Uptake of Oxidized Low Density Lipoprotein by CD36 Occurs by an Actin-dependent Pathway Distinct from Macropinocytosis

The class B scavenger receptor CD36 has numerous ligands that include modified forms of low density lipoprotein, fibrillar amyloid, apoptotic cells, and Plasmodium falciparum-infected red blood cells, linking this molecule to atherosclerosis, Alzheimer disease, malaria, and other diseases. We studied the signaling events that follow receptor engagement and lead to CD36 and ligand internalization. We show that oxidized low density lipoprotein or antibody-induced clustering of CD36 triggers macropinocytosis and internalization of the receptor-ligand complex. Remarkably, however, CD36 internalization is independent of macropinocytosis and occurs by a novel endocytic mechanism that depends on actin, but not dynamin. This actin-driven endocytosis requires the activation Src family kinases, JNK, and Rho family GTPases, but, unlike macropinocytosis, it is not affected by inhibitors of phosphatidylinositol 3-kinase or Na/H exchange. Manipulation of this unique mode of internalization may prove helpful in the prevention and management of the wide range of diseases in which CD36 is implicated. The class B scavenger receptor CD36 has numerous ligands that include modified forms of low density lipoprotein, fibrillar amyloid, apoptotic cells, and Plasmodium falciparum-infected red blood cells, linking this molecule to atherosclerosis, Alzheimer disease, malaria, and other diseases. We studied the signaling events that follow receptor engagement and lead to CD36 and ligand internalization. We show that oxidized low density lipoprotein or antibody-induced clustering of CD36 triggers macropinocytosis and internalization of the receptor-ligand complex. Remarkably, however, CD36 internalization is independent of macropinocytosis and occurs by a novel endocytic mechanism that depends on actin, but not dynamin. This actin-driven endocytosis requires the activation Src family kinases, JNK, and Rho family GTPases, but, unlike macropinocytosis, it is not affected by inhibitors of phosphatidylinositol 3-kinase or Na/H exchange. Manipulation of this unique mode of internalization may prove helpful in the prevention and management of the wide range of diseases in which CD36 is implicated. Uptake and storage of cholesterol by macrophages are key contributors to the formation of atherosclerotic plaque. Endothelial cells, seemingly activated by the deposition of modified forms of low-density lipoprotein (LDL), 3The abbreviations used are: LDLlow density lipoproteinoxLDLoxidized LDLDNdominant negativeGAPDHglyceraldehyde-3-phosphate dehydrogenaseGFPgreen fluorescent proteinM-CSFmacrophage colony-stimulating factorPBDPAK-binding domainPFAparaformaldehydePI3Kphosphatidylinositol 3-kinasePIP3phosphatidylinositol 3,4,5-trisphosphateYFPyellow fluorescent proteinDiI1,1′-didodecyl-3,3,3′,3′-tetramethylindocarbocyanine perchloratePP14-amino-5-(4- methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]-pyrimidine. release chemokines that recruit macrophages into the vascular intima. Infiltrated macrophages can then readily oxidize and take up the modified LDL. Accumulation of lipids derived from oxidized LDL (oxLDL) transforms macrophages into foam cells, which release excess cytokines, triggering an inflammatory cascade. In addition, foam cells express proteases and other factors that contribute to plaque rupture and subsequent thrombosis. low density lipoprotein oxidized LDL dominant negative glyceraldehyde-3-phosphate dehydrogenase green fluorescent protein macrophage colony-stimulating factor PAK-binding domain paraformaldehyde phosphatidylinositol 3-kinase phosphatidylinositol 3,4,5-trisphosphate yellow fluorescent protein 1,1′-didodecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate 4-amino-5-(4- methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]-pyrimidine. OxLDL particles are recognized by a variety of receptors, including the class A scavenger receptor SR-A and the class B scavenger receptor CD36. CD36 is thought to be responsible for ∼50% of oxLDL uptake by murine and human macrophages (1Endemann G. Stanton L.W. Madden K.S. Bryant C.M. White R.T. Protter A.A. J. Biol. Chem. 1993; 268: 11811-11816Abstract Full Text PDF PubMed Google Scholar, 2Nozaki S. Kashiwagi H. Yamashita S. Nakagawa T. Kostner B. Tomiyama Y. Nakata A. Ishigami M. Miyagawa J. Kameda-Takemura K. J. Clin. Invest. 