Regulation of SR-BI-mediated selective lipid uptake in Chinese hamster ovary-derived cells by protein kinase signaling pathways
2006; Elsevier BV; Volume: 48; Issue: 2 Linguagem: Inglês
10.1194/jlr.m600326-jlr200
ISSN1539-7262
AutoresYi Zhang, Ayesha Ahmed, Nicole McFarlane, Christina Capone, Douglas R. Boreham, Ray Truant, Suleiman A. Igdoura, Bernardo L. Trigatti,
Tópico(s)Lipoproteins and Cardiovascular Health
ResumoScavenger receptor, class B, type I (SR-BI) mediates binding and internalization of a variety of lipoprotein and nonlipoprotein ligands, including HDL. Studies in genetically engineered mice revealed that SR-BI plays an important role in HDL reverse cholesterol transport and protection against atherosclerosis. Understanding how SR-BI's function is regulated may reveal new approaches to therapeutic intervention in atherosclerosis and heart disease. We utilized a model cell system to explore pathways involved in SR-BI-mediated lipid uptake from and signaling in response to distinct lipoprotein ligands: the physiological ligand, HDL, and a model ligand, acetyl LDL (AcLDL). In Chinese hamster ovary-derived cells, murine SR-BI (mSR-BI) mediates lipid uptake via distinct pathways that are dependent on the lipoprotein ligand. Furthermore, HDL and AcLDL activate distinct signaling pathways. Finally, mSR-BI-mediated selective lipid uptake versus endocytic uptake are differentially regulated by protein kinase signaling pathways. The protein kinase C (PKC) activator PMA and the phosphatidyl inositol 3-kinase inhibitor wortmannin increase the degree of mSR-BI-mediated selective lipid uptake, whereas a PKC inhibitor has the opposite effect. These data demonstrate that SR-BI's selective lipid uptake activity can be acutely regulated by intracellular signaling cascades, some of which can originate from HDL binding to murine SR-BI itself. Scavenger receptor, class B, type I (SR-BI) mediates binding and internalization of a variety of lipoprotein and nonlipoprotein ligands, including HDL. Studies in genetically engineered mice revealed that SR-BI plays an important role in HDL reverse cholesterol transport and protection against atherosclerosis. Understanding how SR-BI's function is regulated may reveal new approaches to therapeutic intervention in atherosclerosis and heart disease. We utilized a model cell system to explore pathways involved in SR-BI-mediated lipid uptake from and signaling in response to distinct lipoprotein ligands: the physiological ligand, HDL, and a model ligand, acetyl LDL (AcLDL). In Chinese hamster ovary-derived cells, murine SR-BI (mSR-BI) mediates lipid uptake via distinct pathways that are dependent on the lipoprotein ligand. Furthermore, HDL and AcLDL activate distinct signaling pathways. Finally, mSR-BI-mediated selective lipid uptake versus endocytic uptake are differentially regulated by protein kinase signaling pathways. The protein kinase C (PKC) activator PMA and the phosphatidyl inositol 3-kinase inhibitor wortmannin increase the degree of mSR-BI-mediated selective lipid uptake, whereas a PKC inhibitor has the opposite effect. These data demonstrate that SR-BI's selective lipid uptake activity can be acutely regulated by intracellular signaling cascades, some of which can originate from HDL binding to murine SR-BI itself. HDL plays an important role in protection against atherosclerosis, in part by mediating reverse cholesterol transport from macrophage foam cells in atherosclerotic plaque to the liver (1Lewis G.F. Rader D.J. New insights into the regulation of HDL metabolism and reverse cholesterol transport. Circ. Res. 2005; 96: 1221-1232Google Scholar). Elimination of scavenger receptor, class B, type I (SR-BI) expression in knock-out mice reduces hepatic HDL cholesterol clearance and biliary cholesterol