title: Fluid-phase pinocytosis of native low density lipoprotein promotes murine M-CSF differentiated macrophage foam cell formation.
abstract: During atherosclerosis, low-density lipoprotein (LDL)-derived cholesterol accumulates in macrophages to form foam cells. Macrophage uptake of LDL promotes foam cell formation but the mechanism mediating this process is not clear. The present study investigates the mechanism of LDL uptake for macrophage colony-stimulating factor (M-CSF)-differentiated murine bone marrow-derived macrophages. LDL receptor-null (LDLR-/-) macrophages incubated with LDL showed non-saturable accumulation of cholesterol that did not down-regulate for the 24 h examined. Incubation of LDLR-/- macrophages with increasing concentrations of (125)I-LDL showed non-saturable macrophage LDL uptake. A 20-fold excess of unlabeled LDL had no effect on (125)I-LDL uptake by wild-type macrophages and genetic deletion of the macrophage scavenger receptors CD36 and SRA did not affect (125)I-LDL uptake, showing that LDL uptake occurred by fluid-phase pinocytosis independently of receptors. Cholesterol accumulation was inhibited approximately 50% in wild-type and LDLR-/- mice treated with LY294002 or wortmannin, inhibitors of all classes of phosphoinositide 3-kinases (PI3K). Time-lapse, phase-contrast microscopy showed that macropinocytosis, an important fluid-phase uptake pathway in macrophages, was blocked almost completely by PI3K inhibition with wortmannin. Pharmacological inhibition of the class I PI3K isoforms alpha, beta, gamma or delta did not affect macrophage LDL-derived cholesterol accumulation or macropinocytosis. Furthermore, macrophages from mice expressing kinase-dead class I PI3K beta, gamma or delta isoforms showed no decrease in cholesterol accumulation or macropinocytosis when compared with wild-type macrophages. Thus, non-class I PI3K isoforms mediated macropinocytosis in these macrophages. Further characterization of the components necessary for LDL uptake, cholesterol accumulation, and macropinocytosis identified dynamin, microtubules, actin, and vacuolar type H(+)-ATPase as contributing to uptake. However, Pak1, Rac1, and Src-family kinases, which mediate fluid-phase pinocytosis in certain other cell types, were unnecessary. In conclusion, our findings provide evidence that targeting those components mediating macrophage macropinocytosis with inhibitors may be an effective strategy to limit macrophage accumulation of LDL-derived cholesterol in arteries.
authors.affiliation: Section of Experimental Atherosclerosis, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Manoj K
authors.initials: MK
authors.lastname: Barthwal
authors.forename: Joshua J
authors.initials: JJ
authors.lastname: Anzinger
authors.forename: Qing
authors.initials: Q
authors.lastname: Xu
authors.forename: Thomas
authors.initials: T
authors.lastname: Bohnacker
authors.forename: Matthias P
authors.initials: MP
authors.lastname: Wymann
authors.forename: Howard S
authors.initials: HS
authors.lastname: Kruth
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects; D015638:Cytochalasin D / Q000494:pharmacology; D034281:Dynamins / Q000378:metabolism; D005487:Foam Cells / Q000187:drug effects* / Q000378:metabolism*; D006801:Humans; D008077:Lipoproteins, LDL / Q000378:metabolism*; D016173:Macrophage Colony-Stimulating Factor / Q000494:pharmacology*; D008264:Macrophages / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010873:Pinocytosis / Q000502:physiology*; D055503:Protein Multimerization / Q000187:drug effects; D011973:Receptors, LDL / Q000172:deficiency / Q000235:genetics
chemical_list: D000199:Actins; D008077:Lipoproteins, LDL; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011973:Receptors, LDL; D015638:Cytochalasin D; D016173:Macrophage Colony-Stimulating Factor; D034281:Dynamins
references.citation: J Biol Chem. 2006 Jun 9;281(23):15757-62
references.pmid: 16606620
references.citation: Methods Enzymol. 1983;98:241-60
references.pmid: 6321901
references.citation: J Clin Invest. 2005 Aug;115(8):2192-201
references.pmid: 16075060
references.citation: Nat Rev Mol Cell Biol. 2008 Aug;9(8):639-49
references.pmid: 18612320
references.citation: Science. 2000 Feb 11;287(5455):1049-53