1995; 96: 1859-1865Crossref PubMed Scopus (285) Google Scholar, 3Kunjathoor V.V. Febbraio M. Podrez E.A. Moore K.J. Andersson L. Koehn S. Rhee J.S. Silverstein R. Hoff H.F. Freeman M.W. J. Biol. Chem. 2002; 277: 49982-49988Abstract Full Text Full Text PDF PubMed Scopus (769) Google Scholar). In addition, this protein mediates cholesterol uptake from high density lipoprotein (4Graf G.A. Connell P.M. van der Westhuyzen D.R. Smart E.J. J. Biol. Chem. 1999; 274: 12043-12048Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar) and is also a receptor for internalization of oxidized high density lipoprotein (5Thorne R.F. Mhaidat N.M. Ralston K.J. Burns G.F. FEBS Lett. 2007; 581: 1227-1232Crossref PubMed Scopus (72) Google Scholar). CD36 encodes a protein with two transmembrane domains located near the N and C termini, leaving only short cytoplasmic tails at each end. Despite having small intracellular domains, engagement of CD36 by its cognate ligands triggers signaling reactions that lead to the internalization of the resulting complex. However, the precise pathways that are activated and the specific mode of internalization remain unclear. Jones and Willingham (6Jones N.L. Willingham M.C. Anat. Rec. 1999; 255: 57-68Crossref PubMed Scopus (24) Google Scholar) demonstrated that, in macrophages, modified LDL stimulates ruffling activity and the formation of phase-bright macropinosomes. By transmission electron microscopy they found that gold-conjugated modified LDL associated with ruffles and was present within macropinosomes. These observations underlie the widely accepted view that uptake of modified LDL occurs by macropinocytosis. However, Zeng et al. (7Zeng Y. Tao N. Chung K.N. Heuser J.E. Lublin D.M. J. Biol. Chem. 2003; 278: 45931-45936Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar) showed that internalized DiI-oxLDL and CD36 were found in moderately sized cytoplasmic structures that co-localized with a glycosylphosphatidylinositol-anchored protein, suggesting uptake via lipid raft endocytosis. Moreover, Sun et al. (8Sun B. Boyanovsky B.B. Connelly M.A. Shridas P. van der Westhuyzen D.R. Webb N.R. J. Lipid Res. 2007; 48: 2560-2570Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) reported that uptake of oxLDL by CD36 was independent of actin but dependent on dynamin. The results of these two studies are not easily reconciled with mediation by macropinocytosis, an actin-dependent process that generates large vacuoles, and they suggest instead that CD36 is internalized by a more conventional endocytic pathway. It is not clear whether the apparent discrepancy stems from the engagement of different receptors in the different biological systems used in these studies. Jones and Willingham used macrophages, whereas Zeng et al. and Sun et al. studied, respectively, Chinese hamster ovary and COS cells heterologously transfected with CD36. The types and abundance of receptors capable of binding modified LDL in all likelihood differ greatly in these systems, and heterologous (over)expression in immortalized cells is liable to produce results of questionable biological relevance. In view of this uncertainty and considering the important and versatile roles of CD36, we set out to reexamine the mode of internalization of this receptor. We used both primary and cultured macrophages and selectively targeted CD36 using specific antibodies. The responses triggered by selective CD36 cross-linking were also compared with those elicited by oxLDL. We show that clustering CD36 initiates a signaling cascade that results in the activation of both macropinocytosis and internalization of CD36. Remarkably, however, CD36 internalization is largely independent of macropinocytosis and occurs by a novel dynamin-independent, actin-driven process that requires activation of Src family and c-Jun N-terminal kinases. Mouse anti-human CD36 IgG (clone 131.2) was prepared by Narendra N. Tandon. The following antibodies were purchased from the sources indicated: mouse anti-mouse CD36 IgA and mouse anti-GAPDH (Chemicon); anti-mouse IgA Texas Red (Bio/FX); anti-CD36 IgM (BD Pharmingen); anti-mouse Alexa Fluor 488 (Molecular Probes/Invitrogen); anti-mouse IgG Cy3, anti-mouse F(ab′)2 Cy3, anti-rabbit horseradish peroxidase, and anti-mouse horseradish peroxidase (Jackson Immunological Laboratories); Alexa Fluor 488-conjugated anti-phosphotyrosine primary antibody (PY-20 clone) (BioLegend); anti-mouse IgA-fluorescein isothiocyanate (Sigma-Aldrich); mouse anti-phosphotyrosine (4G10) (Upstate); rabbit anti-phospho-JNK (Cell Signaling); and mouse anti-vinculin (Chemicon). Human peripheral macrophages were prepared according to McGilvray et al. (9McGilvray I.D. Serghides L. Kapus A. Rotstein O.D. Kain K.C. Blood. 2000; 96: 3231-3240Crossref PubMed Google Scholar). Wild-type and CD36-knock-out mouse peritoneal macrophages were a gift from Kevin Kain (University Health Network, Toronto, Canada) and prepared according to Patel et al. (10Patel S.N. Serghides L. Smith T.G. Febbraio M. Silverstein R.L. Kurtz T.W. Pravenec M. Kain K.C. J. Infect. Dis. 2004; 189: 204-213Crossref PubMed Scopus (105) Google Scholar). U937 cells (ATCC) were differentiated with 1 μm phorbol 12-myristate 13-acetate (Bioshop Canada) for 2 days. RAW 264.7 cells (ATCC) were grown in Dulbecco's modified Eagle's medium (Wisent) with 10% fetal bovine serum. During live experiments, cells were maintained in HEPES-buffered RPMI 1640 medium (H-RPMI; Wisent). Total CD36 immunostaining was accomplished by fixing RAW 264.7, differentiated U937, or human peripheral macrophages with 4% PFA (Electron Microscopy Sciences), permeabilizing with 0.1% Triton X-100, blocking with 5% serum, and labeling with anti-CD36 IgA or anti-human CD36 IgG, followed by anti-mouse IgA Texas Red or anti-mouse IgG Cy3 secondary antibody, respectively. CD36 receptors were internalized by cross-linking with a primary antibody followed by a secondary antibody and warming, as follows. RAW 264.7 cells were chilled for 3 min in ice-cold H-RPMI followed by a 5-min incubation in ice-cold 5% donkey serum (Sigma-Aldrich), a 10-min incubation in ice-cold anti-CD36 and a 10-min incubation in ice-cold anti-mouse IgA Texas Red or donkey anti-mouse F(ab′)2 were with ice-cold H-RPMI and antibody labeling U937 cells and human peripheral macrophages were using mouse IgG or mouse anti-CD36 followed by anti-mouse IgG In only labeling was to be cells were in ice-cold H-RPMI and in 4% internalization cells were at in H-RPMI for min in a In antibodies to the CD36 were with an were for 2 min with ice-cold followed by in ice-cold H-RPMI for 2 were CD36-knock-out mouse peritoneal macrophages and RAW 264.7 cells were in H-RPMI for 1 and with oxidized LDL cells were cells were in 4% and internalized oxidized LDL was RAW 264.7 cells were in at for with 5% serum, and with anti-CD36 IgA followed by anti-mouse IgG Alexa Fluor experiments, RAW 264.7 cells were transfected with 2 of dominant negative or using of according to the CD36 cells were transfected with for CD36, in H-RPMI for 3 and on a In all cells were RAW 264.7 cells were with inhibitors as B B 3 1 or labeling with antibody were for min and and CD36 internalization was by the an to the of CD36 uptake on actin, Src family kinases, and RAW 264.7 cells were for and in the of oxLDL and the for min Total CD36 was by permeabilizing cells and labeling with anti-CD36 IgA and anti-mouse secondary RAW 264.7 cells were grown to in in H-RPMI for then with or These cells were as or with CD36 primary with or secondary antibody in the with the that the secondary antibody used was anti-mouse CD36 F(ab′)2 in ice-cold H-RPMI for were then to CD36 by in H-RPMI at for 3 with or in (Bioshop 1 (Sigma-Aldrich), (Bioshop 1 (Sigma-Aldrich), and inhibitors of were on a 10% to with bovine serum in and with mouse anti-phosphotyrosine antibody (4G10) and anti-mouse horseradish peroxidase secondary were and with mouse anti-GAPDH primary RAW 264.7 cells were in H-RPMI for 1 and then with inhibitors as or in H-RPMI at macropinocytosis, cells were then in H-RPMI mouse (Sigma-Aldrich), B (Molecular and the for min at The negative but were cells were in 4% and were on a fluorescent using were in by only each the of was and to the whether the of formation can lead to of CD36 cells were with to macropinocytosis for min or μm for were in 