Editorial Commentary Lipid Rafts Take Center Stage in Endothelial Cell Redox Signaling by Death Receptors Rhian M. Touyz
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lial dysfunction.5 Hence, lipid rafts are implicated in both growth and death signaling through NAD(P)H oxidase– generated reactive oxygen species. Lipid rafts are liquid-ordered microdomains enriched in sphingolipids and cholesterol constituting a distinct biophysical plasma membrane compartment.6,9 They are highly dynamic, submicroscopic assemblies that float freely within the liquid bilayer in cell membranes. On the other hand, caveolae, which constitute a subset of rafts, are morphologically defined cell surface invaginations that contain caveolin.10 The most important role of lipid rafts at the cell surface may be their function in cell transduction.6,7 Lipid rafts serve as signaling platforms bringing receptors into proximity with activating kinases, scaffolding proteins, and adaptor molecules that are constituent residents of lipid rafts. Raft-binding proteins recruit proteins to a new microenvironment, where the phosphorylation state can be modified by local enzymes, leading to downstream signaling. Typical examples of raftassociated proteins include the glycosylphosphatidylinositolanchored proteins; the Src family tyrosine kinases; palmitoylated and myristoylated proteins, such as flotillins; cholesterol-binding proteins, such as caveolins and hedgehog; heterotrimeric G proteins; and phospholipids-binding proteins, such as annexins.9 –12 Well characterized examples of signaling pathways that involve lipid rafts include immunoglobulin E signaling, T cell antigen receptor signaling, glial-cell– derived neurotrophic factor signaling, Ras signaling, and Hedgehog signaling.9 –13 Added to this list now is redox signaling in endothelial cells through death receptor ligands and apoptotic factors, including TNF␣, FasL, and endostatin. Zhang et al demonstrate that lipid raft clustering is coupled to gp91phox, the catalytic subunit of NAD(P)H oxidase, and that on FasL stimulation, p47phox and the small G-protein Rac translocate into membrane rafts to assemble the activated oxidase complex that generates superoxide5 (Figure). These novel findings in endothelial cells, together with those described in vascular smooth muscle cells and neutrophils where NAD(P)H oxidase subunits have been detected in lipid raft fractions,8,14 strongly suggest that in addition to the classical raft-associated proteins (mentioned above), NAD(P)H oxidase subunits (at least gp91phox and p47phox) can be classified as lipid raft–associated proteins. However, it still remains unclear how these proteins align spatially and temporally within rafts, how they physically interact with death receptor domains on lipid raft aggregation and how cytoplasmic NAD(P)H oxidase subunits are site-directed to specific cholesterol-rich microdomains. The actin cytoskeleton may play an important role in these events, as we recently reported in vascular smooth muscle cells.15,16
eactive oxygen species are important signaling molecules mediating diverse biological effects in vascular cells ranging from cell growth to cell death.1 The primary enzymatic source of vascular reactive species is the multisubunit NAD(P)H oxidase, expressed and functionally active in endothelial, vascular smooth muscle, and adventitial cells.2 Activation of G protein-coupled receptors by vasoactive agents, such as angiotensin II (Ang II), and receptor tyrosine kinases by growth factors, such as epidermal growth factor (EGF), stimulate NAD(P)H oxidase– derived generation of superoxide and hydrogen peroxide in vascular smooth muscle cells, which activate mitogen-activated protein kinase growth signaling pathways and promote cell cycle progression. In pathological conditions associated with vascular injury and remodeling, increased oxidative stress is now considered a fundamental factor underlying proliferation and hypertrophy of vascular smooth muscle cells.1,2 Paradoxically in endothelial cells, death receptor ligands and proapoptotic agonists, including tumor necrosis factor (TNF) ␣, Fas ligand (FasL), and endostatin, also stimulate NAD(P)H oxidase– mediated production of reactive oxygen species.3–5 These processes trigger endothelial cell apoptosis, anoikis, and impaired dilation. How then can agonists that promote cell growth and cell death trigger the same redox-sensitive pathways to elicit divergent cellular responses, and what are the mechanisms that link growth/death receptors to NAD(P)H oxidase in vascular cells? Emerging evidence indicates that lipid microenvironments on the cell surface, known as lipid rafts, may be critically involved in distal redox-sensitive signaling events and ultimate cell fate.6,7 In vascular smooth muscle cells, Ang II–induced cell growth involves epidermal growth factor receptor (EGFR) transactivation, mediated through redoxsensitive c-Src, which is dependent on angiotensin I type 1 receptor trafficking through caveolin 1– enriched lipid rafts.8 In the current issue of Hypertension, Zhang et al elegantly demonstrate that death receptor ligands and apoptotic factors stimulate lipid raft clustering, which results in aggregation and activation of NAD(P)H oxidase and consequent endotheThe opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association. From the Canada Research Chair in Hypertension, Kidney Research Centre, Ottawa Health Research Institute, University of Ottawa, Ontario, Canada. Correspondence to Rhian M Touyz MD, PhD, Canada Research Chair in Hypertension, Ottawa Health Research Institute, University of Ottawa, 451 Smyth Rd, Ottawa, ON, KIH 8M5. E-mail:
