Myosin VI (Myo6) functions in endocytosis in conjunction with binding partners including adaptor protein (AP)-2, disabled 2 (Dab2), and GAIP interacting protein C terminus 1 (GIPC1). This study aimed to investigate the expression and function of Myo6 in macrophages and its possible role in the endocytosis of lipoproteins during the induction of foam cell formation. Expression of Myo6, AP-2 (α2 subunit), and Dab2 in THP-1 macrophages and primary human monocyte-derived macrophages was demonstrated at the mRNA and protein level, but GIPC1 was only detected at the mRNA level. Immunofluorescence showed that Myo6 was distributed similarly to F-actin in both macrophage types. AP-2α2 was found to have a similar subcellular distribution to Myo6 and Dab2 in THP-1 cells. Myo6 was located within membrane ruffles and protrusions of the plasma membrane. These results suggest that in macrophages Myo6 is required for several functions including cell adhesion, cell progression, and macropinocytosis. Low-density lipoprotein (LDL) and oxidised LDL (oxLDL) decreased Myo6 and GIPC1 mRNA expression in THP-1 cells, but uptake of the fluorescence-labelled lipoproteins was unaffected by knockdown of the expression of Myo6 or associated proteins with siRNA. Our findings, therefore, do not support the idea that Myo6 plays a major role in foam cell formation. 1. Introduction The uptake of large amounts of lipids from plasma lipoproteins via endocytosis by macrophages in the artery wall to form lipid-engorged foam cells is an important initiating event in the development of atherosclerosis, a major cause of heart disease. The main lipoprotein implicated is low-density lipoprotein (LDL), but oxidative modification of the particles, a process which can occur within the vessel wall, is required before foam cell formation is induced [1]. The myosin superfamily is a group of actin-associated motor proteins that use ATP hydrolysis to generate force or directional movement along actin filaments [2, 3]. Many new classes of myosin have been identified since the discovery, over seventy years ago, of myosin II (“conventional” myosin), which produces contractile force in muscle cells; to date, 35 classes of myosin proteins have been characterised in mammalian cells [4]. It is now known that these various classes of myosin motor are involved in a diverse range of intracellular processes, ranging from cell migration and division to cell anchorage and transport of cargo proteins along actin filaments [3]. Myosin VI (Myo6), a member of the “unconventional” myosin classes, is an unusual member of
References
[1]
R. Albertini, R. Moratti, and G. De Luca, “Oxidation of low-density lipoprotein in atherosclerosis from basic biochemistry to clinical studies,” Current Molecular Medicine, vol. 2, no. 6, pp. 579–592, 2002.
[2]
M. A. Hartman, D. Finan, S. Sivaramakrishnan, and J. A. Spudich, “Principles of unconventional Myosin function and targeting,” Annual Review of Cell and Developmental Biology, vol. 27, pp. 133–155, 2011.
[3]
J. R. Sellers, “Myosins: a diverse superfamily,” Biochimica et Biophysica Acta, vol. 1496, no. 1, pp. 3–22, 2000.
[4]
F. Odronitz and M. Kollmar, “Drawing the tree of eukaryotic life based on the analysis of 2,269 manually annotated myosins from 328 species,” Genome Biology, vol. 8, no. 9, article R196, 2007.
[5]
A. L. Wells, A. W. Lin, L. Q. Chen et al., “Myosin VI is an actin-based motor that moves backwards,” Nature, vol. 401, no. 6752, pp. 505–508, 1999.
[6]
F. Buss, S. D. Arden, M. Lindsay, J. P. Luzio, and J. Kendrick-Jones, “Myosin VI isoform localized to clathrin-coated vesicles with a role in clathrin-mediated endocytosis,” EMBO Journal, vol. 20, no. 14, pp. 3676–3684, 2001.
[7]
F. Buss, G. Spudich, and J. Kendrick-Jones, “Myosin VI: cellular functions and motor properties,” Annual Review of Cell and Developmental Biology, vol. 20, pp. 649–676, 2004.
