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High Density Lipoproteins (HDL) Interrupt the Sphingosine Kinase Signaling Pathway

A POSSIBLE MECHANISM FOR PROTECTION AGAINST ATHEROSCLEROSIS BY HDL*
Open AccessPublished:November 12, 1999DOI:https://doi.org/10.1074/jbc.274.46.33143
      The ability of high density lipoproteins (HDL) to inhibit cytokine-induced adhesion molecule expression has been demonstrated in their protective function against the development of atherosclerosis and associated coronary heart disease. A key event in atherogenesis is endothelial activation induced by a variety of stimuli such as tumor necrosis factor-α (TNF), resulting in the expression of various adhesion proteins. We have recently reported that sphingosine 1-phosphate, generated by sphingosine kinase activation, is a key molecule in mediating TNF-induced adhesion protein expression. We now show that HDL profoundly inhibit TNF-stimulated sphingosine kinase activity in endothelial cells resulting in a decrease in sphingosine 1-phosphate production and adhesion protein expression. HDL also reduced TNF-mediated activation of extracellular signal-regulated kinases and NF-κB signaling cascades. Furthermore, HDL enhanced the cellular levels of ceramide which in turn inhibits endothelial activation. Thus, the regulation of sphingolipid signaling in endothelial cells by HDL provides a novel insight into the mechanism of protection against atherosclerosis.
      HDL
      high density lipoproteins
      apoA-I
      apolipoprotein A-I
      ERK
      extracellular signal-regulated kinase
      HUVEC
      human umbilical vein endothelial cell
      ICAM-1
      intercellular adhesion molecule-1
      LDL
      low density lipoproteins
      oxLDL
      oxidized LDL
      S1P
      sphingosine 1-phosphate
      SphK
      sphingosine kinase
      TNF
      tumor necrosis factor-α
      VCAM-1
      vascular cell adhesion molecule-1
      DMS
      N,N-dimethylsphingosine
      POPC
      1-palmityl-2-oleylphosphatidylcholine
      Numerous evidence from epidemiological, clinical, and genetic studies have clearly shown a potential protective role of high density lipoproteins (HDL)1 against the development of atherosclerosis and associated coronary heart disease (
      • Gordon T.
      • Castelli W.P.
      • Hjortland M.C.
      • Kannel W.B.
      • Dawber T.R.
      ,
      • Rubin E.M.
      • Krauss R.M.
      • Spangler E.A.
      • Verstuyft J.G.
      • Clift S.M.
      ,
      • Assmann G.
      • Schulte H.
      ,
      • Silverman D.I.
      • Ginsburg G.S.
      • Pasternak R.C.
      ). Several mechanisms have been proposed for the cardioprotective function of HDL. These include the promotion of the efflux of cholesterol from atherosclerotic plaques and reducing the atherogenicity of LDL by inhibition of their oxidative modification (
      • Silverman D.I.
      • Ginsburg G.S.
      • Pasternak R.C.
      ,
      • Navab M.
      • Hama S.Y.
      • Hough G.P.
      • Hedrick C.C.
      • Sorenson R.
      • La Du B.N.
      • Kobashigawa J.A.
      • Fonarow G.C.
      • Berliner J.A.
      • Laks H.
      • Fogelman A.M.
      ,
      • Fielding C.J.
      • Fielding P.E.
      ). Recently, we and other groups have demonstrated an ability of HDL to inhibit endothelial adhesion protein expression, providing a new mechanistic explanation for its protective effect on atherosclerosis (
      • Cockerill G.W.
      • Rye K.-A.
      • Gamble J.R.
      • Vadas M.A.
      • Barter P.J.
      ,
      • Moudry R.
      • Spycher M.O.
      • Doran J.E.
      ,
      • Calabresi L.
      • Franceschini G.
      • Sirtori C.R.
      • De Palma A.
      • Saresella M.
      • Ferrante P.
      • Taramelli D.
      ,
      • Ashby D.T.
      • Rye K.-A.
      • Clay M.A.
      • Vadas M.A.
      • Gamble J.R.
      • Barter P.J.
      ,
      • Baker P.W.
      • Rye K.-A.
      • Gamble J.R.
      • Vadas M.A.
      • Barter P.J.
      ).
      Atherosclerosis has been definitely characterized as an inflammatory disease (
      • Ross R.