secretion, and increases plasma levels of cholesterol associated with abnormally large, apolipoprotein E (apoE)-enriched HDL-like particles (as reviewed in Ref. 2Trigatti B. Kaur S.C. Role of the scavenger receptor class B type I in lipoprotein metabolism and atherosclerosis: insights from genetically altered mice. In Biochemistry of Atherosclerosis. Springer, New York2006: 53-69Google Scholar). Thus, in mice, SR-BI plays an important role, both in hepatic HDL-cholesterol clearance, and in driving reverse cholesterol transport. Inactivation of SR-BI expression renders mice more susceptible to atherosclerosis induced by high-fat, high-cholesterol diets or disruption of either apoE or LDL receptor expression (as reviewed in Ref. 2Trigatti B. Kaur S.C. Role of the scavenger receptor class B type I in lipoprotein metabolism and atherosclerosis: insights from genetically altered mice. In Biochemistry of Atherosclerosis. Springer, New York2006: 53-69Google Scholar). Mice deficient in both SR-BI and apoE develop severe occlusive coronary artery atherosclerosis and myocardial infarction and exhibit cardiac functional and conductance abnormalities prior to early death by 8 weeks of age (3Braun A. Trigatti B.L. Post M.J. Sato K. Simons M. Edelberg J.M. Rosenberg R.D. Schrenzel M. Krieger M. Loss of SR-BI expression leads to the early onset of occlusive atherosclerotic coronary artery disease, spontaneous myocardial infarctions, severe cardiac dysfunction, and premature death in apolipoprotein E-deficient mice. Circ. Res. 2002; 90: 270-276Google Scholar). Hepatic SR-BI overexpression increases clearance of plasma lipoprotein cholesterol and biliary cholesterol secretion and decreases atherosclerosis (although decreased HDL cholesterol levels at high levels of overexpression may diminish atheroprotection) (as reviewed in Ref. 2Trigatti B. Kaur S.C. Role of the scavenger receptor class B type I in lipoprotein metabolism and atherosclerosis: insights from genetically altered mice. In Biochemistry of Atherosclerosis. Springer, New York2006: 53-69Google Scholar). Elimination of SR-BI in bone marrow-derived cells, including macrophages, also results in increased atherosclerosis but without altering plasma lipoprotein cholesterol levels (4Covey S.D. Krieger M. Wang W. Penman M. Trigatti B.L. Scavenger receptor class B type I-mediated protection against atherosclerosis in LDL receptor-negative mice involves its expression in bone marrow-derived cells. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 1589-1594Google Scholar, 5Zhang W. Yancey P.G. Su Y.R. Babaev V.R. Zhang Y. Fazio S. Linton M.F. Inactivation of macrophage scavenger receptor class B type I promotes atherosclerotic lesion development in apolipoprotein E-deficient mice. Circulation. 2003; 108: 2258-2263Google Scholar, 6Van Eck M. Bos I.S. Hildebrand R.B. Van Rij B.T. Van Berkel T.J. Dual role for scavenger receptor class B, type I on bone marrow-derived cells in atherosclerotic lesion development. Am. J. Pathol. 2004; 165: 785-794Google Scholar). Thus, both hepatic and macrophage SR-BI protect against atherosclerosis, and reverse cholesterol transport may be only one of multiple pathways involved. SR-BI is a multiligand receptor that can bind to a variety of diverse ligands, including native lipoproteins (HDL, LDL, VLDL, and chylomicrons) and modified lipoproteins (acetylated LDL, oxidized LDL, oxidized HDL, and acute-phase HDL containing serum amyloid α instead of apo A-I) (as reviewed in Ref. 2Trigatti B. Kaur S.C. Role of the scavenger receptor class B type I in lipoprotein metabolism and atherosclerosis: insights from genetically altered mice. In Biochemistry of Atherosclerosis. Springer, New York2006: 53-69Google Scholar). Mutational analyses have suggested that different amino acid