Caveolae-dependent Endocytosis Is Required for Class A Macrophage Scavenger Receptor-mediated Apoptosis in Macrophages

SR-A (class A macrophage scavenger receptor) is a transmembrane receptor that can bind many different ligands, including modified lipoproteins that are relevant to the development of vascular diseases. However, the precise endocytic pathways of SR-A/mediated ligands internalization are not fully characterized. In this study, we show that the SR-A/ligand complex can be endocytosed by both clathrin- and caveolae-dependent pathways. Internalizations of SR-A-lipoprotein (such as acLDL) complexes primarily go through clathrin-dependent endocytosis. In contrast, macrophage apoptosis triggered by SR-A-fucoidan internalization requires caveolae-dependent endocytosis. The caveolae-dependent process activates p38 kinase and JNK signaling, whereas the clathrin-mediated endocytosis elicits ERK signaling. Our results suggest that different SR-A endocytic pathways have distinct functional consequences due to the activation of different signaling cascades in macrophages. SR-A (class A macrophage scavenger receptor) is a transmembrane receptor that can bind many different ligands, including modified lipoproteins that are relevant to the development of vascular diseases. However, the precise endocytic pathways of SR-A/mediated ligands internalization are not fully characterized. In this study, we show that the SR-A/ligand complex can be endocytosed by both clathrin- and caveolae-dependent pathways. Internalizations of SR-A-lipoprotein (such as acLDL) complexes primarily go through clathrin-dependent endocytosis. In contrast, macrophage apoptosis triggered by SR-A-fucoidan internalization requires caveolae-dependent endocytosis. The caveolae-dependent process activates p38 kinase and JNK signaling, whereas the clathrin-mediated endocytosis elicits ERK signaling. Our results suggest that different SR-A endocytic pathways have distinct functional consequences due to the activation of different signaling cascades in macrophages. IntroductionEndocytosis is characterized by the internalization of molecules from the cell surface into internal membrane compartments. It is important for diverse cellular signaling events. Two main pathways have been identified for receptor-mediated endocytosis: the clathrin-dependent and the caveolae/lipid raft-dependent endocytic pathways (1Le Roy C. Wrana J.L. Nat. Rev. Mol. Cell Biol. 2005; 6: 112-126Crossref PubMed Scopus (697) Google Scholar, 2Polo S. Di Fiore P.P. Cell. 2006; 124: 897-900Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). Clathrin-dependent endocytosis is the most well characterized mechanism for mediating the internalization of membrane receptors into cells. It is also important for intracellular trafficking at the trans-Golgi network and endosomes (3Young A. Semin. Cell Dev. Biol. 2007; 18: 448-458Crossref PubMed Scopus (47) Google Scholar). Caveolae/raft-dependent endocytosis is involved in multiple biological processes, including mediating virus entry into host cells, internalizing glycophosphatidylinositol-anchored proteins and regulating certain signaling cascades. Caveola are cholesterol and sphingolipid-rich plasma membrane invaginations of a diameter of 60–80 nm, of which caveolin-1 is the main protein component required for caveola biogenesis (4Simons K. Toomre D. Nat. Rev. Mol. Cell Biol. 2000; 1: 31-39Crossref PubMed Scopus (5111) Google Scholar). Trafficking through these two endocytic pathways affects the amplitude and the on/off status of many signaling pathways. Thus, these endocytic processes contribute to the regulations of cell migration, cell cycle, cell polarity, apoptosis, and gene transcription (1Le Roy C. Wrana J.L. Nat. Rev. Mol. Cell Biol. 2005; 6: 112-126Crossref PubMed Scopus (697) Google Scholar, 4Simons K. Toomre D. Nat. Rev. Mol. Cell Biol. 2000; 1: 31-39Crossref PubMed Scopus (5111) Google Scholar, 5Nesterov A. Carter R.E. Sorkina T. Gill G.N. Sorkin A. EMBO J. 1999; 18: 2489-2499Crossref PubMed Scopus (184) Google Scholar).SR-A (class A macrophage scavenger receptor) is a transmembrane receptor expressed mainly in macrophages (6Freeman M. Ashkenas J. Rees D.J. Kingsley D.M. Copeland N.G. Jenkins N.A. Krieger M. Proc. Natl. Acad. Sci. U.S.A. 1990; 87: 8810-8814Crossref PubMed Scopus (257) Google Scholar, 7Suzuki H. Kurihara Y. Takeya M. Kamada N. Kataoka M. Jishage K. Ueda O. Sakaguchi H. Higashi T. Suzuki T. Takashima Y. Kawabe Y. Cynshi O. Wada Y. Honda M. Kurihara H. Aburatani H. Doi T. Matsumoto A. Azuma S. Noda T. Toyoda Y. Itakura H. Yazaki Y. Kodama T. et al.Nature. 