNK blocked oxLDL uptake and dramatically inhibited foam cell formation in vitro and in vivo. These findings show that a specific CD36-dependent signaling pathway initiated by oxLDL is necessary for foam cell formation and identify a potential new target for anti-atherosclerosis therapy.

Introduction

The formation and progression of atherosclerotic lesions is a highly complex inflammatory process that results from interaction of blood components, including monocytes, T cells, and lipoproteins with cellular components of the arterial wall (Glass and Witztum, 2001; Lusis, 2000; Libby, 2002). During atherogenesis lipid-laden macrophages, known as foam cells, accumulate within the arterial neointima and become a major contributor to plaque. A critical step in foam cell formation is recognition and internalization of oxLDL particles by specific macrophage scavenger receptors, including CD36 and scavenger receptor A (SRA) (Platt and Gordon, 2001; Silverstein and Febbraio, 2000). Combined inhibition of these 2 receptors blocks human and murine foam cell formation in vitro (Kunjathoor et al., 2002), and genetic deletion of either SRA or CD36 slows lesion development in atherogenic apoE null mice (Suzuki et al., 1997; Febbraio et al., 2000). Although the ligands and receptors that mediate interaction of modified LDL particles with macrophages have been well defined, the intracellular processes that regulate internalization and trafficking of lipids from oxLDL remain poorly understood. Our lab has shown that CD36 accounts for a large proportion of oxLDL uptake by macrophages (Febbraio et al., 2000), especially when LDL is oxidized by leukocyte myeloperoxidase generated reactive nitrogen species (Podrez et al., 2000), an oxidizing system shown to be highly relevant to the atherosclerotic process (Podrez et al., 2000; Podrez et al., 2002). Since CD36 has also been shown to mediate internalization signals in macrophages, microglial cells, and retinal pigment epithelial cells exposed to apoptotic cells, fibrillar amyloid, photoreceptor outer segments, and staphylococcus (Febbraio et al., 2001; Finnemann and Silverstein, 2001; Fadok et al., 1998; Hoebe et al, 2005), and since CD36 has been shown to transduce signals that regulate apoptotic and inflammatory responses in endothelial cells and macrophages (Moore et al., 2002; Medeiros et al., 2004; Jimenez et al., 2000; Janabi et al., 2000; Bamberger et al., 2003; Hoebe et al, 2005; Stuart et al, 2005), we hypothesized that a CD36 triggered signaling cascade in macrophages may be involved in foam cell formation. Previous studies have implicated non-receptor tyrosine kinases of the src family and serine/threonine kinases of the mitogen activated protein (MAP) kinase family in CD36 signal transduction. For example fyn kinase and p38 MAPK were shown to be necessary for CD36-mediated endothelial cell anti-angiogenic responses to thromobospondin-1 (Jimenez et al., 2000); and fyn, lyn, and syk tyrosine kinases and pERK and p44/42 MAP kinases have been implicated in CD36-dependent THP-1 and microglial cell inflammatory responses to fibrillar amyloid (Moore et al., 2002; Bamberger et al., 2003). In this manuscript we report that exposing peritoneal macrophages from wild type (WT), but not CD36 null (CD36−/−) mice to oxLDL led to activation of the MAP kinases c-Jun N-terminal kinase (JNK)-1 and -2 and that pharmacologic blockade of JNK or Src-family kinases inhibited CD36-dependent foam cell formation in vitro and in vivo. In addition, we showed a direct physical association in monocytic cells between the intra-cytoplasmic carboxy-terminal domain of CD36 and a signaling complex containing lyn kinase and MEKK2, an upstream MAP kinase.

Results

OxLDL induced CD36-dependent activation of JNK in macrophages

We investigated the role of CD36 in activation of specific MAP kinase family members by oxLDL in murine peritoneal macrophages, comparing cells from WT and CD36−/− mice. Cells were treated with 50μg/ml oxLDL and the phosphorylation state of MAP kinase family members determined by immunoblot analysis. Fig. 1A shows that Erk1/Erk2 and p38 became phosphorylated transiently in both cell types, peaking at 5–15min and returning to baseline by 30–60min. Similarly there was no difference in phosphorylation of Erk5 in macrophages from WT and CD36−/− mice (data not shown). In contrast to the behavior of ERK and p38, JNK was phosphorylated only in WT cells, not in CD36−/− cells, phosphorylation peaked at 30min and was sustained for over 60min. CD36 dependent phosphorylation of JNK was dose-dependent, peaking at 50–100 μg/ml (Fig. 1B). Phosphorylation of JNK was also dependent on oxidation of the LDL; native LDL had no effect at doses up to100μg/ml (data not shown).

Figure 1. CD36-dependent phosphorylation of macrophage JNK by oxidized lipoproteins and phospholipids. (A).