[8]
T. Hasson, “Myosin VI: two distinct roles in endocytosis,” Journal of Cell Science, vol. 116, no. 17, pp. 3453–3461, 2003.
[9]
F. Buss and J. Kendrick-Jones, “How are the cellular functions of myosin VI regulated within the cell?” Biochemical and Biophysical Research Communications, vol. 369, no. 1, pp. 165–175, 2008.
[10]
S. D. Conner and S. L. Schmid, “Regulated portals of entry into the cell,” Nature, vol. 422, no. 6927, pp. 37–44, 2003.
[11]
A. L. Dance, M. Miller, S. Seragaki et al., “Regulation of myosin-VI targeting to endocytic compartments,” Traffic, vol. 5, no. 10, pp. 798–813, 2004.
[12]
G. J. Doherty and H. T. McMahon, “Mechanisms of endocytosis,” Annual Review of Biochemistry, vol. 78, pp. 857–902, 2009.
[13]
F. Buss, J. Kendrick-Jones, C. Lionne, A. E. Knight, G. P. C？té, and J. P. Luzio, “The localization of myosin VI at the Golgi complex and leading edge of fibroblasts and its phosphorylation and recruitment into membrane ruffles of A431 cells after growth factor stimulation,” Journal of Cell Biology, vol. 143, no. 6, pp. 1535–1545, 1998.
[14]
A. Inoue, O. Sato, K. Homma, and M. Ikebe, “DOC-2/DAB2 is the binding partner of myosin VI,” Biochemical and Biophysical Research Communications, vol. 292, no. 2, pp. 300–307, 2002.
[15]
S. M. Morris, S. D. Arden, R. C. Roberts et al., “Myosin VI binds to and localises with Dab2, potentially linking receptor-mediated endocytosis and the actin cytoskeleton,” Traffic, vol. 3, no. 5, pp. 331–341, 2002.
[16]
L. Aschenbrenner, T. T. Lee, and T. Hasson, “Myo6 facilitates the translocation of endocytic vesicles from cell peripheries,” Molecular Biology of the Cell, vol. 14, no. 7, pp. 2728–2743, 2003.
[17]
J. P. Holt, K. Bottomly, and M. S. Mooseker, “Assessment of myosin II, Va, VI and VIIa loss of function on endocytosis and endocytic vesicle motility in bone marrow-derived dendritic cells,” Cell Motility and the Cytoskeleton, vol. 64, no. 10, pp. 756–766, 2007.
[18]
J. V. Small, T. Stradal, E. Vignal, and K. Rottner, “The lamellipodium: where motility begins,” Trends in Cell Biology, vol. 12, no. 3, pp. 112–120, 2002.
[19]
D. S. Reis, M. A. Souza, J. R. Mineo, and F. S. Espindola, “Myosin V and iNOS expression is enhanced in J774 murine macrophages treated with IFN-γ,” Brazilian Journal of Medical and Biological Research, vol. 34, no. 2, pp. 221–226, 2001.
[20]
F. Rosenbauer, A. Kallies, M. Scheller et al., “Disabled-2 is transcriptionally regulated by ICSBP and augments macrophage spreading and adhesion,” EMBO Journal, vol. 21, no. 3, pp. 211–220, 2002.
[21]
V. Braun, C. Deschamps, G. Raposo et al., “AP-1 and ARF1 control endosomal dynamics at sites of FcR-mediated phagocytosis,” Molecular Biology of the Cell, vol. 18, no. 12, pp. 4921–4931, 2007.
[22]
S. S. Bohlson, M. Zhang, C. E. Ortiz, and A. J. Tenner, “CD93 interacts with the PDZ domain-containing adaptor protein GIPC: implications in the modulation of phagocytosis,” Journal of Leukocyte Biology, vol. 77, no. 1, pp. 80–89, 2005.
[23]
T. T. Tuomisto, M. S. Riekkinen, H. Viita, A. L. Levonen, and S. Yl？-Herttuala, “Analysis of gene and protein expression during monocyte-macrophage differentiation and cholesterol loading-cDNA and protein array study,” Atherosclerosis, vol. 180, no. 2, pp. 283–291, 2005.