      ). An important event in the initiation of atherosclerosis is adhesion of circulating monocytes to activated endothelial cells and their subsequent transendothelial migration to the subendothelium. This process is mediated by adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin (
      • Springer T.A.
      ,
      • Vadas M.A.
      • Gamble J.R.
      ). The inappropriate expression of these adhesion proteins in response to the “injury” are induced by various inflammatory stimuli, including cytokines and noncytokines such as interleukin-1, tumor necrosis factor-α (TNF), and oxidized or native LDL (
      • Vadas M.A.
      • Gamble J.R.
      ,
      • Libby P.
      • Ross R.
      ,
      • Allen S.
      • Khan S.
      • Al-Mohanna F.
      • Batten P.
      • Yacoub M.
      ). Pathological studies have shown increased adhesion molecule expression in several components of the atherosclerotic plaque (
      • Cybulsky M.I.
      • Gimbrone Jr., M.A.
      ,
      • Poston R.N.
      • Haskard D.O.
      • Coucher J.R.
      • Gall N.P.
      • Johnson-Tidey R.R.
      ,
      • Van der Wal A.C.
      • Das P.K.
      • Tigges A.J.
      • Becker A.E.
      ,
      • O'Brien K.D.
      • McDonald T.O.
      • Chait A.
      • Allen M.D.
      • Alpers C.E.
      ), and there is also evidence for a role of adhesion molecules in the acute atherothrombotic process (
      • Jang Y.
      • Lincoff A.M.
      • Plow E.F.
      • Topol E.J.
      ). Furthermore, a direct association between an increased plasma concentration of soluble adhesion molecules and the increase in risk of future cardiovascular diseases has recently been reported (
      • Hwang S.J.
      • Ballantyne C.M.
      • Sharrett A.R.
      • Smith L.C.
      • Davis C.E.
      • Gotto Jr., A.M.
      • Boerwinkle E.
      ,
      • Ridker P.M.
      • Hennekens C.H.
      • Roitman-Johnson B.
      • Stampfer M.J.
      • Allen J.
      ).
      The ability of HDL to inhibit the cytokines-induced adhesion protein expression has been well documented. It has been reported that human HDL profoundly inhibit the expression of VCAM-1, ICAM-1, and E-selectin in human umbilical vein endothelial cells (HUVEC) activated by TNF or interleukin-1 (
      • Cockerill G.W.
      • Rye K.-A.
      • Gamble J.R.
      • Vadas M.A.
      • Barter P.J.
      ). Total native HDL together with both HDL2and HDL3 subfractions, or the reconstituted HDL particles showed the inhibitory effect in a concentration-dependent manner, although considerable variation existed among different experiments (
      • Cockerill G.W.
      • Rye K.-A.
      • Gamble J.R.
      • Vadas M.A.
      • Barter P.J.
      ,
      • Moudry R.
      • Spycher M.O.
      • Doran J.E.
      ,
      • Calabresi L.
      • Franceschini G.
      • Sirtori C.R.
      • De Palma A.
      • Saresella M.
      • Ferrante P.
      • Taramelli D.
      ,
      • Ashby D.T.
      • Rye K.-A.
      • Clay M.A.
      • Vadas M.A.
      • Gamble J.R.
      • Barter P.J.
      ,
      • Baker P.W.
      • Rye K.-A.
      • Gamble J.R.
      • Vadas M.A.
      • Barter P.J.
      ). The phenotype of inhibition on adhesion molecule expression by HDL differs from their well known function in promoting cholesterol efflux and protecting against lipid peroxidation, suggesting a distinct mechanism exists. We recently demonstrated a novel signaling pathway, sphingosine kinase (SphK) pathway, through the generation of sphingosine 1-phosphate (S1P), which is critically involved in mediating adhesion protein expression and endothelial cell activation after TNF stimulation (
      • Xia P.
      • Gamble J.R.
      • Rye K.-A.
      • Wang L.
      • Hii C.S.T.
      • Cockerill P.
      • Khew-Goodall Y.
      • Bert A.G.
      • Barter P.J.
      • Vadas M.A.
      ). The SphK pathway has also emerged as a signaling pathway in mediating a variety of cellular functions such as cell growth, proliferation, and inflammatory reaction (
      • Xia P.
      • Gamble J.R.
      • Rye K.-A.
      • Wang L.