residues of SR-BI are involved in its binding to distinct ligands (7Gu X. Kozarsky K. Krieger M. Scavenger receptor class B, type I-mediated [3H]cholesterol efflux to high and low density lipoproteins is dependent on lipoprotein binding to the receptor. J. Biol. Chem. 2000; 275: 29993-30001Google Scholar, 8Gu X. Lawrence R. Krieger M. Dissociation of the high density lipoprotein and low density lipoprotein binding activities of murine scavenger receptor class B type I (mSR-BI) using retrovirus library-based activity dissection. J. Biol. Chem. 2000; 275: 9120-9130Google Scholar, 9Connelly M.A. De La Llera-Moya M. Peng Y. Drazul-Schrader D. Rothblat G.H. Williams D.L. Separation of lipid transport functions by mutations in the extracellular domain of scavenger receptor class B, type I. J. Biol. Chem. 2003; 278: 25773-25782Google Scholar). SR-BI mediates the bidirectional exchange of lipids between cells and bound lipoproteins, with the direction of net transfer (cellular uptake versus efflux) determined by the lipid concentration gradient (as reviewed in Ref. 10Yancey P.G. Bortnick A.E. Kellner-Weibel G. de la Llera-Moya M. Phillips M.C. Rothblat G.H. Importance of different pathways of cellular cholesterol efflux. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 712-719Crossref PubMed Scopus (436) Google Scholar). Lipoprotein binding and lipid transfer appear to be separable activities of SR-BI, because small molecules (e.g., BLT-1 and related compounds) can block SR-BI-mediated lipid transfer without blocking lipoprotein binding (11Nieland T.J. Penman M. Dori L. Krieger M. Kirchhausen T. Discovery of chemical inhibitors of the selective transfer of lipids mediated by the HDL receptor SR-BI. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 15422-15427Google Scholar). SR-BI is heavily glycosylated, fatty acylated, and localized to lipid rafts and/or caveolae in a variety of cell types (as reviewed in Ref. 2Trigatti B. Kaur S.C. Role of the scavenger receptor class B type I in lipoprotein metabolism and atherosclerosis: insights from genetically altered mice. In Biochemistry of Atherosclerosis. Springer, New York2006: 53-69Google Scholar). It undergoes internalization and recycling back to the cell surface. In polarized cells, cholesterol depletion induces SR-BI redistribution from the basal to apical membrane surface in a protein kinase A-dependent manner (12Burgos P.V. Klattenhoff C de la Fuente E. Rigotti A. Gonzalez A. Cholesterol depletion induces PKA-mediated basolateral-to-apical transcytosis of the scavenger receptor class B type I in MDCK cells. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 3845-3850Google Scholar). In differentiating 3T3-L1 adipocytes and HepG2 human hepatoma cells, recruitment of SR-BI to the cell surface from internal sites is induced by insulin and/or serum and dependent on the phosphatidyl inositol 3-kinase (PI3K)/Akt (protein kinase B) signaling pathway (13Tondu A.L. Robichon C Yvan-Charvet L. Donne N. Le Liepvre X. Hajduch E. Ferre P. Dugail I. Dagher G. Insulin and angiotensin II induce the translocation of scavenger receptor class B, type I from intracellular sites to the plasma membrane of adipocytes. J. Biol. Chem. 2005; 280: 33536-33540Google Scholar, 14Shetty S. Eckhardt E.R. Post S.R. van der Westhuyzen D.R. Phosphatidylinositol-3-kinase regulates scavenger receptor class B type I subcellular localization and selective lipid uptake in hepatocytes. Arterioscler. Thromb. Vasc. Biol. 2006; 26: 2125-2131Google Scholar). Murine scavenger receptor, class B, type I (mSR-BI)-mediated HDL lipid uptake does not, however, appear to require endocytosis, because purified, reconstituted mSR-BI is active (15Liu B. Krieger M. Highly purified scavenger receptor class B, type I reconstituted into