1997; 386: 292-296Crossref PubMed Scopus (999) Google Scholar). SR-A can bind with an unusually broad range of polyanionic ligands, which includes modified lipoproteins, LPS of Gram-negative bacteria, and advanced glycation end products. Its broad specificity in binding with the ligands supports the multiple functions of SR-A in macrophage growth, adhesion to the substratum, cell-cell interactions, phagocytosis, and host defense (8Murphy J.E. Tedbury P.R. Homer-Vanniasinkam S. Walker J.H. Ponnambalam S. Atherosclerosis. 2005; 182: 1-15Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar, 9Moore K.J. Freeman M.W. Arterioscler. Thromb. Vasc. Biol. 2006; 26: 1702-1711Crossref PubMed Scopus (413) Google Scholar, 10Bowdish D.M. Gordon S. Immunol. Rev. 2009; 227: 19-31Crossref PubMed Scopus (96) Google Scholar). However, the molecular mechanisms enabling SR-A to exert multiple functions are still not well understood. It is reported that acetylated low density lipoprotein (acLDL), 2The abbreviations used are: acLDLacetylated low density lipoproteinDiI1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchloratePIpropidium iodide. a specific ligand of SR-A (17Goldstein J.L. Ho Y.K. Basu S.K. Brown M.S. Proc. Natl. Acad. Sci. U.S.A. 1979; 76: 333-337Crossref PubMed Scopus (1925) Google Scholar), is internalized via coated pit-mediated endocytosis (14Chen Y. Wang X. Ben J. Yue S. Bai H. Guan X. Bai X. Jiang L. Ji Y. Fan L. Chen Q. Arterioscler. Thromb. Vasc. Biol. 2006; 26: 1317-1322Crossref PubMed Scopus (30) Google Scholar, 18Mommaas-Kienhuis A.M. van der Schroeff J.G. Wijsman M.C. Daems W.T. Vermeer B.J. Histochemistry. 1985; 83: 29-35Crossref PubMed Scopus (17) Google Scholar, 19Jones N.L. Reagan J.W. Willingham M.C. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 773-781Crossref PubMed Scopus (33) Google Scholar). Macropinocytosis may also contribute to the uptake of acLDL at a low level (19Jones N.L. Reagan J.W. Willingham M.C. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 773-781Crossref PubMed Scopus (33) Google Scholar). On the other hand, the VirB-dependent bacterial internalization induces localization of SR-A into the detergent-resistant membrane lipid rafts (20Kim S. Watarai M. Suzuki H. Makino S. Kodama T. Shirahata T. Microb. Pathog. 2004; 37: 11-19Crossref PubMed Scopus (82) Google Scholar), which are sterol- and sphingolipid-enriched, and caveolin-containing domains that compartmentalize cellular processes. We and others (11Fong L.G. Le D. J. Biol. Chem. 1999; 274: 36808-36816Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 12Morimoto K. Wada Y. Hinagata J. Imanishi T. Kodama T. Doi T. Biol. Pharm. Bull. 1999; 22: 1022-1026Crossref PubMed Scopus (23) Google Scholar, 13Kosswig N. Rice S. Daugherty A. Post S.R. J. Biol. Chem. 2003; 278: 34219-34225Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 14Chen Y. Wang X. Ben J. Yue S. Bai H. Guan X. Bai X. Jiang L. Ji Y. Fan L. Chen Q. Arterioscler. Thromb. Vasc. Biol. 2006; 26: 1317-1322Crossref PubMed Scopus (30) Google Scholar) previously identified a few unique signal motives in the cytoplasmic domain of SR-A required for its internalization. We also showed that the internalization of SR-A and its ligand complex into cell is regulated by interaction of SR-A to its coupler (15Wang X. Zheng Y. Xu Y. Ben J. Gao S. Zhu X. Zhuang Y. Yue S. Bai H. Chen Y. Jiang L. Ji Y. Xu Y. Fan L. Sha J. He Z. Chen Q. Biochim. Biophys. Acta. 2009; 1791: 76-83Crossref PubMed Scopus (11) Google Scholar, 16Ben J. Gao S. Zhu X. Zheng Y. Zhuang Y. Bai H. Xu Y. Ji Y. Sha J. He Z. Chen Q. J. Mol. Cell. Cardiol. 