[email protected] (Hypertension. 2006;47:16-18.) © 2005 American Heart Association, Inc. Hypertension is available at http://www.hypertensionaha.org DOI: 10.1161/01.HYP.0000196730.13216.f3
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Model for lipid raft aggregation and redox signaling in endothelial cells in response to death receptor activation by TNF␣, FasL, and endostatin. In resting cells, lipid rafts are in an inactive disassociated conformation. On stimulation of death receptors by death ligands, glycosylphosphatidylinositol (GPI)-anchored proteins may become clustered19 with associated lipid raft aggregate formation. NAD(P)H oxidase subunits, gp91phox and p47phox, together with p22phox, p67phox, and Rac, translocate to the lipid raft aggregates to assemble the NAD(P)H oxidase complex. Activated NAD(P)H oxidase leads to generation of reactive oxygen species, which influences redox-dependent signaling molecules and ultimately endothelial function.
Two questions arise from the study of Zhang et al.5 First, what is the need for lipid rafts in redox signaling by death receptors in endothelial cells, and second, what is the functional significance of lipid raft:NAD(P)H oxidase interactions in the endothelium? Formation of lipid raft aggregates in response to TNF␣ and FasL may provide a dynamic scaffold to ensure efficient assembly and activation of NAD(P)H oxidase, thereby providing a platform for redox signaling through reactive oxygen species. Redox signaling complexes formed within rafts may be protected from non-raft enzymes, such as membrane phosphatases and antioxidant enzymes that otherwise could affect the signaling process. Hence, such microenvironments determine distal signaling events, which ultimately influence cell fate. This is particularly relevant to TNF receptor signaling, which triggers diverse biological responses.9 The presence of a particular TNF receptor on the cell surface does not necessarily predict efficacy or biological outcomes of signal transduction by that receptor, because signaling is dependent on cell type and environmental factors. For example, TNFR1 and Fas, members of TNF receptor superfamily, stimulate distinct signaling cascades, which trigger alternative fates of cells depending on receptor localization in lipid raft microdomains.9,17 Both antiapoptotic and proapoptotic effects of TNF signaling have been demonstrated depending on the integrity of rafts. In some cell types, when lipid rafts are disrupted, phosphorylation of IB␣ in response to TNF␣ is inhibited and apoptosis is induced, whereas in other conditions death signaling complexes depend on intact lipid rafts.9,17 Whether such divergent effects occur in endothelial cells and whether reactive oxygen species influence life and death decisions following TNF receptor stimulation remains unclear.
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The functional significance of death receptor signaling in endothelial cells seems to extend beyond apoptosis. FasL and TNF␣ impair endothelium-dependent vasodilation and promote endothelial injury. These processes occur through lipid raft clustering, activation of NAD(P)H oxidase, and formation of reactive oxygen species, since lipid raft disruption with nystatin and inhibition of NAD(P)H oxidase with apocynin improved vasodilatory responses and endothelial function.5 Such events may represent early functional responses to death receptor signaling, whereas apoptosis may be the consequence of long-term signaling. The novel findings of Zhang et al5 certainly contribute to the further understanding of signaling mechanisms mediating the early response of death receptors in endothelial cells and highlight the importance of lipid rafts as central players linking death receptor domains to NAD(P)H oxidase and reactive oxygen species. However, there are some limitations that warrant attention. First, the relative importance of lipid rafts versus non-lipid rafts (caveolae) is not fully addressed. This is particularly important in the context of endothelial function, because caveolae are richly endowed with endothelial NOS, the major source of nitric oxide, a potent vasodilator.4,10,18 Second, studies were performed in cultured coronary endothelial cells. Whether findings are particular to coronary endothelial cells or to all endothelial cells, whether effects are cell-type specific, and whether similar responses occur in in vivo conditions remain unknown. Third, the experimental paradigm was designed to mimic pathological conditions where death receptors are stimulated. The physiological significance of lipid raft clustering and redox signaling platform formation in endothelial cells still awaits clarification. It is now becoming clear that lipid microdomains on the cell surface participate in signal transduction and that they may constitute a missing link between death receptor domains and NAD(P)H oxidase in endothelial cells. Findings that NAD(P)H oxidase subunits are raft-associated proteins and dependency of intact lipid rafts in efficient death receptor and redox signaling highlights the importance of these cholesterol-rich domains in endothelial cells. Future challenges will be to identify the significance of lipid rafts as key players in redox signaling in the physiological regulation of vascular function and in the pathophysiological effects of oxidative stress in vascular disease.