phosphatidylcholine/cholesterol liposomes mediates high affinity high density lipoprotein binding and selective lipid uptake. J. Biol. Chem. 2002; 277: 34125-34135Google Scholar) and mSR-BI-mediated HDL lipid uptake in cells is not affected by inhibition of endocytosis (16Nieland T.J. Ehrlich M. Krieger M. Kirchhausen T. Endocytosis is not required for the selective lipid uptake mediated by murine SR-BI. Biochim. Biophys. Acta. 2005; 1734: 44-51Crossref PubMed Scopus (58) Google Scholar, 17Harder C.J. Vassiliou G. McBride H.M. McPherson R. Hepatic SR-BI-mediated cholesteryl ester selective uptake occurs with unaltered efficiency in the absence of cellular energy. J. Lipid Res. 2006; 47: 492-503Google Scholar). Similarly, SR-BI-mediated sterol uptake by hepatocytes and trafficking to the bile canalicular (apical) space does not appear to be energy dependent (17Harder C.J. Vassiliou G. McBride H.M. McPherson R. Hepatic SR-BI-mediated cholesteryl ester selective uptake occurs with unaltered efficiency in the absence of cellular energy. J. Lipid Res. 2006; 47: 492-503Google Scholar, 18Wustner D. Mondal M Huang A. Maxfield F.R. Different transport routes for high density lipoprotein and its associated free sterol in polarized hepatic cells. J. Lipid Res. 2004; 45: 427-437Google Scholar). Furthermore, the low selective uptake activities of SR-BII, a splice variant of SR-BI with a different carboxy-terminal cytoplasmic tail, and of a mutant form of SR-BI with an endocytic sequence inserted into its carboxy-terminal tail appear to be due to their increased endocytosis (19Eckhardt E.R. Cai L. Sun B. Webb N.R. van der Westhuyzen D.R. High density lipoprotein uptake by scavenger receptor SR-BII. J. Biol. Chem. 2004; 279: 14372-14381Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 20Eckhardt E.R. Cai L. Shetty S. Zhao Z. Szanto A. Webb N.R. Van der Westhuyzen D.R. High density lipoprotein endocytosis by scavenger receptor SR-BII is clathrin-dependent and requires a carboxyl-terminal dileucine motif. J. Biol. Chem. 2006; 281: 4348-4353Google Scholar, 21Sun B. Eckhardt E.R. Shetty S. van der Westhuyzen D.R. Webb N.R. Quantitative analysis of SR-BI-dependent HDL retroendocytosis in hepatocytes and fibroblasts. J. Lipid Res. 2006; 47: 1700-1713Google Scholar). On the other hand, mSR-BI does mediate the endocytosis and intracellular accumulation of a variety of other ligands, including serum amyloid α, lipopolysaccharide, and apoptotic cells (22Cai L. de Beer M.C. de Beer F.C. van der Westhuyzen D.R. Serum amyloid A is a ligand for scavenger receptor class B type I and inhibits high density lipoprotein binding and selective lipid uptake. J. Biol. Chem. 2005; 280: 2954-2961Google Scholar, 23Baranova I.N. Vishnyakova T.G. Bocharov A.V. Kurlander R. Chen Z. Kimelman M.L. Remaley A.T. Csako G. Thomas F. Eggerman T.L. Serum amyloid A binding to CLA-1 (CD36 and LIMPII analogous-1) mediates serum amyloid A protein-induced activation of ERK1/2 and p38 mitogen-activated protein kinases. J. Biol. Chem. 2005; 280 (et al.): 8031-8040Google Scholar, 24Vishnyakova T.G. Bocharov A.V. Baranova I.N. Chen Z. Remaley A.T. Csako G. Eggerman T.L. Patterson A.P. Binding and internalization of lipopolysaccharide by Cla-1, a human orthologue of rodent scavenger receptor B1. J. Biol. Chem. 2003; 278: 22771-22780Google Scholar, 25Kawasaki Y. Nakagawa A. Nagaosa K. Shiratsuchi A. Nakanishi Y. Phosphatidylserine binding of class B scavenger receptor type I, a phagocytosis receptor of testicular sertoli cells. J. Biol. Chem. 2002; 277: 27559-27566Google Scholar, 26Osada Y. Shiratsuchi A. Nakanishi Y. Involvement of mitogen-activated protein kinases in class B scavenger receptor type I-induced phagocytosis of apoptotic cells. Exp. Cell Res. 2006; 312: 1820-1830Google Scholar). SR-BI-mediated