2009; 47: 646-655Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). In this study, we want to address the question of whether different functions of SR-A are carried out by different endocytosis pathways. We show that SR-A can be internalized by both clathrin- and caveolae-dependent pathways. The clathrin-dependent SR-A endocytosis activates ERK signaling. The caveolae-dependent pathway is required for p38 kinase and JNK signaling as well as caspase activation. Our results indicate for the first time that SR-A-mediated macrophage apoptosis may be selectively regulated by the caveolae-dependent endocytosis.DISCUSSIONThe uptake of modified lipoproteins by SR-A is thought to be central to foam cell formation, from which atherosclerotic lesion is generated and develops. However, the conflicting outcomes from multiple studies on the impact of SR-A in mouse models of atherosclerosis suggest the existence of pathogenesis mechanisms beyond lipid uptake (7Suzuki H. Kurihara Y. Takeya M. Kamada N. Kataoka M. Jishage K. Ueda O. Sakaguchi H. Higashi T. Suzuki T. Takashima Y. Kawabe Y. Cynshi O. Wada Y. Honda M. Kurihara H. Aburatani H. Doi T. Matsumoto A. Azuma S. Noda T. Toyoda Y. Itakura H. Yazaki Y. Kodama T. et al.Nature. 1997; 386: 292-296Crossref PubMed Scopus (999) Google Scholar, 9Moore K.J. Freeman M.W. Arterioscler. Thromb. Vasc. Biol. 2006; 26: 1702-1711Crossref PubMed Scopus (413) Google Scholar, 31Moore K.J. Kunjathoor V.V. Koehn S.L. Manning J.J. Tseng A.A. Silver J.M. McKee M. Freeman M.W. J. Clin. Invest. 2005; 115: 2192-2201Crossref PubMed Scopus (316) Google Scholar). Foam cell formation is believed to represent one of the major events that activate the proinflammatory phenotype of lesional macrophages. SR-A has been found to initiate signaling cascades regulating not only lipid metabolism but also macrophage activation and inflammatory programs that may influence the development and stability of the atherosclerotic plaque. More importantly, SR-A has roles in the induction of apoptosis that may differentially impact early versus later more complex lesions (25Devries-Seimon T. Li Y. Yao P.M. Stone E. Wang Y. Davis R.J. Flavell R. Tabas I. J. Cell Biol. 2005; 171: 61-73Crossref PubMed Scopus (285) Google Scholar, 32Tabas I. Arterioscler. Thromb. Vasc. Biol. 2005; 25: 2255-2264Crossref PubMed Scopus (534) Google Scholar). The molecular background underlying these multiple functions of SR-A is its unique ability to bind with broad array of ligands. As a member of the group of pattern recognition receptors that mediate the innate immune host response, SR-A can bind and “scavenge” modified forms of LDL, apoptotic cells, anionic phospholipids, β-amyloid peptide, and advanced glycation end products, as well as pathogens and pathogen-associated molecules (8Murphy J.E. Tedbury P.R. Homer-Vanniasinkam S. Walker J.H. Ponnambalam S. Atherosclerosis. 2005; 182: 1-15Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar, 10Bowdish D.M. Gordon S. Immunol. Rev. 2009; 227: 19-31Crossref PubMed Scopus (96) Google Scholar). Here, we demonstrated that SR-A can endocytose various ligands via different pathways. Multiple endocytic pathways are utilized by many other receptors. For example, bone morphogenic protein receptor, epidermal growth factor receptor, and platelet-derived growth factor receptor have been shown to exert distinct functions by choosing different endocytic pathways (21Hartung A. Bitton-Worms K. Rechtman M.M. Wenzel V. Boergermann J.H. Hassel S. Henis Y.I. Knaus P. Mol. Cell. Biol. 2006; 26: 7791-7805Crossref PubMed Scopus (198) Google Scholar, 33De Donatis A. Comito G. Buricchi F. Vinci M.C. Parenti A. Caselli A. Camici G. Manao G. Ramponi G. Cirri P. J. Biol. Chem. 2008; 283: 19948-19956Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). We found that the distinct endocytic pathways of SR-A are independent on the concentration of ligand. Presence of ligand fucoidan did not influence SR-A choosing its endocytic routes. This is different from the epidermal growth factor receptor, which enters into the cell via different routes based on the dose of ligand (24Sigismund S. Woelk T. Puri C. Maspero E. Tacchetti C. Transidico P. Di Fiore P.P. Polo S. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 2760-2765Crossref PubMed Scopus (653) Google Scholar, 34Chen H. De Camilli P. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 2766-2771Crossref PubMed Scopus (122) Google Scholar).Endocytosis is originally regarded as a mechanism to terminate signaling through receptor internalization and subsequent lysosomal degradation. But it is now widely accepted as a fundamental organizer of cell signaling events (2Polo S. Di Fiore P.P. Cell. 2006; 124: 897-900Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 35Maxfield F.R. McGraw T.E. Nat. Rev. Mol. Cell. Biol. 2004; 5: 121-132Crossref PubMed Scopus (1463) Google Scholar, 36Miaczynska M. Stenmark H. J. Cell Biol. 2008; 180: 7-11Crossref PubMed Scopus (58) Google Scholar). As we showed different signaling responses to SR-A ligands at 4 and 37 °C, signaling persists throughout the endocytic route of SR-A. Thus, endocytic signaling is not merely a “passive” extension of binding to the ligands by SR-A at the plasma membrane but a tool to achieve signal diversification and specificity (37Mills I.G. Semin Cell Dev. Biol. 2007; 18: 459-470Crossref PubMed Scopus (38) Google Scholar, 38Kholodenko B.N. Trends Cell Biol. 2002; 12: 173-177Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 39Kholodenko B.N. Nat. Rev. Mol. Cell. Biol. 2006; 7: 165-176Crossref PubMed Scopus (1003) Google Scholar). This may constitute the molecular mechanisms for the multiple functions of SR-A.Macrophage apoptosis occurs at all stages of atherosclerosis. In early lesions, it is mildly dangerous because apoptotic cells are efficiently cleared by neighboring macrophage. In advanced atherosclerotic lesions, macrophage cell death leads to necrotic core formation and plaque destabilization (32Tabas I. Arterioscler. Thromb. Vasc. Biol. 2005; 25: 2255-2264Crossref PubMed Scopus (534) Google Scholar, 40Tabas I. Cell Death Differ. 2004; 11: S12-S16Crossref PubMed Scopus (142) Google Scholar, 41Tabas I. Nat. Rev. Immunol. 2010; 10: 36-46Crossref PubMed Scopus (811) Google Scholar). SR-A plays an important role in both the induction of macrophage apoptosis and the clearance of these dying cells. Under conditions of hypercholesterolemia macrophages in the vessel wall are overloaded by modified lipoproteins, leading to a toxic accumulation of free cholesterol in the cell that result in endoplasmic reticular stress. Subsequently, engagement of SR-A pathways by modified lipoproteins or fucoidan triggers apoptotic cell death, indicating that SR-A signaling contributes to macrophage death and necrotic core formation (25Devries-Seimon T. Li Y. Yao P.M. Stone E. Wang Y. Davis R.J. Flavell R. Tabas I. J. Cell Biol. 2005; 171: 61-73Crossref PubMed Scopus (285) Google Scholar, 32Tabas I. Arterioscler. Thromb. Vasc. Biol. 2005; 25: 2255-2264Crossref PubMed Scopus (534) Google Scholar). Caveolin-1 is believed to play a role in macrophage apoptosis (42Gargalovic P. Dory L. J. Lipid Res. 2003; 44: 1622-1632Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). We showed that the SR-A-engaged apoptosis is primarily mediated by the caveolae-dependent endocytosis in macrophages. Furthermore, Frank et al. (43Frank P.G. Lee H. Park D.S. Tandon N.N. Scherer P.E. Lisanti M.P. Arterioscler. Thromb. Vasc. Biol. 2004; 24: 98-105Crossref PubMed Scopus (182) Google Scholar) demonstrated that the loss of caveolin-1 gene expression is protective against the development of aortic atheromas, with up to an 70% reduction in the atherosclerotic lesion area. It is possible that blockade of caveolae-mediated endocytic pathway may diminish macrophage apoptosis and thus inhibit atherosclerosis lesion development.In the present study, we further showed that fucoidan/thapsigargin-induced macrophage apoptosis is dependent on activation of p38 kinase