Figure 1

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Immunoblot analysis of MAP kinases ERK, p38 and JNK in WT and CD36−/− mouse peritoneal macrophages treated with 50μg/ml copper oxidized LDL (oxLDL) for times indicated. The prefix ‘p’ denotes blots probed with antibodies specific for phosphorylated forms of the proteins while the corresponding unmarked rows are blots using antibodies that recognize all forms to indicate total protein loaded. (B) Immunoblot analysis of phosphorylated JNK (p-JNK) from macrophages exposed to oxLDL at different concentrations for 30min. The membrane was then stripped and re-probed with anti-JNK antibody to indicate total JNK loaded. (C) Macrophages from WT (dark bars), SRA−/− (hatched bars) or CD36−/− (clear bars) mice were treated with indicated ligands at 50μg/ml for 30min and assayed for JNK activation by immunoblot. Data are graphed as the fold change in phosphorylation compared to untreated cells. (D) 50μg/ml PAPC oxidized with the MPO system in the presence (+NO2) or absence (−NO2) of nitrite was exposed to macrophages from WT (squares) or CD36−/− (triangles) mice and phosphorylation of JNK assayed by immunoblot. Data are plotted as percent maximal response. (E) Immunoblot showing activation of JNK1 and JNK2 by +NO2LDL in macrophages from WT, SRA−/− and CD36−/− mice. Expression of total JNK1 and JNK2 is shown as loading control.

As further evidence for CD36 dependence of oxLDL-induced JNK phosphorylation we examined a form of oxLDL generated by myeloperoxidase (MPO) in the presence of nitrite (+NO2LDL) that we previously demonstrated to be a high-affinity ligand for CD36, but that is not recognized by SRA (Podrez et al., 2000). We found that +NO2LDL stimulated phosphorylation of JNK similarly to copper oxidized LDL in cells from WT but not CD36−/− mice (Fig. 1C). Similarly, 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphatidylcholine (PAPC) vesicles oxidized by the same MPO/nitrite system (+NO2PAPC) when incubated with macrophages also activated JNK in WT, but not CD36−/− cells (Fig. 1D). Control LDL or PAPC preparations exposed to all components of the MPO system except nitrite (−NO2LDL and −NO2PAPC) had minimal JNK stimulatory activity (Fig. 1C and D), similar to results seen with native LDL (not shown).

To assess the role of the other major macrophage scavenger receptor, SRA, in JNK activation we compared macrophages from SRA−/− mice with those from WT and CD36−/− mice, using acLDL, a specific ligand for SRA that is not recognized by CD36 and +NO2LDL, a specific ligand for CD36 that is not recognized by SRA. AcLDL did not activate JNK in any of the cells, while +NO2LDL activated JNKs in both WT and SRA−/− cells, but not in CD36−/− cells (Fig. 1C). In these studies we also examined specific members of the JNK family of MAP kinases, focusing on the more widely expressed members JNK1 and JNK2 (JNK3 expression is restricted to brain, heart and testis) (Davis, 2000). We found that both JNK1 and JNK2 in peritoneal mouse macrophages were phosphorylated in response to treatment with +NO2LDL but that activation of JNK2 was significantly more predominant than JNK1 (Fig. 1E).

To demonstrate that oxLDL treatment of macrophages induced CD36-dependent JNK activity along with phosphorylation we examined downstream targets of pJNK. oxLDL exposure led to increased phosphorylation of the transcription factor c-Jun and ATF2 in WT, but not CD36−/− cells (data not shown).

JNK2 was activated in vivo in hyperlipidemic mice in a CD36 dependent manner

To evaluate the in vivo relevance of our observations we utilized a macrophage transfer assay described by Li et al (2004). Peritoneal macrophages from donor mice (WT, CD36−/− or SRA−/−) were transferred into the peritoneal cavities of pro-atherogenic apoE−/− mice maintained on a “Western” diet for six weeks to induce hyperlipidemia. Cells were removed 3d later and analyzed. We hypothesized that the modified LDL particles produced in these mice would activate JNK in the donor macrophages depending on the availability of CD36. We found markedly higher levels of activation of JNK2 in WT and SRA−/− macrophages compared to CD36−/− cells (Fig. 2A). These finding suggest that oxLDL-mediated activation of JNK2 occurs in vivo in the hyperlipidemic pro-atherogenic mice and that is primarily dependent on CD36 expression. We did not find any significant difference in activation of p38 and Erk1/2 between WT and CD36−/− macrophages (Fig. 2B), demonstrating that the in vitro systems faithfully mimicked the in vivo model. We also examined activation of upstream MAP kinase kinases, including MKK4 and MKK3/6 in this model and found higher activation of MKK4, not MKK3/6, in WT cells compared to CD36−/− cells (Fig. 2C), raising the possibility that activated MKK4 might be involved as an upstream MAPKK in activation of JNK2.

Figure 2. CD36-dependent JNK2 phosphorylation in vivo in macrophages transferred into hyperlipidemic mice.

Figure 2

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Thioglycollate-elicited peritoneal macrophages were collected from WT, CD36−/− or SRA−/− mice and injected intraperitoneally into apoE−/− recipient mice maintained on Western or chow diets for 6 wks. Cells were recovered from the peritoneal cavity after 3d and analyzed by immunoblot. (A) Bar graphs show the mean SD levels of phopho-JNK for WT (n=6), CD36−/− (n=6) and SRA−/− (n=4) cells transferred into western diet fed recipients. A representative immunoblot with antibodies to phospho-JNK1 and 2 (top) and total JNK1 and 2 (bottom) is shown below the graph. (B) Immunoblots with antibodies to phosphorylated and total p38 and Erk1/2. RAW cells treated with LPS were used as a positive control. ND indicates normal chow diet fed recipients. (C) Immunoblots with antibodies to phosphorylated MKK4 and MKK3/6 show CD36-dependent activation of MKK4 in cells transferred into western diet-fed recipients.