free cholesterol efflux from cells also appears to be dependent on the transient internalization and resecretion of HDL (27Pagler T.A. Rhode S. Neuhofer A. Laggner H. Strobl W. Hinterndorfer C. Volf I. Pavelka M. Eckhardt E.R. van der Westhuyzen D.R. SR-BI-mediated high density lipoprotein (HDL) endocytosis leads to HDL resecretion facilitating cholesterol efflux. J. Biol. Chem. 2006; 281 (et al.): 11193-11204Google Scholar). HDL binding to SR-BI leads to activation of diverse signaling pathways. These include ras-dependent activation of mitogen-activated protein kinases (MAPKs) (28Grewal T. de Diego I. Kirchhoff M.F. Tebar F. Heeren J. Rinninger F. Enrich C. High density lipoprotein-induced signaling of the MAPK pathway involves scavenger receptor type BI-mediated activation of Ras. J. Biol. Chem. 2003; 278: 16478-16481Google Scholar, 29Rentero C. Evans R. Wood P. Tebar F. Vila de Muga S. Cubells L. de Diego I. Hayes T.E. Hughes W.E. Pol A. Inhibition of H-Ras and MAPK is compensated by PKC-dependent pathways in annexin A6 expressing cells. Cell. Signal. 2006; 18 (et al.): 1006-1016Google Scholar), activation of the PI3K/protein kinase B (Akt) pathway (30Li X.A. Titlow W.B. Jackson B.A. Giltiay N. Nikolova-Karakashian M. Uittenbogaard A. Smart E.J. High density lipoprotein binding to scavenger receptor, Class B, type I activates endothelial nitric-oxide synthase in a ceramide-dependent manner. J. Biol. Chem. 2002; 277: 11058-11063Google Scholar, 31Mineo C. Shaul P.W. HDL stimulation of endothelial nitric oxide synthase: a novel mechanism of HDL action. Trends Cardiovasc. Med. 2003; 13: 226-231Google Scholar, 32Mineo C. Yuhanna I.S. Quon M.J. Shaul P.W. High density lipoprotein-induced endothelial nitric-oxide synthase activation is mediated by Akt and MAP kinases. J. Biol. Chem. 2003; 278: 9142-9149Google Scholar, 33Cao W.M. Murao K. Imachi H. Yu X. Abe H. Yamauchi A. Niimi M. Miyauchi A. Wong N.C. Ishida T. A mutant high-density lipoprotein receptor inhibits proliferation of human breast cancer cells. Cancer Res. 2004; 64: 1515-1521Google Scholar), and activation of protein kinase C (PKC) (28Grewal T. de Diego I. Kirchhoff M.F. Tebar F. Heeren J. Rinninger F. Enrich C. High density lipoprotein-induced signaling of the MAPK pathway involves scavenger receptor type BI-mediated activation of Ras. J. Biol. Chem. 2003; 278: 16478-16481Google Scholar). HDL signaling through SR-BI mediates the activation of endothelial nitric oxide synthase in endothelial cells (30Li X.A. Titlow W.B. Jackson B.A. Giltiay N. Nikolova-Karakashian M. Uittenbogaard A. Smart E.J. High density lipoprotein binding to scavenger receptor, Class B, type I activates endothelial nitric-oxide synthase in a ceramide-dependent manner. J. Biol. Chem. 2002; 277: 11058-11063Google Scholar, 31Mineo C. Shaul P.W. HDL stimulation of endothelial nitric oxide synthase: a novel mechanism of HDL action. Trends Cardiovasc. Med. 2003; 13: 226-231Google Scholar, 32Mineo C. Yuhanna I.S. Quon M.J. Shaul P.W. High density lipoprotein-induced endothelial nitric-oxide synthase activation is mediated by Akt and MAP kinases. J. Biol. Chem. 2003; 278: 9142-9149Google Scholar, 34Gong M. Wilson M. Kelly T. Su W. Dressman J. Kincer J. Matveev S.V. Guo L. Guerin T. Li X.A. HDL-associated estradiol stimulates endothelial NO synthase and vasodilation in an SR-BI-dependent manner. J. Clin. Invest. 