      • Hii C.S.T.
      • Cockerill P.
      • Khew-Goodall Y.
      • Bert A.G.
      • Barter P.J.
      • Vadas M.A.
      ,
      • Olivera A.
      • Spiegel S.
      ,
      • Spiegel S.
      • Merrill Jr., A.H.
      ,
      • Igarashi Y.
      • Yatomi Y.
      ,
      • Lee M.J.
      • Van Brocklyn J.R.
      • Thangada S.
      • Liu C.H.
      • Hand A.R.
      • Menzeleev R.
      • Spiegel S.
      • Hla T.
      ,
      • Rakhit S.
      • Conway A.M.
      • Tate R.
      • Bower T.
      • Pyne N.J.
      • Pyne S.
      ). In the present report we show that HDL profoundly inhibit the TNF-induced SphK activity and S1P generation, and subsequently reduce the activation of ERK and NF-κB signal cascades. We thus demonstrate that HDL interrupt a signaling cascade, the SphK pathway, which results in inhibition of endothelial activation. This could provide a new potential mechanism by which HDL protect against atherosclerosis, a cardiovascular inflammatory disease.

      DISCUSSION

      In this report, we show a novel mechanism of atheroprotection by HDL. In this model, HDL interrupt a signal transduction pathway, the SphK pathway, which is critically involved in endothelial cell activation and adhesion protein expression. The expression of adhesion proteins on activated endothelial cells plays an essential role for the inflammatory processes in the pathogenesis of atherosclerosis (
      • Ross R.
      ). The importance of adhesion molecules in atherogenesis is strongly supported by several lines of evidence: (i) adhesion molecules are present in atherosclerotic plaques (
      • Cybulsky M.I.
      • Gimbrone Jr., M.A.
      ,
      • Poston R.N.
      • Haskard D.O.
      • Coucher J.R.
      • Gall N.P.
      • Johnson-Tidey R.R.
      ,
      • Van der Wal A.C.
      • Das P.K.
      • Tigges A.J.
      • Becker A.E.
      ,
      • O'Brien K.D.
      • McDonald T.O.
      • Chait A.
      • Allen M.D.
      • Alpers C.E.
      ,
      • Jang Y.
      • Lincoff A.M.
      • Plow E.F.
      • Topol E.J.
      ); (ii) increased plasma levels of adhesion molecules are associated with the risks of atherosclerosis (
      • Hwang S.J.
      • Ballantyne C.M.
      • Sharrett A.R.
      • Smith L.C.
      • Davis C.E.
      • Gotto Jr., A.M.
      • Boerwinkle E.
      ,
      • Ridker P.M.
      • Hennekens C.H.
      • Roitman-Johnson B.
      • Stampfer M.J.
      • Allen J.
      ); and (iii) a deficiency of adhesion molecules significantly reduces the formation of atherosclerotic fatty streaks in knockout mice lacking the genes of ICAM-1, P-selectin, or both ICAM-1 and P-selectin (
      • Johnson R.C.
      • Chapman S.M.
      • Dong Z.M.
      • Ordovas J.M.
      • Mayadas T.N.
      • Herz J.
      • Hynes R.O.
      • Schaefer E.J.
      • Wagner D.D.
      ,
      • Nageh M.F.
      • Sandberg E.T.
      • Marotti K.R.
      • Lin A.H.
      • Melchior E.P.
      • Bullard D.C.
      • Beaudet A.L.
      ).
      The nature of the inflammatory signals and associated molecular mechanisms that activate adhesion molecule expression in endothelial cells in the atherogenic lesion are unknown. Factors such as TNF and interleukin-1 that are commonly found in inflammatory atherogenic lesions induce the expression of adhesion molecules in cultured endothelial cells. Thus, TNF-stimulated adhesion molecule expression on HUVEC provides a useful model to investigate the signaling pathways involved in the regulation of endothelial cell activation. In this model we have recently identified a novel signaling pathway, the SphK pathway, in mediating TNF-induced adhesion protein expression and endothelial cell activation (
      • Xia P.
      • Gamble J.R.
      • Rye K.-A.
      • Wang L.
      • Hii C.S.T.
      • Cockerill P.
      • Khew-Goodall Y.
      • Bert A.G.
      • Barter P.J.
      • Vadas M.A.