The carboxy-terminal cytoplasmic tail of monocyte CD36 interacts with a signaling complex containing MEKK2 and lyn

To understand potential mechanisms by which CD36 activates intracellular kinases we sought to determine a role for the intracellular domain(s) of CD36. Several laboratories have reported that the Src-family nonreceptor protein tyrosine kinases fyn, lyn, and yes co-immunoprecipitated with CD36 from human platelet, endothelial cell, and monocytic cell line detergent lysates (Medeiros et at., 2004; Huang et al., 1991; Bull et al., 1994). The structural basis for this association is not known and has been speculated to be due to CD36 interactions with other membrane proteins, CD36 localization in caveolae-like membrane microdomains, or direct interactions of CD36 intracellular domains with kinases. Although CD36 has intracellular domains at both its amino and carboxy terminal regions, we hypothesized that the carboxy terminal domain was the better candidate to function as a binding site for intracellular signaling proteins based on its homology to similar sites in CD4, CD8 and other proteins (Shattil and Brugge, 1991; Shaw et al., 1990; Turner et al., 1990), and because the amino-terminal domain is extremely short. Using affinity chromatography with recombinant GST/CD36 fusion proteins we now report a direct association between the CD36 carboxy-terminal domain and a signaling complex in human monocytic cells. In pilot studies (not shown) using metabolically labeled THP-1 monocytic cells, we identified 9 bands by SDS-PAGE autoradiography ranging from 40–120kD that bound to an immobilized GST-CD36 cytoplasmic tail fusion protein (P36I), but that did not bind to GST alone or to a GST fusion protein containing a scrambled CD36 tail sequence (P36S). The recombinant CD36 cytoplasmic tail peptide purified after cleavage from the fusion protein effectively competed for 7 of the 9 proteins. To identify the P36I binding proteins, similar studies were performed using a larger number of unlabeled cells and the eluted material was analyzed by western blot using antibodies to candidate antigens and by 2-D gel electrophoresis. Based on the size and pI of one of the more prominent dots on the 2-D gels we hypothesized that one of the CD36 binding proteins was MEKK2, a component of the MAP kinase pathway. Fig. 3A shows a western blot confirming this; anti-MEKK2 IgG recognized a protein of the appropriate MW in the material eluted from P36I, but not the GST control. Further control studies showed that MEKK2 could be eluted from P36I with recombinant CD36 cytoplasmic tail peptide but not the scrambled peptide and that MEKK2 did not bind to P36S. To validate the physical association of CD36 with MEKK2 we also demonstrated by immunoblot analysis that MEKK2 was precipitated from human cells by an anti-CD36 monoclonal antibody (Fig. 3B).

Figure 3. Interaction of the carboxy-terminal cytoplasmic tail of monocyte CD36 with a signaling complex containing MEKK2 and lyn.

Figure 3

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(A) GST or GST/CD36 cytoplasmic tail fusion protein (P36I)-bound complexes from THP-1 cell lysates were separated by SDS-PAGE and analyzed by immunoblot with anti-MEKK2 antibody. The specific MEKK2 band is shown with an arrow. (B) IP with anti-CD36 IgG or isotype control from human platelet lysates. Complexes were analyzed by immunoblot as in panel A. (C) GST or GST/CD36 cytoplasmic tail fusion protein (P36I)-bound complexes from THP-1 cell lysates were assayed for tyrosine kinase activity in the presence of 32P-γATP. Labeled proteins were separated by SDS-PAGE and visualized by autoradiography. Arrows indicate proteins that became specifically phosphorylated in P36I complexes. (D) GST or GST/CD36 cytoplasmic tail fusion protein (P36I)-bound complexes from endothelial cell (EC) or THP-1 cell lysates were analyzed by immunoblot with anti-Lyn antibody. Cell extracts from EC and THP-1 were used as negative and positive controls respectively. (E) 293T cells were transiently transfected with CD36 and/or lyn cDNA. Cells were immunoprecipitated using anti-CD36 antibody, and complexes were probed for the presence of CD36 (top) and MEKK2 (bottom) by immunoblot analysis. In all cases, blots or gels are representative of at least 3 experiments.

To determine if the association of src-family kinases with CD36 also involves the C-terminal cytoplasmic tail we analyzed the GST/P36I and control eluates by in vitro kinase assay and western blot with src-specific antibodies. Fig. 3C shows that the bound complex contained an active kinase with multiple substrates of 25–150kD (arrows). Using antibodies to specific members of the src kinase family we found that lyn was present in the P36I eluate, but not the control GST eluate (Fig. 3D, right lanes). In contrast, eluates from a lysate made from a transformed human microvascular endothelial cell line did not contain lyn (Fig. 3D, left lanes), but did contain fyn (not shown), suggesting that CD36 associates with different kinases in different cell types.