2003; 111 (et al.): 1579-1587Google Scholar, 35Yuhanna I.S. Zhu Y. Cox B.E. Hahner L.D. Osborne-Lawrence S. Lu P. Marcel Y.L. Anderson R.G. Mendelsohn M.E. Hobbs H.H. High-density lipoprotein binding to scavenger receptor-BI activates endothelial nitric oxide synthase. Nat. Med. 2001; 7 (et al.): 853-857Google Scholar), endothelial cell migration (36Seetharam D. Mineo C. Gormley A.K. Gibson L.L. Vongpatanasin W. Chambliss K.L. Hahner L.D. Cummings M.L. Kitchens R.L. Marcel Y.L. High-density lipoprotein promotes endothelial cell migration and reendothelialization via scavenger receptor-B type I. Circ. Res. 2006; 98 (et al.): 63-72Google Scholar), and the suppression of apoptosis in diverse cells (33Cao W.M. Murao K. Imachi H. Yu X. Abe H. Yamauchi A. Niimi M. Miyauchi A. Wong N.C. Ishida T. A mutant high-density lipoprotein receptor inhibits proliferation of human breast cancer cells. Cancer Res. 2004; 64: 1515-1521Google Scholar, 37Li X.A. Guo L. Dressman J.L. Asmis R. Smart E.J. A novel ligand-independent apoptotic pathway induced by scavenger receptor class B, type I and suppressed by endothelial nitric-oxide synthase and high density lipoprotein. J. Biol. Chem. 2005; 280: 19087-19096Google Scholar). The signaling events immediately following mSR-BI binding to its ligands, including HDL, remain unclear but may include transfer of signaling lipids (ceramides, sphingosine 1 phosphate, estradiol) and/or cholesterol either into or out of cells (30Li X.A. Titlow W.B. Jackson B.A. Giltiay N. Nikolova-Karakashian M. Uittenbogaard A. Smart E.J. High density lipoprotein binding to scavenger receptor, Class B, type I activates endothelial nitric-oxide synthase in a ceramide-dependent manner. J. Biol. Chem. 2002; 277: 11058-11063Google Scholar, 34Gong M. Wilson M. Kelly T. Su W. Dressman J. Kincer J. Matveev S.V. Guo L. Guerin T. Li X.A. HDL-associated estradiol stimulates endothelial NO synthase and vasodilation in an SR-BI-dependent manner. J. Clin. Invest. 2003; 111 (et al.): 1579-1587Google Scholar, 38Kimura T. Sato K. Malchinkhuu E. Tomura H. Tamama K. Kuwabara A. Murakami M. Okajima F. High-density lipoprotein stimulates endothelial cell migration and survival through sphingosine 1-phosphate and its receptors. Arterioscler. Thromb. Vasc. Biol. 2003; 23: 1283-1288Google Scholar, 39Assanasen C. Mineo C. Seetharam D. Yuhanna I.S. Marcel Y.L. Connelly M.A. Williams D.L. de la Llera-Moya M. Shaul P.W. Silver D.L. Cholesterol binding, efflux, and a PDZ-interacting domain of scavenger receptor-BI mediate HDL-initiated signaling. J. Clin. Invest. 2005; 115: 969-977Google Scholar). Alternatively, signaling may require the C-terminal cytoplasmic region of SR-BI and may involve the binding of adaptor proteins (31Mineo C. Shaul P.W. HDL stimulation of endothelial nitric oxide synthase: a novel mechanism of HDL action. Trends Cardiovasc. Med. 2003; 13: 226-231Google Scholar, 32Mineo C. Yuhanna I.S. Quon M.J. Shaul P.W. High density lipoprotein-induced endothelial nitric-oxide synthase activation is mediated by Akt and MAP kinases. J. Biol. Chem. 2003; 278: 9142-9149Google Scholar), possibly including PDZK1 (39Assanasen C. Mineo C. Seetharam D. Yuhanna I.S. Marcel Y.L. Connelly M.A. Williams D.L. de la Llera-Moya M. Shaul P.W. Silver D.L. Cholesterol binding, efflux, and a PDZ-interacting domain of scavenger receptor-BI mediate HDL-initiated signaling. J. Clin. Invest. 2005; 115: 969-977Google Scholar, 40Yesilaltay A. Kocher O. Rigotti A. Krieger M. Regulation of SR-BI-mediated high-density lipoprotein metabolism by the tissue-specific adaptor protein PDZK1. Curr. Opin. Lipidol. 2005; 16: 147-152Google Scholar). Despite SR-BI's importance for HDL lipid uptake, reverse cholesterol transport, and protection against atherosclerosis in mice, little is known about how its activity is regulated at the posttranslational level. It has recently been demonstrated that SR-BI-dependent binding of phosphatidyl serine-containing liposomes and apoptotic cells leads to the activation of MAPK signaling cascades (p38 MAPK, extracellular signal-regulated kinase (ERK)1/2, and JNK), which feed back to regulate SR-BI-dependent engulfment (26Osada Y. Shiratsuchi A. Nakanishi