4% and min with (Molecular CD36 uptake for electron RAW 264.7 cells were with CD36 antibodies and for 3 min at ruffles by cells were with mouse and B for 3 min at were in and Microscopy was on an electron those for macropinocytosis were on a using were on a fluorescent using electron were at a of for the in were by with a were were using to a using the at an range from to were in of to an of CD36 of These were to the were and the were in We the of CD36 in and we the murine RAW the human U937 differentiated to macrophages by phorbol 12-myristate and primary human in microscopy that all of the CD36 at the of The primary macrophages a on the that of CD36 as in However, this was to the of these cells, and of the each demonstrated that the was at the CD36 was in Chinese hamster ovary cells (7Zeng Y. Tao N. Chung K.N. Heuser J.E. Lublin D.M. J. Biol. Chem. 2003; 278: 45931-45936Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). whether antibody-induced cross-linking signaling CD36 and its live cells were in the in the of anti-CD36 followed by a secondary maintained at CD36 at the but a more in all types cells that in the were to a of CD36 labeling was CD36 was found in a within intracellular the intracellular of the and to the of the internalization cells were as and then and with primary and a different secondary antibody to CD36. CD36 at the the used not of that the internalization was not by engagement of receptors, which are on the of internalization was CD36 was in RAW cells using a primary IgA antibody followed by anti-mouse F(ab′)2 secondary antibodies internalization was by U937 and primary human macrophages to anti-human CD36 IgM which not with CD36 is thought to be responsible for ∼50% of oxLDL uptake by murine and human macrophages (1Endemann G. Stanton L.W. Madden K.S. Bryant C.M. White R.T. Protter A.A. J. Biol. Chem. 1993; 268: 11811-11816Abstract Full Text PDF PubMed Google Scholar, 2Nozaki S. Kashiwagi H. Yamashita S. Nakagawa T. Kostner B. Tomiyama Y. Nakata A. Ishigami M. Miyagawa J. Kameda-Takemura K. J. Clin. Invest. 1995; 96: 1859-1865Crossref PubMed Scopus (285) Google Scholar, 3Kunjathoor V.V. Febbraio M. Podrez E.A. Moore K.J. Andersson L. Koehn S. Rhee J.S. Silverstein R. Hoff H.F. Freeman M.W. J. Biol. Chem. 2002; 277: 49982-49988Abstract Full Text Full Text PDF PubMed Scopus (769) Google Scholar). whether oxLDL internalization of CD36, RAW cells were in the of oxLDL for min and then and for CD36. in CD36 was internalized into in the following to the antibodies triggered internalization. results were reported in cells transfected with CD36 (7Zeng Y. Tao N. Chung K.N. Heuser J.E. Lublin D.M. J. Biol. Chem. 2003; 278: 45931-45936Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). that CD36 is at responsible for oxLDL uptake in these cells, primary macrophages from or were with oxLDL. in and macrophages and internalized a of oxLDL of independent that macrophages only internalized as oxLDL as these results suggest that the CD36 is responsible for the of oxLDL uptake and that the internalization of CD36 triggered by oxLDL can be by cross-linking the receptors with anti-CD36 antibodies. The by Sun et al. (8Sun B. Boyanovsky B.B. Connelly M.A. Shridas P. van der Westhuyzen D.R. Webb N.R. J. Lipid Res. 2007; 48: 2560-2570Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) that in cells, uptake of oxLDL by CD36 depends on dynamin. We whether the uptake of CD36 in macrophages also on dynamin. RAW cells were transfected with or dominant negative forms of cells were in the in the of anti-CD36 and a secondary antibody and then to for and for that in we also the cells with a of fluorescent Uptake of is to by and endocytosis J.S. J. Biol. 1993; PubMed Scopus Google Scholar). in the cells transfected with internalized both and CD36. was in the of these two the that they were internalized by different cells transfected with dominant negative were to take up but internalized CD36 to with and that the that is not The results of independent experiments, and in that internalization of CD36 is largely independent of in macropinocytosis as a mechanism for oxLDL uptake (6Jones N.L. Willingham M.C. Anat. Rec. 1999; 255: 57-68Crossref PubMed Scopus (24) Google we whether internalization of CD36 were for