Interestingly, when CD36 cDNA was transfected into 293T cells and the cells immunoprecipitated with anti-CD36 IgG (Fig. 3E) MEKK2 was found in the precipitates only if lyn cDNA was co-transfected, suggesting that the CD36-lyn interaction may be necessary to recruit MEKK2 into the complex.

OxLDL treatment leads to activation and recruitment of signaling molecules to CD36

To assess the effects of CD36 ligands on the cytoplasmic tail signaling complex murine macrophages were exposed to the CD36-specific ligand +NO2LDL and analyzed by immunoprecipitation (IP)/western blot. We found 2.5–3 fold more lyn and MEKK2 in the anti-CD36 IP from +NO2LDL treated cells compared to cells exposed to non-oxidized LDL (Fig. 4A). Anti-lyn IP of +NO2LDL exposed macrophages were examined by immunoblot with an anti-phosphotyrosine antibody revealing a ~3 fold increase in tyrosine phosphorylation in WT compared to CD36−/− cells (Fig. 4B). This increase was blocked completely by co-incubation with a src inhibitor AG1879. As an additional validation of the cytoplasmic pull down assays, we also performed IP with anti-MEKK2 and analyzed the precipitates by immunoblot with anti-lyn and anti-phosphotyrosine. As shown in Fig. 4C, +NO2LDL treated WT macrophages showed increased phosphorylation of MEKK2 and lyn in the MEKK2 IP compared to CD36−/− cells. Together these studies show that lyn, MEKK2 and CD36 co-precipitated from macrophages with antibodies to either CD36 or MEKK2 and that the amount of MEKK2 and lyn and the degree of their phosphorylation was increased by exposure of the cells to +NO2LDL.

Figure 4. Co-precipitation of CD36, lyn and MEKK2 from macrophages treated with +NO2LDL.

Figure 4

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(A) WT and CD36−/− macrophages were treated with 50μg/ml +NO2LDL or control −NO2LDL and CD36 immunoprecipitated with monoclonal anti-CD36. Co-precipitated lyn (top blot) and MEKK2 (middle blot) were detected by immunoblot using specific monoclonal antibodies. The blot was stripped and re-probed with anti-CD36 antibody (lower blot). No CD36-immunoreactive band was detected in CD36−/− sample. Total lyn and MEKK2 were quantified and expressed as fold-change relative to CD36−/− lane. (B) WT and CD36−/−macrophages were treated with 50μg/ml +NO2LDL for 20min and then lyn was immunoprecipitated. Phosphorylation of Lyn was assayed by immunoblot with an anti-phosphotyrosine antibody (top blot). The blot was stripped and re-probed with anti-lyn (lower blot). A lysate from WT cells pretreated with the src kinase inhibitor AG1879 was used as a control. (C) WT and CD36−/− macrophages were treated with 50μg/ml +NO2LDL for 20min and then MEKK2 was immunoprecipitated. The precipates were then analyzed by immunoblot with antibodies to p-MEKK2, p-lyn and total lyn. Top blot shows increased phosphorylation of MEKK2 in WT compared to CD36−/− cells. Lower blot shows co-precipitation of lyn with the anti-MEKK2 and middle blot shows increased phosphorylation of lyn in the WT cells compared to CD36−/− cells. Total whole cell lysate (WCL) was used as a control.

Inhibition of JNK or Src pathways prevented oxLDL uptake and foam cell formation in murine peritoneal macrophages

Having shown that oxLDL induced CD36-dependent macrophage JNK activation as well as increased physical association between CD36, lyn and MEKK2 we next assessed the biological role of this system in macrophage scavenging function. We examined oxLDL uptake and foam cell formation in the presence of specific pharmacologic inhibitors, using a quantitative assay based on Oil-Red-O staining (Febbraio et al., 2000; Podrez et al., 2000). Fig. 5A shows that inhibition of JNK activation by either a small molecular weight chemical inhibitor, SP600125, or an HIV-TAT based peptide inhibitor, JNKi, led to up to 80% reduction in foam cell formation in peritoneal macrophages exposed to either oxLDL or +NO2LDL. Similar results were found when cell were treated with a broadly active Src family kinase inhibitor, AG1879 (Fig. 5B). Inhibition of Erk1/2 activity by U0126, Jak by AG490, PI3Kinase by LY294002, or Protein Kinase C by Go6983 had no significant effect on foam cell formation, demonstrating specificity. In these studies we found that JNK inhibitors did not affect expression of PPARγ or CD36, and that both JNK and Src inhibitors significantly decreased the level of c-Jun phosphorylation in macrophages exposed to +NO2LDL (data not shown). Interestingly, the src inhibitors did not affect phosphorylation of JNK itself even though phosphorylation of the downstream JNK target c-Jun was inhibited.

Figure 5. Inhibition of foam cell formation by pharmacologic inhibitors of JNK and Src-family kinases. A).