Y. Involvement of mitogen-activated protein kinases in class B scavenger receptor type I-induced phagocytosis of apoptotic cells. Exp. Cell Res. 2006; 312: 1820-1830Google Scholar). In contrast, little is known about the effects of signaling pathways stimulated by SR-BI's lipoprotein ligands on SR-BI-mediated lipid uptake. Here we demonstrate in a model cell system that SR-BI-mediated lipid uptake from distinct lipoprotein ligands, acetyl LDL (AcLDL) and HDL, occurs via distinct endocytic and nonendocytic pathways. We also demonstrate that the physiological ligand, HDL, but not the model ligand, AcLDL, stimulates PKC activity in an SR-BI-dependent manner, whereas both ligands stimulate PI3K and MAPK pathways. HDL also stimulates the retention of SR-BI on the cell surface in a manner that is not dependent on PKC, PI3K, or ERK signaling. Finally, we demonstrate that PKC activation and PI3K inactivation lead to an increased ratio of uptake of lipids to uptake of protein components of lipoproteins, suggesting increased selective lipid uptake activity of SR-BI. These data support the idea that endocytosis may be a point at which SR-BI's activity is regulated and that PKC and PI3K may play important roles in that regulation. Antibodies were obtained from the following suppliers. Rabbit anti-SR-BI antibody (NB400-101) and anti-phospho-myristoylated alanine-rich C-kinase substrate (MARCKS) (NB500-140) were from Novus Biologicals, Inc. (distributed by Cedarlane Laboratories Ltd, Hornby, Ontario, Canada). Phospho- and total Akt, ERK1/2, and p38 MAPK (generously provided by Karen Mossman) were from Cell Signaling Technologies, Inc. (Danvers, MA). Mouse-anti-β-actin was from MP Biomedicals (Aurora, OH). Horseradish peroxidase-conjugated goat anti-rabbit antibody and streptavidin-agarose were from Amersham Biosciences (Piscataway, NJ). Horseradish peroxidase-conjugated donkey anti-mouse antibody was from Jackson Immunoresearch (distributed by BioCan Scientific, Inc., Etobicoke, Ontario). The anti-SR-BI blocking antiserum was generously provided by Karen Kozarsky. Western Lighting Enhanced Chemiluminescence assay reagent was from PerkinElmer Life and Analytical Sciences (Boston, MA). Alexa 488 cholera toxin subunit B (CTX-B), alexa 488 protein-labeling reagent, alexa 594-transferrin, and 1,1′-dioctadecyl-3,3,3′,3′- tetramethylindocarbocyanine perchlorate [DiI (C18)] were from Invitrogen/Molecular Probes (Eugene, OR). The Pierce Biotechnology, Inc. bicinchoninic acid protein assay reagent and EZ-Link sulfo-NHS-biotin were from MJS Biolinx (Brockville, Ontario, Canada). BLT-1 (ID 5234221) was from ChemBridge Corp. (San Diego, CA). RO31-8220 (3-[1-[3-(amidinothio)propyl]-1H-indol-3-yl]3-(1-methyl-1H-indol-3-yl)maleimide) and wortmannin were from EMD Biosciences (La Jolla, CA), and PMA, cytochalasin D and colchicine were from Sigma Chemical Co. (St. Louis, MO). HDL and LDL were prepared from human plasma by KBr density gradient ultracentrifugation and labeled with DiI as previously described (41Chapman M.J. Goldstein S Lagrange D. Laplaud P.M. A density gradient ultracentrifugal procedure for the isolation of the major lipoprotein classes from human serum. J. Lipid Res. 1981; 22: 339-358Google Scholar, 42Reynolds G.D. St Clair R.W. A comparative microscopic and biochemical study of the uptake of fluorescent and 125I-labeled lipoproteins by skin fibroblasts, smooth muscle cells, and peritoneal macrophages in culture. Am. J. Pathol. 1985; 121: 200-211Google Scholar, 43Acton S. Rigotti A. Landschulz K.T. Xu S. Hobbs H.H. Krieger M. Identification of scavenger receptor SR-BI as a high density lipoprotein receptor. Science. 