min with B or cross-linking CD36 with primary and secondary antibodies. that internalization in and CD36 to into large on the A and results were with B activation of Rho family GTPases, and has in large ruffles of the in macropinocytosis. whether or activation is CD36 RAW cells were transfected with to the binding domain of the This protein to and only in the but not to In cells, is the but is to the cross-linking of CD36 in This that is activated CD36 engagement and the that the may contribute to the internalization of the this cells were with which and Rho family with this CD36 internalization was and the of Rho family we transfected RAW cells with that express dominant negative forms of or and internalization in transfected in and dominant negative CD36 uptake by and in both CD36 is also to with we whether its internalization may by the in T. J. J. PubMed Scopus Google Scholar). negative only CD36 internalization by this was also The which and actin are with the that CD36 is internalized by macropinocytosis, as by Jones and Willingham (6Jones N.L. Willingham M.C. Anat. Rec. 1999; 255: 57-68Crossref PubMed Scopus (24) Google Scholar). of phosphatidylinositol 3,4,5-trisphosphate is an of macropinocytosis, we whether this also CD36 The of was by RAW cells with a of the domain of to In cells, this is found in the that are low However, following cross-linking of CD36, to the of phosphatidylinositol which is a of of and is also recognized by the that the of the was to activation of phosphatidylinositol 3-kinase cells were with the specific This the of at the that CD36 clustering to activation of of and followed by actin are of macropinocytosis Biol. PubMed Scopus Google Scholar). We whether CD36 internalization via the formation of macropinosomes. macropinocytosis we RAW cells in the of as a The macrophages were with a of macropinocytosis, or with anti-CD36 primary secondary antibodies to as can also be up by only structures μm in were as macropinosomes. in were in In the of in a of the cells with in primary macrophages J. PubMed Google Scholar). CD36 cross-linking also the formation of in a of the cells that the large structures the are we in the of This of in cells that activity is for macropinocytosis N. J. Biol. PubMed Scopus Google Scholar). The of the was also by with mediation by macropinocytosis. formation is also to this has its on macropinocytosis is by of it is by low this is a selective of the of the L. A. J. 1993; PubMed Scopus Google Scholar). We that both and the formation of elicited by and to the of these on in both the formation of large vacuoles, that the structures engagement of CD36 are macropinosomes. The of and as inhibitors of macropinocytosis to whether this process is responsible for the internalization of CD36. Remarkably, that the of CD36 was only to a These results that, CD36 macropinocytosis, this process for only a small of the internalization of the receptor. The occurs by a that, unlike macropinocytosis, is independent of and to of The results that in macrophages CD36 is for the by a unique endocytic process that is from macropinocytosis, dependent on actin The also that is not an of the to actin-dependent endocytosis. We to the for this endocytic In CD36 is associated with and can Src family kinases. the of these kinases, we by both and RAW cells with the antibody showed In cells CD36 immunostaining of the This was not in cells with suggesting that it from activation of Src family of these A in the of was apparent following CD36 The which for at was apparent at the of Src to at the by was also by whether activation of Src is for the internalization of CD36, we the of on receptor endocytosis. in the internalization of CD36 following that activation of Src family is for the studies that is activated in macrophages following to modified a of CD36, and that this activity is for foam formation Febbraio M. Podrez E.A. Silverstein R.L. Full Text Full Text PDF PubMed Scopus Google Scholar). whether the activation of is for CD36 we cells with a that with internalization of CD36. of these results is in activation of JNK, of Src kinases, is for the actin-dependent endocytosis of CD36. found by and also in oxLDL can the internalization of CD36, the of cross-linking of CD36 with specific