Figure 5

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Peritoneal macrophages were cultured for 48h in RPMI-1640 containing 10% FBS and were then treated with SP600125 (SP), U0126 (U), JNK1 peptide inhibitor (JNKi), or control HIV-TAT peptide for 3h prior to incubation for an additional 18h with 50μg/ml native LDL, copper oxidized LDL (ox LDL) or +NO2LDL. Cells were then fixed and stained with oil-red-O to quantify foam cell formation. The bar graphs show the mean SD of triplicate determinations. B) Cells as in A were pretreated with inhibitors AG1879, AG490, LY294002 (LY) or Go6983 for 3h prior to incubation with modified LDL and staining with oil-red-O. C) Thioglycollate-elicited peritoneal macrophages were collected from WT and TLR2−/− mice and analyzed by Oil-red-O staining to quantify the number of foam cells as above. The corresponding bar graphs show the mean SD for WT and TLR2−/− mice.

Recently it was reported that CD36 functions as a co-receptor with TLR2 to mediate recognition and internalization of phospholipid components of the cell wall of staphylococcus bacteria. Since TLR2 activation can result in activation of JNK it was important to determine if CD36-dependent foam cell formation and JNK activation involved TLR2. To address this possibility peritoneal macrophages were obtained from TLR2−/− mice on the same C57Bl background as CD36−/−. We found that macrophages from TLR2−/− mice formed foam cells in response to various forms of oxidized LDL (copper oxidized or MPO/NO2 oxidized) to the same extent as WT cells (Fig 5C). We also found no difference in JNK phosphorylation in TLR2−/− null cells exposed to the various forms of oxLDL compared to WT cells (data not shown). Importantly, western blots showed no difference in levels of CD36 expression in the TLR2−/− null cells compared to WT. These studies show that CD36-dependent foam cell formation does not require signaling through TLR2.

Macrophage scavenging function requires binding of ligand to receptor followed by ligand internalization. To assess the role of the CD36 signaling system in these processes we exposed cells to a fluorophore (DiI)-tagged form of +NO2LDL (DiI-NO2LDL) and examined them by confocal fluorescence microscopy after short term incubations at 4°C to measure binding and 37°C to measure internalization. No significant reduction in binding of +NO2LDL was observed in JNK-inhibited cells, while treatment with the src inhibitor caused a small inhibition of binding (Fig. 6A). As reported previously (Febbraio et al, 2000), no binding was seen to CD36−/− cells. Fig. 6B however, shows that blockade of JNK kinase activation by SP600125 or src kinase by AG1879 significantly inhibited +NO2LDL uptake by peritoneal macrophages. Similar results were seen with the JNKi peptide (not shown).

Figure 6. DiI-labeled +NO2LDL uptake but not binding is blocked in presence of JNK and Src family kinase inhibitors.

Figure 6

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Peritoneal macrophages from WT or CD36−/− mice were pretreated with vehicle or inhibitors for 18h and then exposed to DiI-labeled +NO2LDL for 3h at 4°C (A) or for 5min, 30min and 60min at 37°C (B) to assess binding and uptake of +NO2LDL respectively by confocal microscopy.

To show in vivo relevance for these observations we examined foam cell formation in the macrophage transfer model described above. Approximately 75% of WT or SRA−/− donor macrophages recovered from western diet-fed apoE−/− mice stained strongly with Oil red O, indicating foam cell formation, compared to ~25% of those from CD36−/− donors (Fig. 7). WT cells transferred to apoE−/− mice maintained on a chow diet did not form foam cells. These results correlate well with the degree of JNK2 and MKK4 phosphorylation (Fig. 2) and are consistent with a model in which interaction of CD36 with specific oxidized phospholipids within oxLDL results in recruitment of lyn and MEKK2 to the C-terminal cytoplasmic tail of CD36. This then facilitates activation of these kinases and then downstream activation of JNK2 (presumably via MKK4) followed by internalization of the bound ligands leading to formation of foam cells.

Figure 7. CD36-dependent in vivo foam cell formation in hyperlipidemic apoE−/− mice.

Figure 7

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Thioglycollate-elicited peritoneal macrophages were collected from WT, CD36−/− or SRA−/− mice and injected intraperitoneally into apoE−/− recipient mice maintained on Western or chow diets for 6 wks. Cells were recovered from the peritoneal cavity after 3d and analyzed by Oil-red-O staining to quantify the number of foam cells. The corresponding bar graphs show the mean SD for WT (n=6), CD36−/− (n=9) and SRA−/− (n=6) on Western diet. As a control we have shown WT cells transferred into normal chow fed animals (n = 3).

Discussion

Macrophage foam cell formation is an essential, yet incompletely understood component of the atherogenic process. Critical steps in foam cell formation include recognition and internalization of modified lipoprotein particles, including oxLDL and aggregated LDL, trapped in the arterial intima in response to inflammatory injury (Glass and Witztum, 2001; Lusis, 2000; Libby, 2002). This leads to accumulation of cholesterol and other lipids within the macrophages, presumably due to insufficient activity of lipid efflux and/or catabolic pathways. In this manuscript we have identified an intracellular signaling pathway triggered by interaction of oxLDL with a specific scavenger receptor, CD36 that is necessary for foam cell formation in vitro and in an in vivo model involving cell transfer into hyperlipidemic mice. Using a combination of specific pharmacologic inhibitors, scavenger receptor null macrophages, and ligands with high degree of specificity for CD36 we showed that the MAP kinases JNK-1 and JNK-2 were phosphorylated and activated in a CD36-dependent manner by exposure of macrophages to oxLDL, and that blockade of JNK activation potently inhibited foam cell formation. We also showed that the src-family kinase lyn is part of this pathway by demonstrating an interaction between the cytoplasmic domain of CD36 with lyn and MAPK pathway upstream components, and by showing that pharmacologic blockade of src kinases also inhibited foam cell formation.