[24]
Z. Qin, “The use of THP-1 cell as a model for mimicking the function and regulation of monocytes and macrophages in the vasculature,” Atherosclerosis, vol. 221, no. 1, pp. 2–11, 2012.
[25]
J. V. Small, “Organization of actin in the leading edge of cultured cells: influence of osmium tetroxide and dehydration on the ultrastructure of actin meshworks,” Journal of Cell Biology, vol. 91, no. 3 I, pp. 695–705, 1981.
[26]
M. H. Symons and T. J. Mitchison, “Control of actin polymerization in live and permeabilized fibroblasts,” Journal of Cell Biology, vol. 114, no. 3, pp. 503–513, 1991.
[27]
L. P. Cramer and T. J. Mitchison, “Myosin is involved in postmitotic cell spreading,” Journal of Cell Biology, vol. 131, no. 1, pp. 179–189, 1995.
[28]
U. P. Steinbrecher, S. Parthasarathy, and D. S. Leake, “Modification of low density lipoprotein by endothelial cells involves lipid peroxidation and degradation of low density lipoprotein phospholipids,” Proceedings of the National Academy of Sciences of the United States of America, vol. 81, no. 12, pp. 3883–3887, 1984.
[29]
O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, “Protein measurement with the Folin phenol reagent,” The Journal of Biological Chemistry, vol. 193, no. 1, pp. 265–275, 1951.
[30]
G. L. Peterson, “A simplification of the protein assay method of Lowry et al. Which is more generally applicable,” Analytical Biochemistry, vol. 83, no. 2, pp. 346–356, 1977.
[31]
C. Puri, “Loss of myosin VI no insert isoform (NoI) induces a defect in clathrin-mediated endocytosis and leads to caveolar endocytosis of transferrin receptor,” Journal of Biological Chemistry, vol. 284, no. 50, pp. 34998–35014, 2009.
[32]
X. X. Xu, W. Yang, S. Jackowski, and C. O. Rock, “Cloning of a novel phosphoprotein regulated by colony-stimulating factor 1 shares a domain with the Drosophila disabled gene product,” Journal of Biological Chemistry, vol. 270, no. 23, pp. 14184–14191, 1995.
[33]
W. M. Bement, T. Hasson, J. A. Wirth, R. E. Cheney, and M. S. Mooseker, “Identification and overlapping expression of multiple unconventional myosin genes in vertebrate cell types,” Proceedings of the National Academy of Sciences of the United States of America, vol. 91, no. 14, pp. 6549–6553, 1994.
[34]
H. Kirikoshi and M. Katoh, “Expression of human GIPC1 in normal tissues, cancer cell lines, and primary tumors,” International Journal of Molecular Medicine, vol. 9, no. 5, pp. 509–513, 2002.
[35]
H. Yoshida, W. Cheng, J. Hung et al., “Lessons from border cell migration in the Drosophila ovary: a role for myosin VI in dissemination of human ovarian cancer,” Proceedings of the National Academy of Sciences of the United States of America, vol. 101, no. 21, pp. 8144–8149, 2004.
[36]
M. V. Chibalina, C. Puri, J. Kendrick-Jones, and F. Buss, “Potential roles of myosin VI in cell motility,” Biochemical Society Transactions, vol. 37, no. 5, pp. 966–970, 2009.
[37]
D. Valdembri, P. T. Caswell, K. I. Anderson et al., “Neuropilin-1/GIPC1 signaling regulates α5β1 integrin traffic and function in endothelial cells,” PLoS Biology, vol. 7, no. 1, Article ID e1000025, 2009.
[38]
J. Z. Rappoport, “Focusing on clathrin-mediated endocytosis,” Biochemical Journal, vol. 412, no. 3, pp. 415–423, 2008.
[39]
M. S. Robinson, “Adaptable adaptors for coated vesicles,” Trends in Cell Biology, vol. 14, no. 4, pp. 167–174, 2004.
[40]
M. E. Maurer and J. A. Cooper, “The adaptor protein Dab2 sorts LDL receptors into coated pits independently of AP-2 and ARH,” Journal of Cell Science, vol. 119, no. 20, pp. 4235–4246, 2006.