and JNK. The proapoptosis properties of p38 kinase and JNK have also been reported in some other apoptosis models (44Deng Y. Ren X. Yang L. Lin Y. Wu X. Cell. 2003; 115: 61-70Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar, 45Porras A. Zuluaga S. Black E. Valladares A. Alvarez A.M. Ambrosino C. Benito M. Nebreda A.R. Mol. Biol. Cell. 2004; 15: PubMed Scopus Google Scholar, Lin Wu J.J. Lin J. Biol. Chem. 2009; Full Text Full Text PDF PubMed Scopus (17) Google Scholar). In is shown to be required for SR-A-mediated foam cell formation and R. G. I. I. M. A. M. F.R. J. Chen M. K.J. Freeman M.W. 2004; PubMed Scopus Google Scholar). as a many signaling one plasma membrane domain including and T. A. Scherer P.E. Lisanti M.P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of by caveolin-1 the p38 kinase pathway R. A.M. J. Cell. Mol. Biol. 2006; PubMed Scopus Google caveolin-1 and may to apoptosis in endoplasmic demonstrated that SR-A/ligand internalization occurs through two endocytosis clathrin- and caveolae-dependent pathways. of the ligands not to influence of the route by SR-A. of modified by SR-A primarily go through the apoptosis requires endocytosis through the caveola which is to p38 kinase and JNK activation SR-A may exert multiple functions by choosing distinct endocytic routes and the signaling in macrophages. IntroductionEndocytosis is characterized by the internalization of molecules from the cell surface into internal membrane compartments. It is important for diverse cellular signaling events. Two main pathways have been identified for receptor-mediated endocytosis: the clathrin-dependent and the caveolae/lipid raft-dependent endocytic pathways (1Le Roy C. Wrana J.L. Nat. Rev. Mol. Cell Biol. 2005; 6: 112-126Crossref PubMed Scopus (697) Google Scholar, 2Polo S. Di Fiore P.P. Cell. 2006; 124: 897-900Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). Clathrin-dependent endocytosis is the most well characterized mechanism for mediating the internalization of membrane receptors into cells. It is also important for intracellular trafficking at the trans-Golgi network and endosomes (3Young A. Semin. Cell Dev. Biol. 2007; 18: 448-458Crossref PubMed Scopus (47) Google Scholar). Caveolae/raft-dependent endocytosis is involved in multiple biological processes, including mediating virus entry into host cells, internalizing glycophosphatidylinositol-anchored proteins and regulating certain signaling cascades. Caveola are cholesterol and sphingolipid-rich plasma membrane invaginations of a diameter of 60–80 nm, of which caveolin-1 is the main protein component required for caveola biogenesis (4Simons K. Toomre D. Nat. Rev. Mol. Cell Biol. 2000; 1: 31-39Crossref PubMed Scopus (5111) Google Scholar). Trafficking through these two endocytic pathways affects the amplitude and the on/off status of many signaling pathways. Thus, these endocytic processes contribute to the regulations of cell migration, cell cycle, cell polarity, apoptosis, and gene transcription (1Le Roy C. Wrana J.L. Nat. Rev. Mol. Cell Biol. 2005; 6: 112-126Crossref PubMed Scopus (697) Google Scholar, 4Simons K. Toomre D. Nat. Rev. Mol. Cell Biol. 2000; 1: 31-39Crossref PubMed Scopus (5111) Google Scholar, 5Nesterov A. Carter R.E. Sorkina T. Gill G.N. Sorkin A. EMBO J. 1999; 18: 2489-2499Crossref PubMed Scopus (184) Google Scholar).SR-A (class A macrophage scavenger receptor) is a transmembrane receptor expressed mainly in macrophages (6Freeman M. Ashkenas J. Rees D.J. Kingsley D.M. Copeland N.G. Jenkins N.A. Krieger M. Proc. Natl. Acad. Sci. U.S.A. 1990; 87: 8810-8814Crossref PubMed Scopus (257) Google Scholar, 7Suzuki H. Kurihara Y. Takeya M. Kamada N. Kataoka M. Jishage K. Ueda O. Sakaguchi H. Higashi T. Suzuki T. Takashima Y. Kawabe Y. Cynshi O. Wada Y. Honda M. Kurihara H. Aburatani H. Doi T. Matsumoto A. Azuma S. Noda T. Toyoda Y. Itakura H. Yazaki Y. Kodama T. et al.Nature. 