CD36-dependent signal transduction can proceed by at least two pathways. By facilitating internalization of bioactive lipids from oxLDL that activate nuclear receptors, such as PPARγ, CD36 can participate in the initiation of a transcriptional program that includes up-regulation of its own gene as well as those of other critical genes involved in lipid metabolism (Tontonoz et al., 1998; Nagy et al., 1998; Moore et al., 2001; Chinetti et al., 2001; Li et al., 2004). Other data, however suggests that CD36 can function as traditional signal transduction receptor initiating a signaling cascade conferred upon ligand binding (Moore et al., 2002; Medeiros et al., 2004; Jimenez et al., 2000; Janabi et al., 2000; Bamberger et al., 2003). For example, the anti-angiogenic activity of CD36 in microvascular endothelial cells requires specific src and MAP kinases (Jimenez et al., 2000) as do CD36-dependent generation of oxidative bursts in macrophages and microglial cells (Moore et al., 2002; Medeiros et al., 2004; Cho et al., 2005; El Khoury et al., 2003). It is interesting that endothelial cell responses to CD36 are mediated by fyn kinase and p38 MAP kinase, while the responses noted here involve lyn and JNK, suggesting that different cell types may respond differently to CD36 engagement, perhaps depending on their predominant src-family member.

Work reported here sheds light on some of the molecular mechanisms linking CD36, JNK and src-family kinases. Others have speculated that CD36 signal transduction may relate to indirect effects due to CD36 interactions with other transmembrane signaling receptors, such as CD9, CD47, β1 integrins, SRA, and TLR2 (Miao et al., 2001; Thorne et al., 2000; Hoebe et al, 2005; Stuart et al., 2005) or to CD36 membrane localization in lipid rafts (Bamberger et al., 2003; Miao et al., 2001). We now show, however, that CD36 signaling by oxLDL proceeds in the absence of TLR2 and SRA and that the short 13 amino acid carboxy terminal intracellular domain of CD36 was sufficient to precipitate a multi-component signaling complex from monocyte cell lysates. This domain contains a CXCX5K motif homologous to the sequence of the cytoplasmic domain in CD4 and CD8 that serves as a docking site for signaling molecules including the src kinase lck (Shattil and Brugge, 1991; Shaw et al., 1990; Turner et al., 1990). Others, however, have shown that lyn and fyn do not bind with high affinity to this site. Interestingly, Stuart et al recently reported (2005) that Y468 and C464 in the carboxy-terminal CD36 cytoplasmic domain were necessary for TLR2/CD36-dependent internalization and inflammatory responses to phospholipids in staph bacteria. The precise role of these residues in TLR2-independent CD36 functions, such as foam cell formation remains to be determined.

Our biochemical data suggests that the lyn-CD36 interaction is necessary for recruitment of the MAP kinase component, MEKK2 (Fig. 3E). The observation that src inhibitors blocked macrophage foam cell formation and c-Jun phosphorylation, but did not inhibit JNK phosphorylation is not easily explained. Perhaps activation of lyn in a CD36-dependent manner results in transient recruitment of pJNK to a signaling complex that then allows oxLDL internalization. Blockade of lyn activity might then alter intracellular localization of pJNK without changing its level, and thereby inhibit foam cell formation. This is supported by data (Fig. 4) showing increased lyn phosphorylation in macrophages exposed to CD36 ligands and increased recruitment of both p-lyn and MEKK2 to CD36 in response to oxLDL.

Our results, although based on in vitro experiments are highly relevant to recent in vivo findings. Oxidation of LDL by the myeloperoxidase system in vivo is supported by mass spectroscopic analysis of lipids in atheromatous plaque and by epidemiologic data linking circulating MPO and nitrotyrosine levels with atherosclerotic risk (Podrez et al., 2002; Ricci et al., 2004; Yla-Herttuala et al., 1989; Palinski et al., 1989; Nicholls and Hazen, 2005). Furthermore, lyn-deficient mice fed a high fat diet have been shown to have hyperlipidemia without increased levels of atherosclerosis (Miki et al., 2001), JNK activity is up regulated in atheromas (Ricci et al., 2004; Metzler et al., 2000), and macrophages closest to the lipid core of lesions express the most CD36 (Nakata et al., 1999). Ricci et al. recently reported studies showing decreased atherosclerosis in JNK2 null mice. They also showed decreased phosphorylation of SRA in these mice, suggesting a link between JNK2 and SRA activity. Here we showed that acLDL, a specific ligand for SRA, did not activate JNK, while +NO2LDL, a specific ligand for CD36, activated JNKs in wild type but not CD36 null macrophages (Fig. 1), suggesting that there may be “cross talk” between CD36 and SRA pathways.