1996; 271: 518-520Google Scholar). DiI-AcLDL and unlabeled AcLDL were prepared by acetylation of either DiI-LDL or unlabeled LDL (44Basu S.K. Goldstein J.L. Anderson G.W. Brown M.S. Degradation of cationized low density lipoprotein and regulation of cholesterol metabolism in homozygous familial hypercholesterolemia fibroblasts. Proc. Natl. Acad. Sci. U.S.A. 1976; 73: 3178-3182Google Scholar). DiI/alexa 488 double-labeled HDL was prepared by alexa 488 labeling of DiI-HDL. DiI/alexa 488 double-labeled AcLDL was prepared from DiI-LDL by alexa 488 labeling, followed by acetylation (44Basu S.K. Goldstein J.L. Anderson G.W. Brown M.S. Degradation of cationized low density lipoprotein and regulation of cholesterol metabolism in homozygous familial hypercholesterolemia fibroblasts. Proc. Natl. Acad. Sci. U.S.A. 1976; 73: 3178-3182Google Scholar). Labeled lipoproteins were dialyzed against 0.9% NaCl and 1 mM EDTA, sterilized by 0.2 μm filtration, and stored under N2 gas at 4°C. Newborn calf lipoprotein-deficient serum was prepared by KBr density gradient centrifugation of newborn calf serum (45Krieger M. Brown M.S. Goldstein J.L. Isolation of Chinese hamster cell mutants defective in the receptor-mediated endocytosis of low density lipoprotein. J. Mol. Biol. 1981; 150: 167-184Google Scholar). Maleyl-BSA was prepared as described by Goldstein et al. (46Goldstein J.L. Ho Y.K. Basu S.K. Brown M.S. Binding site on macrophages that mediates uptake and degradation of acetylated low density lipoprotein, producing massive cholesterol deposition. Proc. Natl. Acad. Sci. U.S.A. 1979; 76: 333-337Google Scholar). The following cell lines were kindly provided by Monty Krieger (Massachusetts Institute of Technology): The ldlA7 mutant of Chinese hamster ovary (CHO) cells, lacking functional LDL receptor (47Kozarsky K.F. Brush H.A. Krieger M. Unusual forms of low density lipoprotein receptors in hamster cell mutants with defects in the receptor structural gene. J. Cell Biol. 1986; 102: 1567-1575Google Scholar); CHO-K1; ldlA7 cells overexpressing mouse SR-BI (ldlA[mSR-BI] cells) (43Acton S. Rigotti A. Landschulz K.T. Xu S. Hobbs H.H. Krieger M. Identification of scavenger receptor SR-BI as a high density lipoprotein receptor. Science. 1996; 271: 518-520Google Scholar); and CHO cells overexpressing murine scavenger receptor class A, type I (CHO[mSR-AI] cells) (48Ashkenas J. Penman M. Vasile E. Acton S. Freeman M. Krieger M. Structures and high and low affinity ligand binding properties of murine type I and type II macrophage scavenger receptors. J. Lipid Res. 1993; 34: 983-1000Google Scholar). Cells were cultured in Ham's F12 medium containing 5% FBS (Hyclone), 2 mM l-glutamine, 50 U/ml penicillin, and 50 μg/ml streptomycin (medium A). For experiments, cells were seeded in medium B (same as medium A except that 5% FBS was replaced with 3% newborn calf lipoprotein-deficient serum). CHO[mSR-AI] and ldlA[mSR-BI] cells were enriched by fluorescence-activated cell sorting after incubation with DiI-AcLDL or DiI-HDL, respectively (see below). Cells, cultured for 2 days in medium B, were released from dishes by mild trypsinization, washed once in Ham's F12 medium, and suspended (2 × 106 cells/ml) in Ham's F12 medium without additions. Cells were incubated for 20 min at 37°C with rocking to recover. The cells were then treated as described for the indicated times in the absence or presence of 25 μM monensin. The cells were chilled on ice, and 1 ml of ice-cold PBS was added. Cells were washed twice in 2 ml and suspended in 0.5 ml of ice-cold PBS (pH 8.0) containing 2 mM sulfo-NHS-biotin and incubated for 60 min at 4°C with rocking. Biotinylation was quenched by addition of ice-cold PBS containing 100 mM glycine, and cells were pelleted by centrifugation for 5 min at 4,000 rpm in a Spectramax microcentrifuge. The cell pellets were washed twice with PBS with 100
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