Modulation of Membrane Lipid Raft by Omega-3 Fatty Acids and Possible

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R EVIEW

E. SOTTOCORNOLA, Modulation of membrane lipid raft by B. BERRA omega-3 fatty acids and possible functional implication in receptor tyrosine-kinase activation

PROGRESS IN NUTRITION Summary VOL. 10,N.4, 210-213, 2008 Current understanding of biologic membrane structure and function is lar- gely based on the concept of lipid rafts. Lipid rafts are composed primarily TITOLO of tightly packed, liquid-ordered sphingolipids/cholesterol/saturated pho- Modulazione dei raft lipidici spholipids that float in a sea of more unsaturated and loosely packed, li- di membrana da parte degli quid-disordered lipids. Lipid rafts have important clinical implications be- acidi grassi omega-3 e possibili cause many important membrane-signalling proteins are located within implicazioni funzionali sulla the raft regions of the membrane, and alterations in the raft structure can attivazione di recettori tirosin- alter the activity of these signalling proteins. Because rafts are lipid-based, chinasici their composition, structure, and function are susceptible to manipulation by dietary components such as omega-3 polyunsaturated fatty acids and by KEY WORDS cholesterol depletion. Important components of lipid rafts are receptor Lipid raft, omega-3 PUFA, tyrosine-kinase, such as the ErbB receptor superfamily. Here we review receptor tyrosine-kinase, ErbB how alteration of raft lipids affects the raft/nonraft localization and hence receptor, mammary the function of several proteins involved in cell signalling, focusing our dis- adenocarcinoma cussion on two members of the ErbB receptors: EGFR and ErbB2, that are specifically involved in the development of many solid tumors, such as PAROLE CHIAVE mammary adenocarcinomas. Raft lipidici, omega-3 PUFA, recettori tirosin-chinasici, recettori Riassunto ErbB, adenocarcinoma mammario Ad oggi, la comprensione della struttura e della funzionalità della membrana plasmatica non può prescindere dal concetto dei ‘raft’ lipidici. I raft lipidici sono costituiti principalmente da sfingolipidi/colesterolo/fosfolipidi saturi strettamente impaccati in una fase liquido-ordinata che flottano al- Institute of General Physiology and l’interno della restante massa dei lipidi di membrana, maggiormente insa- Biological Chemistry “G. Esposito” University of Milan turi e costituenti la fase lipidica liquido-disordinata. I raft lipidici presenta- no importanti implicazioni cliniche poiché numerose importanti proteine

Indirizzo per la corrispondenza: deputate alla trasduzione del segnale sono in essi localizzate, e alterazioni Prof. Bruno Berra della struttura dei raft possono alterare la trasduzione del segnale da parte Institute of General Physiology and Biological and Chemistry “G. Esposito” di queste proteine. Dal momento che la massa critica dei raft è fondamen- University of Milan talmente costitutita da lipidi, la loro composizione, struttura e funzione è Via Trentacoste, 2 - 20134 Milano Tel. 02.503.15781 suscettibile a manipolazioni da parte di lipidi di derivazione dietetica quali Fax 02.503.15775 gli acidi grassi poliinsaturi omega-3 e dalla deplezione di colesterolo. I re- E-mail: [email protected] cettori della super-famiglia ErbB sono tra le tirosin-chinasi recettoriali che

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segregano nei raft. Sarà in questa sede trattato come l’alterazione dei lipidi dei raft possa alterare la localizzazione raft/non-raft e, di conseguenza, la funzione di numerose proteine coinvolte nella trasduzione del segnale, con particolare attenzione proprio a due recettori della famiglia ErbB: EGFR ed ErbB2, che sono specificamente coinvolti nello sviluppo di numerosi tumori solidi, tra i quali l’adenocarcinoma mammario.