references.pmid: 10669418
references.citation: J Lipid Res. 1978 Nov;19(8):1068-70
references.pmid: 731127
references.citation: EMBO J. 2008 Apr 9;27(7):970-81
references.pmid: 18354494
references.citation: Arterioscler Thromb Vasc Biol. 2000 Dec;20(12):2593-9
references.pmid: 11116058
references.citation: Mol Pharm. 2009 Jul-Aug;6(4):1170-9
references.pmid: 19492848
references.citation: Am J Pathol. 1992 Feb;140(2):301-16
references.pmid: 1739124
references.citation: Circulation. 1999 Apr 6;99(13):1740-6
references.pmid: 10190885
references.citation: Nat Cell Biol. 2009 May;11(5):510-20
references.pmid: 19404330
references.citation: Cell Signal. 2012 Sep;24(9):1753-61
references.pmid: 22575736
references.citation: J Cell Sci. 2009 Jun 1;122(Pt 11):1863-71
references.pmid: 19461075
references.citation: Curr Opin Lipidol. 2011 Oct;22(5):386-93
references.pmid: 21881499
references.citation: Proc Natl Acad Sci U S A. 1995 Aug 29;92(18):8264-8
references.pmid: 7667279
references.citation: Curr Drug Targets. 2007 Dec;8(12):1249-63
references.pmid: 18220702
references.citation: J Cell Sci. 1992 Aug;102 ( Pt 4):867-80
references.pmid: 1429898
references.citation: Mol Biol Cell. 2000 Oct;11(10):3341-52
references.pmid: 11029040
references.citation: J Biol Chem. 2002 Dec 20;277(51):49982-8
references.pmid: 12376530
references.citation: Trends Biochem Sci. 2005 Apr;30(4):194-204
references.pmid: 15817396
references.citation: J Biol Chem. 1957 May;226(1):497-509
references.pmid: 13428781
references.citation: Nat Rev Mol Cell Biol. 2010 May;11(5):329-41
references.pmid: 20379207
references.citation: J Biol Chem. 2005 Jan 21;280(3):2352-60
references.pmid: 15533943
references.citation: J Biol Chem. 2002 Sep 13;277(37):34573-80
references.pmid: 12118008
references.citation: Cell. 1992 Aug 7;70(3):401-10
references.pmid: 1643658
references.citation: Eur Heart J. 1990 Aug;11 Suppl E:72-81
references.pmid: 2226536
references.citation: J Biochem. 2002 Apr;131(4):495-501
references.pmid: 11926985
references.citation: J Cell Sci. 2003 Jan 15;116(Pt 2):247-57
references.pmid: 12482911
references.citation: J Cell Sci. 2009 Sep 15;122(Pt 18):3250-61
references.pmid: 19690049
references.citation: J Clin Invest. 2009 May;119(5):1373-81
references.pmid: 19363293
references.citation: Biochim Biophys Acta. 1983 Dec 20;754(3):249-57
references.pmid: 6197091
references.citation: J Lipid Res. 2012 Jan;53(1):34-42
references.pmid: 22058424
references.citation: Curr Drug Targets. 2011 Jun;12(7):1056-81
references.pmid: 21291386
references.citation: Curr Oncol Rep. 2010 Mar;12(2):87-94
references.pmid: 20425592
references.citation: Cell. 2004 Aug 6;118(3):375-87
references.pmid: 15294162
references.citation: Int J Med Microbiol. 2002 Feb;291(6-7):487-94
references.pmid: 11890548
references.citation: J Cell Biol. 1996 Dec;135(5):1249-60
references.pmid: 8947549
references.citation: J Clin Invest. 2000 Apr;105(8):1049-56
references.pmid: 10772649
references.citation: Arterioscler Thromb Vasc Biol. 1998 Apr;18(4):631-40
references.pmid: 9555870
references.citation: Trends Cell Biol. 1995 Nov;5(11):424-8
references.pmid: 14732047
references.citation: Traffic. 2006 May;7(5):589-603
references.pmid: 16643281
references.citation: Arterioscler Thromb Vasc Biol. 2009 Jan;29(1):19-26
references.pmid: 18948635
references.citation: J Lipid Res. 2010 Jul;51(7):1962-70
references.pmid: 20194110
references.citation: J Lipid Res. 2007 Nov;48(11):2411-8
references.pmid: 17693624
references.citation: J Cell Sci. 1996 Aug;109 ( Pt 8):2005-12
references.pmid: 8856496
references.citation: J Cell Sci. 2007 Dec 1;120(Pt 23):4167-77
references.pmid: 18003703
references.citation: Am J Pathol. 1997 May;150(5):1687-99
references.pmid: 9137093
references.citation: J Clin Invest. 1998 Jun 15;101(12):2702-10
references.pmid: 9637704
references.citation: Arterioscler Thromb Vasc Biol. 2010 Oct;30(10):2022-31
references.pmid: 20634472
references.citation: J Biol Chem. 1951 Nov;193(1):265-75
references.pmid: 14907713
references.citation: J Cell Physiol. 2007 Apr;211(1):220-32
references.pmid: 17167779
references.citation: Biochem Biophys Res Commun. 1978 Dec 29;85(4):1424-30
references.pmid: 217387