The precise role of JNK in foam cell formation remains to be defined, but our data showing blockade of oxLDL uptake by JNK inhibitors suggest that JNK is affecting a proximal step in the process. These data are consistent with other studies from our lab showing that CD36 signaling plays a role in internalization of shed photoreceptor outer segments by retinal pigment epithelial cells (Finnemann and Silverman, 2001). We thus propose a model in which oxLDL binding to the extracellular domains of CD36 leads to recruitment and activation of lyn kinase and the upstream MAP kinase MEKK2 at the carboxy terminal CD36 cytoplasmic tail. This interaction results in phosphorylation and activation of JNK. Activation of JNK has been reported to regulate numerous cellular processes via phosphorylation of substrates involved in gene transcription, apoptosis and survival pathways, metabolic pathways, cytoskeletal dynamics, and membrane trafficking. Recent genome wide siRNA based “forward genetic” screens designed to define the role of kinases in basic cellular processes have shown that kinases (including JNK) are required for clathrin- and raft-mediated endocytosis (Pelkmans et al, 2005). It is thus likely that CD36-mediated activation of JNK is required to deliver an internalization signal perhaps related to cytoskeletal or membrane dynamics. In addition, the work of Ricci et al. (2004) suggests a role for JNK2 in macrophage cholesterol efflux to apoA1. Thus CD36 signaling through JNK may modulate foam cell formation via inhibition of both influx and efflux of lipids. Although the studies in this manuscript focus on foam cell formation, JNK signaling has been implicated in many other processes of relevance to macrophage function, including cytoskeletal dynamics, NFκB inflammatory signaling, apoptosis and insulin resistance. The role of oxLDL and CD36 in these events remains to be determined.

Experimental procedures

Antibodies, cells and other reagents

Antibodies to phosphorylated forms of Erk1/2, p38, JNK1/2, c-Jun, MKK4, MKK3/6, MKK3/6, and to native Erk1/2, p38, and Jnk1/2 were from Cell Signaling, Beverly, MA. Antibodies to phosphorylated Erk5, Erk5, MEKK2, MKK4 and PPARγ were from Santa Cruz Biotechnology (Beverly, MA). Anti-Lyn was from BD-Transduction (Franklin Lakes, NJ), anti-actin from Sigma (St. Louis, Missouri), and anti-HA from Berkeley Antibodies Co. (Richmond, CA). Monoclonal anti-human CD36 was from Immunotech (Westbrook, ME) and polyclonal anti-murine CD36 was provided by Huy Ong, University of Montreal, Canada. Mouse monoclonal anti-mouse CD36 was prepared as previously described (Finneman and Silverstein, 2001) and anti-p-tyrosine antibody was from Upstate (Charlottesville, VA).

AcLDL was purchased from Intracell (Issaquah, WA). PAPC was obtained from Avanti polar lipids (Alabaster, Alabama) and small unilammelar liposomes were prepared as previously described (Podrez et al., 2000). Oxidized lipoproteins and phospholipids and their non-oxidized controls were prepared as described previously (Podrez et al., 2000). DiI was from Molecular probes (Eugene, OR), and U0126 from Promega (Madison, WI). AG490, SP600125, LY294002, Go6983, AG1879, and JNKi (HIV-TAT-JNKi, JNK inhibitory peptide) and HIV-TAT (control peptide) were purchased from Calbiochem (La Jolla, CA). All chemicals were obtained from Sigma (St. Louis, MO) unless otherwise indicated.

CD36 (Febbraio et al., 1999), SRA (Suzuki et al., 1997), and TLR2 null mice (Takeuchi et al., 1999) were described previously. CD36 null mice were backcrossed 6 times to C57Bl/6 and a background matched WT control line was used in all studies. Peritoneal macrophages were obtained by lavage 4d after injection with thioglycollate and adherent cells maintained in culture as described (Febbraio et al., 2000). Cells from TLR2−/− mice were generously provided by C. Harding (Case Western Reserve University). THP-1, RAW, and 293T cells were obtained from ATCC. All cell culture reagents were purchased from Life Technologies (Rockville, MD). Human platelets were obtained from healthy, medication-free, volunteer donors following IRB approved protocol. Platelets were separated from plasma by chromatography of platelet-rich plasma on a sepharose 2B column as previously described (Silverstein and Nachman, 1987) and were used immediately.

Foam cell formation

Thioglycolate-elicited peritoneal macrophages from background matched control wild type (WT), SRA−/−, TLR2−/− and CD36−/− mice were plated on coverslips in 12 well plates in RPMI 1640 medium supplied with 10% FCS. After 2h non-adherent cells were washed out and fresh medium added for 24h. Cells were then incubated with 50μg/ml native or modified LDL overnight in the presence or absence of various inhibitors in the same media. Cells were fixed with 4% formaldehyde and stained with oil-red-O.

Binding and uptake of +NO2LDL

Peritoneal macrophages from WT and CD36−/− mice on coverslips were pretreated either with SP600125, AG1879 or vehicle for 15h, and incubated with DiI+NO2LDL (10μg/ml) for 3h at 4°C to assess binding. To assess uptake cells were incubated with DiI+NO2LDL(10μg/ml) for timed points at 37°C before analysis. Nonspecific binding to free DiI was measured using CD36−/− cells. Fluorescence intensity was examined by confocal microscopy (63X).