What lipid rafts are? gent-resistant characteristics of the composed of tiny (on the nanometer glycosphingolipid clusters (4, 5), scale) and unstable (existing for less A provisional contemporary defini- suggesting that lipid lateral hetero- than microseconds) microdomains tion of rafts that embodies their geneity occurs spontaneously as a (like rafts, caveolae, and glycosynap- complexity is: “Membrane rafts are function of the lipid composition of ses), and, as a result, their investiga- small (10-200 nm), heterogeneous, the membrane. Simons and Ikonen tion and comprehension is very diffi- highly dynamic, sterol- and sphingo- originally proposed that lipid “…la- cult. lipid-enriched domains that com- teral organization probably results Lipid rafts are in a liquid-ordered partmentalize cellular processes. from preferential packing of sphin- state and are envisioned to float in a Small rafts can sometimes be stabili- golipids and cholesterol into moving sea of liquid-disordered phospholi- zed to form larger platforms through platforms, or rafts, onto which speci- pids, poor of cholesterol and sphin- protein-protein and protein-lipid in- fic proteins attach within the bilayer” golipids. They are enriched in tightly teractions”. The origin of the raft (1). packed, saturated fatty acyl chains, hypothesis (1) can be traced to the It is now well established that all cel- whereas the non-raft regions are en- perplexing discovery that glyco- lular membranes of mammalian as riched in loosely packed, polyunsatu- sphingolipids cluster in the Golgi well as of Drosophila, Dictyostelium, rated chains. apparatus before being sorted to the and yeast, are not homogeneous in It is established that rafts incorpora- apical surface of polarized epithelial their lipid and protein distribution, te many diverse signalling receptors, cells (2). Subsequent studies establis- but instead are composed of diverse, downstream signal molecules, and hed that glycosphingolipid clusters ever-changing patches, called do- signaling associated adaptor pro- tend to be insoluble in Triton X-100 mains. Domains exist in a bewilde- teins. Included in an ever expanding at 4°C, forming detergent-resistant rind array of sizes, stabilities, lipid list of raft-associated signalling pro- membranes (DRM), have a light and protein compositions, and func- teins are high-affinity IgE receptors buoyant density on sucrose gradients tionalities. Some domains are ma- (FcR), insulin receptors, T-cell anti- and are rich in both cholesterol and croscopic and stable for extended ti- gen receptors, G-protein-coupled re- glycosylphosphatidyl inositol (GPI)- me periods (such as the apical–baso- ceptors, epidermal growth factor anchored proteins (3). The raft lateral specializations and junctional (EGF) receptors, and several kinases hypothesis was bolstered by the ob- complexes in epithelial cells and and phosphatases. Lipid rafts are servation that synthetic membranes clathrin-coated pits) and so are isola- therefore postulated to serve as plat- composed of glycosphingolipids and ted and well defined, but the major forms for protein activity by accu- cholesterol recapitulate the deter- proportions of most membranes are mulating specific proteins usually in-

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volved in cell signalling. Recent in- and to modulate expression of many properties of cell membranes, inclu- vestigations have shown that move- gene products involved in inflamma- ding acyl chain order and fluidity, ment of proteins into or out of lipid tory and immunologic processes. phase behaviour, elastic compressibi- rafts can be modified by changes in These and other long-chain fatty lity, ion permeability, fusion, flip- the lipid microenvironment. acids are known to localize to cell flop, and the activity of many resi- membranes and to alter their pro- dent proteins (11). perties. The exact mechanisms whe- The shorter chain omega-3 PUFA, Lipid rafts and omega-3 long chain reby long-chain fatty acids modulate alfa-linolenic acid, can be desatura- fatty acids cell functions are not entirely clear. ted and elongated to DHA and EPA However, an extensive body of scien- in vivo. Therefore alfa-linolenic acid The lipid composition of cancer and tific evidence suggests that PUFAs may also exhibit beneficial effects on non-cancer cell membranes are no- affect cellular functions by modula- health similar to EPA and DHA. tably different (6). It has been sugge- ting the structure and function of However, there is concern that pa- sted that increased proportions of sa- specific lipid domains, such as lipid tients with a variety of diseases may turated fatty acid in cancer cells alter rafts, within the plasma membrane have decreased capacity to elongate lipid raft structure and protein com- (8). alfa-linolenic acid to EPA and position in a manner that enhances A number of recent in vitro studies DHA. Therefore, alfa-linolenic acid cancer cell survival by promoting using model membranes and cell should be used with caution as sole growth, escaping immune surveillan- culture systems have demonstrated source of omega-3 PUFAs. ce, or preventing apoptosis (7). In- that PUFAs can greatly reduce raft Recent studies have demonstrated deed, increased activity levels of the formation and can displace signal- that EPA and DHA can displace se- fatty acid synthase (FAS) has been ling proteins (9, 10). Changes in the veral raft-associated signalling pro- found in numerous malignancies (7). nature and abundance of functional teins from membrane rafts (12, 13). Moreover, cancer cells have higher proteins in lipid rafts, can alter cell levels of cholesterol, that is perhaps physiology and hence the develop- the most important component of ment and progression of various dis- Lipid raft and ErbB rafts. eases. Experimental evidences indi- tyrosine-kinase receptors As rafts are lipid-based, their com- cate that lipid raft signalling proteins position, structure and function are can be modulated in inflammation The ErbB superfamily of receptors, susceptible to manipulation by die- and cancer models by the omega-3 particularly EGFR and ErbB2, are tary components such as omega-3 PUFAs, particularly docosahexae- actively involved in mammary gland polyunsaturated fatty acids (PU- noic acid (DHA) and eicosapentae- development and differentiation du- FAs), cholesterol depletion, manipu- noic acid (EPA), which are found in ring pregnancy and lactation and in lation of glycosphingolipid expres- high concentrations in fish oils. the uncontrolled growth of mam- sion. DHA is of special interest because it mary adenocarcinomas characterized Dietary lipids and lipid emulsions is the longest (22 carbons) and the by poor prognosis and drug resistan- are known to have many effects most unsaturated (6 double bonds) ce. Many are the literature data de- upon cellular functions. PUFAs have fatty acid commonly found in mem- scribing ErbB receptor compart- been reported to alter synthesis of li- branes. DHA is a perturbing fatty mentalization in and out raft and the pid mediators such as prostaglandins acid, significantly altering the basic mechanisms of their homo-/hetero-