antibodies. It is that oxLDL internalization the novel actin-dependent internalization This was using and actin in the of the endocytosis of CD36 triggered by oxLDL was by A and Src and the uptake of CD36 by oxLDL C and In affected CD36 internalization. These that, both modified LDL (6Jones N.L. Willingham M.C. Anat. Rec. 1999; 255: 57-68Crossref PubMed Scopus (24) Google Scholar) and CD36 clustering can macropinocytosis, this process not contribute to the internalization of the This was to an the mode of oxLDL uptake by macrophages, a process of studies that oxLDL was internalized by macropinocytosis (6Jones N.L. Willingham M.C. Anat. Rec. 1999; 255: 57-68Crossref PubMed Scopus (24) Google whereas that it by a to conventional dependent on but not Remarkably, studies using both oxLDL and anti-CD36 antibodies demonstrated that of the was CD36 engagement triggers internalization of the receptor by an mechanism that activation of and and the subsequent of actin to small endocytic that are from macropinosomes. The internalization process from macropinocytosis not only in the of the but also in that is for CD36 that Src family are for CD36 endocytosis. These are with the reported the receptors and J.S. PubMed Scopus Google Scholar). The linking activity with actin however, is In which via CD36, Src family to In these cells, clustering CD36 the activation of and to actin and the is responsible for CD36 internalization to be It is however, that CD36 with in a variety of including D.R. J. J. 2003; PubMed Google Scholar, J. G. J. 2004; PubMed Scopus Google Scholar). The sources of apparent results and those Sun et al. (8Sun B. Boyanovsky B.B. Connelly M.A. Shridas P. van der Westhuyzen D.R. Webb N.R. J. Lipid Res. 2007; 48: 2560-2570Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) and Zeng et al. (7Zeng Y. Tao N. Chung K.N. Heuser J.E. Lublin D.M. J. Biol. Chem. 2003; 278: 45931-45936Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar) used of CD36 in to oxLDL We that in the of for the studies and CD36 to in with kinases, J.S. PubMed Scopus Google Scholar, D.R. J. J. 2003; PubMed Google Scholar, J. G. J. 2004; PubMed Scopus Google Scholar, J. Blood. PubMed Scopus Google Scholar). of the of by heterologous is to in forms of CD36 that may internalized by that we that of cells that express CD36 as macrophages, more Jones and Willingham (6Jones N.L. Willingham M.C. Anat. Rec. 1999; 255: 57-68Crossref PubMed Scopus (24) Google Scholar) macrophages to the uptake of modified LDL. reported that of the cells to or but not LDL the formation of ruffles and large the modified LDL were within vacuoles, macropinocytosis was as the mechanism internalization. studies the of macropinocytosis but demonstrated that the of CD36 internalized by this process is the of the CD36 was in small by a process that was to the conventional inhibitors of macropinocytosis. modified LDL were also by Jones and and are not and only the of and differ The and of modified LDL may the primary of internalization. that the are to be by a process to to internalization. low or modified LDL be up by the actin-dependent endocytosis process in this these the of macropinocytosis is to be However, as the of the protein of modified LDL may be within the of macropinosomes. LDL uptake can lead to foam formation macropinocytosis is activated by phorbol Jones N.L. B. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) or B. Y. Jones N.L. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google of scavenger receptor the of foam cells in a of low of modified LDL internalization by a process also macropinocytosis. This in in the uptake of the more the and to foam this the oxLDL uptake and LDL uptake may an to selective to by the of modified LDL the uptake of LDL in macropinosomes. In we found that in to macropinocytosis, engagement of CD36 triggers a novel of actin-dependent endocytosis. The precise mechanism CD36 to the is but we that may a a signaling In this it is that in cells with the and also with CD36. A may also contribute to the of red cells and to the of by We H. J. for the gift of and for Toronto, Canada) for with Wild-type and were by L. was from A. (University of was from negative and were by

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