References 1. Touyz RM. Reactive oxygen species, oxidative stress and redox signaling in hypertension–what is the clinical significance? Hypertension. 2004;44: 248 –252. 2. Brandes RP, Kreuzer J. Vascular NADPH oxidases: molecular mechanisms of activation. Cardiovasc Res. 2005;65:16 –27. 3. Devadas S, Hinshaw JA, Zaritskaya L, Williams MS. Fas-stimulated generation of reactive oxygen species or exogenous oxidative stress sensitize cells to Fas-mediated apoptosis. Free Radic Biol Med. 2003;35: 648 – 661. 4. Zhang AY, Teggatz EG, Zou AP, Campbell WB, Li PL. Endostatin uncouples NO and Ca2⫹ response to bradykinin through enhanced superoxide production in the intact coronary endothelium. Am J Physiol. 2005;288:686 – 694. 5. Zhang AY, Yi F, Zhang G, Gulbins E, Li P-L. Lipid raft clustering and redox signaling platform formation in coronary arterial endothelial cells. Hypertension. 2006;47:74 – 80. 6. Simons K, Toomre D. Lipid rafts and signal transduction. Nature Rev. 2000;1:31–39.
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7. Holthius JCM, Levine TP. Lipid traffic: floppy drives and a superhighway. Nature Rev. 2005;6:209 –220. 8. Zuo l, Ushio-Fukai M, Ikeda S, Hilenski L, Patrushev SM Alexander RW. Caveolin1 is essential for activation of Rac1 and NAD(P)H oxidase after angiotensin I type 1 receptor stimulation in vascular smooth muscle cells: role in redox signaling and vascular hypertrophy. Arterioscler Thromb Vasc Biol. 2005;25:1824 –1830. 9. Muppidi JR, Tschopp J, Siegel RM. Life and death decisions: secondary complexes and lipid rafts in TNF receptor family signal transduction. Immunity. 2004;21:461– 465. 10. Gratton J-P, Bernatchez P, Sessa WC. Caveolae and caveolins in the cardiovascular system. Circ Res. 2004;94:1408 –1417. 11. Lin D, Takemoto DJ. Oxidative activation of protein kianse C gamma through the C1 domain. Effects on gap junctions. J Biol Chem. 2005; 280:13682–13693. 12. Rajendran L, Simons K. Lipid rafts and membrane dynamics. J Cell Sci. 2005;118:1099 –1102. 13. Schwartz EA, Reavan E, Topper JN, Tsao PS. Transforming growth factor -receptor localize to caveolae and regulate endothelial nitric oxide synthase in normal human endothelail cells. Biochem J. 2005;390:199–206.
14. Shao D, Segal AW, Dekker LV. Lipid rafts determine efficiency of NADPH oxidase activation in neutrophils. FEBS Lett. 2003;550: 101–106. 15. Touyz RM, Yao G, Quinn MT, Pagano PJ, Schiffrin EL. p47phox associates with the cytoskeleton through cortactin in human vascular smooth muscle cells. Role in NAD(P)H oxidase regulation by angiotensin II. Arterioscler Thromb Vasc Biol. 2005;25:512–518. 16. Touyz RM, Yao G, Schiffrin EL. c-Src induces phosphorylation and translocation of p47phox: Role in Superoxide Generation by Ang II in human vascular smooth muscle cells. Arterioscler Thromb Vasc Biol. 2003;23:981–987. 17. Natoli G, Costanzo A, Guido F, Moretti F, Levrero M. Apoptotic, nonapoptotic and anti-apoptotic pathways of tumor necrosis factor signaling. Biochem Pharmacol. 1998;56:915–920. 18. Peterson TE, Poppa V, Ueba H, Wu A, Yan C, Berk BC. Opposing effects of reactive oxygen species and cholesterol on endothelial nitric oxide synthase and endothelial cell caveolae. Circ Res. 1999;85:29 –37. 19. Gekara NO, Weiss S. Lipid rafts clustering and signaling by listeriolysin O. Biochem Soc Transact. 2004;32:712–715.