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dimerization and activation (14), but decanoylamino-3-morpholino-1- LC, Bennett MJ. Increased ratio of saturated to unsaturated C18 fatty acids in very little is known about the regula- propanol hydrochloride (PDMP) in- colonic adenocarcinoma: implications for tory effect of omega-3 PUFA sup- duced a drastic cell-surface redistri- cryotherapy and lipid raft function. Med plementation. Shley et al. (15) de- bution of ErbB2, EGFR and Shc- Hypothesis 2005; 65: 1120-3. monstrated that treatment of the p66, within the Triton-soluble frac- 8. Chen W, Jump DB, Esselman WJ, Bu- sik JV. Inihibition of cytokine signalling MDA-MB-231 human breast can- tions. This redistribution was par- in human retinal endothelial cells cer cell line with a mixture of EPA tially reverted when exogenous through modification of caveolae/lipid and DHA induced the decrease of GM3 was added to ganglioside-de- rafts by docosaexaenoic acid. Invest. sphingomyelin, cholesterol and pleted HC11 cells. The results point Ophthalmol. Vis Sci 2007; 48: 18-26. 9. Stulnig TM, Huber J, Leitinger N. Pol- diacylglicerol as well as EGFR in li- out the key role of ganglioside GM3 yunsaturated eicosapentaenoic acid dis- pid raft, although phosphorylation in retaining ErbB2/EGFR and si- places proteins from membrane rafts by of both EGFR and p38 MAPK we- gnal-transduction-correlated pro- altering raft lipid composition. J Biol re sensibly increased. Activation of teins in lipid rafts, suggesting the Chem 2001; 276: 37335-40. 10. Stulnig TM, Berger M, Sigmund T, EGFR and p38 MAPK was associa- possible existence of different pool of Raederstorff D, Stockinger H, Walhausl ted with apoptosis of MDA-MB- receptors at the plasma membrane W. Polyunseturated fatty acids inhibit T 231 cells, suggesting that the altera- levels. cell signal transduction by modification tion of lipid composition of mem- of detergent-insoluble membrane domains. J Cell Biol 1998; 143: 637-44. brane raft is able to modify EGFR 11. Stillwell W, Shaikh SR, Zerouga M, signalling transduction pathways and Bibliografia Siddiqui R, Wassal SR. Docosaexaenoic cell fate. acid affects cell signalling by altering li- We analyzed the role of gangliosi- 1. Simons K, Ikonen E. Functional rafts in pid rafts. Reprod Nutr Dev 2005; 45: cell membranes. Nature 1997; 387: 569- 559-79. des, an important lipid component 72. 12. Ma DW, Seo J, Davidson LA, et al. N-3 of rafts, in the association of the 2. Simons K, van Meer G. Lipid sorting in PUFA alter caveolae lipid composition ErbB2, EGFR and shc-p66 (protein epithelial cells. Biochemistry 1988; 27: and resident protein localization in mou- correlated with the ErbB2 signal 6197–202. se colon. FASEB J 2004; 18: 1040-2. 3. Brown D. GPI-anchored proteins and 13. Li Q, Wang M, Tan L, et al. Docosahe- transduction pathway) with lipid detergent-resistant membrane domains. xaenoic acid changes lipid composition rafts in mammary epithelial HC11 Braz J Med Biol Res. 1994; 27: 309–15. and interleukin-2 receptor signalling in cells (16). Scanning confocal micro- 4. Dietrich C, Bagatolli LA, Volovyk ZN, et membrane rafts. J Lipid Res 2005; 46: scopy experiments together with the al. Lipid rafts reconstituted in model 1904-13. membranes. Biophys J 2001; 80: 1417–28. 14. Yarden Y, Sliwkowski MX. Untangling analysis of membrane fractions ob- 5.Dietrich C, Volovyk ZN, Levi M, the ErbB signalling network. Nature tained by linear sucrose gradient ul- Thompson NL, Jacobson K. Partitioning 2001; 2: 127-37. tracentrifugation revealed the prefe- of Thy-1, GM1, and cross-linked pho- 15. Schley PD, Brindley DN, Field CJ. (n- rential association of ErbB2/EGFR spholipids into lipid rafts reconstituted in 3) PUFA alter lipid raft composition and supported model membrane monolayers. decrease epidermal growth factor recep- and ganglioside GM3 in lipid raft. Proc Natl Acad Sci USA 2001; 98: tor levels in lipid rafts of human breast In addition, after activation of 10642–7. cancer cells. J Nutr 2007; 137: 548-53. EGFR with EGF, shc-p66 was pre- 6. Meng X, Riordan NH, Riordan HD. Cell 16. Sottocornola E, Misasi R, Mattei V, et ferentially enriched in lipid rafts. membrane fatty acid composition differs al. Role of gangliosides in the association between normal and malignant cell lines. of ErbB2 with lipid rafts in mammary Blocking of endogenous ganglioside P R Health Sci J 2004; 23: 103-6. epithelial HC11 cells. FEBS J 2006; synthesis by (+/-)-threo-1-phenyl-2- 7. Rakheja D, Kapur P, Hoang MP, Roy 273; 1821-30.

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