1997; 386: 292-296Crossref PubMed Scopus (999) Google Scholar). SR-A can bind with an unusually broad range of polyanionic ligands, which includes modified lipoproteins, LPS of Gram-negative bacteria, and advanced glycation end products. Its broad specificity in binding with the ligands supports the multiple functions of SR-A in macrophage growth, adhesion to the substratum, cell-cell interactions, phagocytosis, and host defense (8Murphy J.E. Tedbury P.R. Homer-Vanniasinkam S. Walker J.H. Ponnambalam S. Atherosclerosis. 2005; 182: 1-15Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar, 9Moore K.J. Freeman M.W. Arterioscler. Thromb. Vasc. Biol. 2006; 26: 1702-1711Crossref PubMed Scopus (413) Google Scholar, 10Bowdish D.M. Gordon S. Immunol. Rev. 2009; 227: 19-31Crossref PubMed Scopus (96) Google Scholar). However, the molecular mechanisms enabling SR-A to exert multiple functions are still not well understood. It is reported that acetylated low density lipoprotein (acLDL), 2The abbreviations used are: acLDLacetylated low density lipoproteinDiI1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchloratePIpropidium iodide. a specific ligand of SR-A (17Goldstein J.L. Ho Y.K. Basu S.K. Brown M.S. Proc. Natl. Acad. Sci. U.S.A. 1979; 76: 333-337Crossref PubMed Scopus (1925) Google Scholar), is internalized via coated pit-mediated endocytosis (14Chen Y. Wang X. Ben J. Yue S. Bai H. Guan X. Bai X. Jiang L. Ji Y. Fan L. Chen Q. Arterioscler. Thromb. Vasc. Biol. 2006; 26: 1317-1322Crossref PubMed Scopus (30) Google Scholar, 18Mommaas-Kienhuis A.M. van der Schroeff J.G. Wijsman M.C. Daems W.T. Vermeer B.J. Histochemistry. 1985; 83: 29-35Crossref PubMed Scopus (17) Google Scholar, 19Jones N.L. Reagan J.W. Willingham M.C. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 773-781Crossref PubMed Scopus (33) Google Scholar). Macropinocytosis may also contribute to the uptake of acLDL at a low level (19Jones N.L. Reagan J.W. Willingham M.C. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 773-781Crossref PubMed Scopus (33) Google Scholar). On the other hand, the VirB-dependent bacterial internalization induces localization of SR-A into the detergent-resistant membrane lipid rafts (20Kim S. Watarai M. Suzuki H. Makino S. Kodama T. Shirahata T. Microb. Pathog. 2004; 37: 11-19Crossref PubMed Scopus (82) Google Scholar), which are sterol- and sphingolipid-enriched, and caveolin-containing domains that compartmentalize cellular processes. We and others (11Fong L.G. Le D. J. Biol. Chem. 1999; 274: 36808-36816Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 12Morimoto K. Wada Y. Hinagata J. Imanishi T. Kodama T. Doi T. Biol. Pharm. Bull. 1999; 22: 1022-1026Crossref PubMed Scopus (23) Google Scholar, 13Kosswig N. Rice S. Daugherty A. Post S.R. J. Biol. Chem. 2003; 278: 34219-34225Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 14Chen Y. Wang X. Ben J. Yue S. Bai H. Guan X. Bai X. Jiang L. Ji Y. Fan L. Chen Q. Arterioscler. Thromb. Vasc. Biol. 2006; 26: 1317-1322Crossref PubMed Scopus (30) Google Scholar) previously identified a few unique signal motives in the cytoplasmic domain of SR-A required for its internalization. We also showed that the internalization of SR-A and its ligand complex into cell is regulated by interaction of SR-A to its coupler (15Wang X. Zheng Y. Xu Y. Ben J. Gao S. Zhu X. Zhuang Y. Yue S. Bai H. Chen Y. Jiang L. Ji Y. Xu Y. Fan L. Sha J. He Z. Chen Q. Biochim. Biophys. Acta. 2009; 1791: 76-83Crossref PubMed Scopus (11) Google Scholar, 16Ben J. Gao S. Zhu X. Zheng Y. Zhuang Y. Bai H. Xu Y. Ji Y. Sha J. He Z. Chen Q. J. Mol. Cell. Cardiol. 2009; 47: 646-655Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). In this study, we want to address the question of whether different functions of SR-A are carried out by different endocytosis pathways. We show that SR-A can be internalized by both clathrin- and caveolae-dependent pathways. The clathrin-dependent SR-A endocytosis activates ERK signaling. The caveolae-dependent pathway is required for p38 kinase and JNK signaling as well as caspase activation. Our results indicate for the first time that SR-A-mediated macrophage apoptosis may be selectively regulated by the caveolae-dependent endocytosis.