[41]
E. R. Eden, D. D. Patel, X. M. Sun et al., “Restoration of LDL receptor function in cells from patients with autosomal recessive hypercholesterolemia by retroviral expression of ARH1,” Journal of Clinical Investigation, vol. 110, no. 11, pp. 1695–1702, 2002.
[42]
D. Chetrit, N. Ziv, and M. Ehrlich, “Dab2 regulates clathrin assembly and cell spreading,” Biochemical Journal, vol. 418, no. 3, pp. 701–715, 2009.
[43]
H. S. Kruth, W. Huang, I. Ishii, and W. Y. Zhang, “Macrophage foam cell formation with native low density lipoprotein,” Journal of Biological Chemistry, vol. 277, no. 37, pp. 34573–34580, 2002.
[44]
H. S. Kruth, N. L. Jones, W. Huang et al., “Macropinocytosis is the endocytic pathway that mediates macrophage foam cell formation with native low density lipoprotein,” Journal of Biological Chemistry, vol. 280, no. 3, pp. 2352–2360, 2005.
[45]
X. Z. Ruan, Z. Varghese, S. H. Powis, and J. F. Moorhead, “Dysregulation of LDL receptor under the influence of inflammatory cytokines: a new pathway for foam cell formation,” Kidney International, vol. 60, no. 5, pp. 1716–1725, 2001.
[46]
J. Han, D. P. Hajjar, M. Febbraio, and A. C. Nicholson, “Native and modified low density lipoproteins increase the functional expression of the macrophage class B scavenger receptor, CD36,” Journal of Biological Chemistry, vol. 272, no. 34, pp. 21654–21659, 1997.
[47]
L. Nagy, P. Tontonoz, J. G. A. Alvarez, H. Chen, and R. M. Evans, “Oxidized LDL regulates macrophage gene expression through ligand activation of PPARγ,” Cell, vol. 93, no. 2, pp. 229–240, 1998.
[48]
T. Nakagawa, S. Nozaki, M. Nishida et al., “Oxidized LDL increases and interferon-γ decreases expression of CD36 in human monocyte-derived macrophages,” Arteriosclerosis, Thrombosis, and Vascular Biology, vol. 18, no. 8, pp. 1350–1357, 1998.
[49]
K. Tsukamoto, M. Kinoshita, K. Kojima et al., “Synergically increased expression of CD36, CLA-1 and CD68, but not of SR-A and LOX-1, with the progression to foam cells from macrophages,” Journal of Atherosclerosis and Thrombosis, vol. 9, no. 1, pp. 57–64, 2002.
[50]
J. R. Smith, T. F. Osborne, J. L. Goldstein, and M. S. Brown, “Identification of nucleotides responsible for enhancer activity of sterol regulatory element in low density lipoprotein receptor gene,” Journal of Biological Chemistry, vol. 265, no. 4, pp. 2306–2310, 1990.
[51]
D. Steinberg, “Low density lipoprotein oxidation and its pathobiological significance,” Journal of Biological Chemistry, vol. 272, no. 34, pp. 20963–20966, 1997.
[52]
V. V. Kunjathoor, M. Febbraio, E. A. Podrez et al., “Scavenger receptors class A-I/II and CD36 are the principal receptors responsible for the uptake of modified low density lipoprotein leading to lipid loading in macrophages,” Journal of Biological Chemistry, vol. 277, no. 51, pp. 49982–49988, 2002.
[53]
Y. Zeng, N. Tao, K. N. Chung, J. E. Heuser, and D. M. Lublin, “Endocytosis of oxidized low density lipoprotein through scavenger receptor CD36 utilizes a lipid raft pathway that does not require caveolin-1,” Journal of Biological Chemistry, vol. 278, no. 46, pp. 45931–45936, 2003.