Macrophage transfer experiments

Peritoneal macrophages were collected as described above from male donor mice of various genotypes. Cells (12 x 106) were then injected intraperitoneally into each recipient apoE−/− mouse maintained on a diet containing 0.15% added cholesterol and 42% milk fat (TD88137, Harlan-Teklad) or normal chow diet for 6 wks. Recipient mice were sacrificed after 3d and peritoneal macrophages collected for analysis.

Plasmids, Transfections and Fusion Proteins

Full length CD36 and lyn cDNAs were cloned into pCDNA3.1 expression plasmid (Invitrogen, Carlsbad, CA). 293T cells were transfected using LipofectAMINE (Life Technologies, Rockville, MD) according to manufacturer’s protocol. For fusion proteins, sequences encoding the putative intracellular carboxy-terminal domain of CD36 (MISYCACRSKTIK) or a scrambled sequence of the same amino acids were synthesized and cloned into the pGEX6P1 vector (Pharmacia) immediately 3/ to the glutathione-S-transferase (GST) cDNA. All clones were confirmed by direct DNA sequencing. Fusion protein expression in transformed bacteria was induced by IPTG and then the bacterial cells were lysed by sonication. Fusion proteins were purified by GST affinity chromatography (GSTrap; Pharmacia) by elution with 10mM reduced glutathione. In some experiments the bound fusion proteins were treated with PreScission Protease® (Pharmacia) to cleave the CD36 peptide from the beads. Purified peptides or fusion proteins were dialyzed extensively prior to use.

Purification of CD36 cytoplasmic tail binding proteins

5–10mg of GST or GST/CD36 fusion proteins were loaded onto 5ml of Fast Flow glutathione sepharose cartridges (Pharmacia) and columns washed in a minimum of 30 column volumes of PBS and 10 volumes of lysis buffer. Lysates from THP-1 monocytic cells were then loaded at 1ml/min, recirculating overnight. In initial pilot experiments, cells were metabolically labeled with 200μCi/ml 35S-methionine (Translabel, NEN) for 5h prior to lysis. Columns were then washed in 30 vol lysis buffer and 10 vol 10mM Tris-HCl, pH7.5, 150mM NaCl prior to elution with 10mM glutathione. Eluted proteins were then loaded onto a 5ml anion exchange column (Q-sepharose), washed in 10 vol of 10mM Tris-HCl, pH 7.5, 150mM NaCl, and proteins were eluted with a 0.15–1M NaCl gradient. Fractions (0.5–1.0ml) were analyzed by SDS-PAGE/autoradioagraphy, silver stained 2D-gel electrophoresis or by immunologic or kinase activity as described below.

Immunoprecipitation, immunoblot and kinase assays

For IP studies cells were lysed in 20mM Tris-HCl (pH7.5), 150mM NaCl, 1mM EDTA, 1mM EGTA, 1%NP-40, 2.5mM sodium pyrophosphate, 1mM β-glycerophosphate, 1mM Na3VO4, and 1μg/ml leupeptide. The cleared supernatant containing 500μg protein was incubated with 3μg of anti-CD36 antibody immobilized on agarose beads overnight at 4°C. Beads were extensively washed, boiled in SDS-PAGE loading buffer and the bound material analyzed by immunoblot using antibodies for CD36, lyn, fyn or MEKK2. For co-IP assays cells were treated with DSP (Dithiobis-succinimidylpropionate) before harvesting in lysis buffer containing 1% CHAPS. Lysate was immunoprecipitated using monoclonal anti-CD36 antibody and Protein-L-Agarose. For immunoblot analysis of signaling proteins, macrophages were plated at 80–95% confluency and cells were placed in serum-free or 1% serum supplied media (RPMI 1640) for a minimum of 2h before treatment. Cells were then treated with modified LDL at doses and durations indicated. Cells were then washed once in ice-cold PBS, lysed in sample buffer and processed as above. After chemiluminescence detection, blots were stripped and re-probed with antibodies to control proteins (actin or non-phosphorylated MAPK) to assess loading. For in vitro kinase assays washed glutathione sepharose-bound proteins were incubated in buffer containing leupeptin, aprotinin, pepstatin, PMSF, Na3VO4, 5μM ATP, 1mM DTT, and 10μCi γ32P-ATP (NEN, Boston, MA) at 22°C for 30min. Reactions were stopped by the addition 12μl 5X sample buffer at 100°C and the samples then analyzed by SDS/PAGE autoradioagraphy.

Footnotes

Grant Support: This work was supported by R01 HL070083 (MF), P01 HL072942 (RLS), P01 HL046403 (RLS) and RO1 HL70621 (SLH)

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Contributor Information

S.O. Rahaman, Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH,.

D.J. Lennon, Department of Medicine, Division of Hematology and Medical Oncology, Weill Medical College of Cornell University, New York, NY..

M. Febbraio, Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH,

E.A. Podrez, Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH,

S.L. Hazen, Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH,

R.L. Silverstein, Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH,

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