      ). We found that TNF consistently stimulated SphK activity and the generation of S1P, and blockage of SphK by its inhibitor, DMS, inhibited NF-κB activation and adhesion protein expression. An inhibitory effect of HDL3 was clearly seen in this pathway: HDL3 inhibited (i) SphK activity, (ii) S1P generation, (iii) S1P levels, (iv) ERK activation and (v) nuclear translocation of NF-κB. Moreover, HDL3-induced inhibition of SphK activity is linear correlating with the reduction of adhesion protein expression, and the inhibitory effects of HDL3 were reversed by the addition of S1P (Fig. 5). Taken together, these results strongly indicated that the inhibition of SphK activation by HDL3 could account for its inhibitory effect on adhesion protein expression and endothelial activation.
      The finding that HDL3 not only inhibited the activity and the V max of SphK in a dose dependent manner but also the generation of S1P and its levels in intact cells (Fig. 2) indicated a primary inhibitory effect of HDL3 on the SphK pathway. On the other hand, it is possible that HDL3 may affect endothelial phenotype by an effect on the sphingomyelin-ceramide turnover since HDL3 increased the TNF-dependent ceramide generation and inhibited the reaccumulation of sphingomyelin (Fig. 3A). It is uncertain whether the ceramide accumulation is primarily due to prolonged hydrolysis by sphingomyelinase or to inefficient metabolism by downstream catalysis. The inhibition of adhesion molecule expression by exogenous ceramide (Fig. 3B) indicated a two-pronged inhibition on endothelial activation by HDL: reduction of S1P formation and increase in ceramide levels. Thus, it is assumed that HDL may reset the ‘biostat’ of ceramide and/or S1P to modulate cellular responses to TNF stimulation and to inhibit endothelial cell activation. This sphingolipid biostat has been proposed in regulating a variety of cellular functions such as cell growth, proliferation and cell death (
      • Hannun Y.A.
      ,
      • Cuvillier O.
      • Pirianov G.
      • Kleuser B.
      • Vanek P.G.
      • Coso O.A.
      • Gutkind S.
      • Spiegel S.
      ).
      HDL may exert the protective effect against atherosclerosis by several mechanisms including (i) promoting cholesterol efflux from the peripheral tissues, (ii) reducing LDL oxidation, or (iii) protecting the vasculature against the cytotoxic effect of oxLDL (
      • Navab M.
      • Hama S.Y.
      • Hough G.P.
      • Hedrick C.C.
      • Sorenson R.
      • La Du B.N.
      • Kobashigawa J.A.
      • Fonarow G.C.
      • Berliner J.A.
      • Laks H.
      • Fogelman A.M.
      ,
      • Fielding C.J.
      • Fielding P.E.
      ,
      • Hajjar D.P.
      • Haberland M.E.
      ). In addition to these effects of HDL, we now demonstrate a novel mechanism whereby HDL interrupt intracellular signaling involved in the pathogenesis of atherosclerosis. The inhibitory effect of HDL on the SphK pathway is very likely independent of the above-mentioned known antiatherogenic ability of HDL, since (i) an intact conformation of HDL particle is required for the inhibition; (ii) the delipidated apoA-I is unable to mimic HDL effect; (iii) the inhibition is serum-independent; and (iv) the interaction of LDL or oxLDL is unlikely to be involved in the inhibition of SphK activation (Fig. 4).
      HDL may function on cells in either a receptor-dependent or -independent manner (
      • Hajjar D.P.
      • Haberland M.E.
      ). Our preliminary data that HDL particles have no direct inhibitory effect on SphK activity in vitro(results not shown) suggested that the access of HDL to cell membrane and putative HDL-binding proteins could be necessary. However, the fact that lipid-free apoA-I is able to access putative HDL receptors such as SR-B1 (
      • Acton S.
      • Rigotti A.
      • Landschulz K.T.
      • Xu S.
      • Hobbs H.H.
      • Krieger M.
      ,
      • Mendez A.J.
      • Anantharamaiah G.M.
      • Segrest J.P.
      • Oram J.F.
      ) suggests that binding per se may not completely explain the inhibition of SphK pathway. It is possible that the apolipoprotein is critical to the effect but only when it is in an intact conformation that results from its association with phospholipids. Alternately, HDL induced inhibition of SphK pathway may act through other putative HDL-binding proteins in endothelial cells.
      In conclusion, this is the first demonstration that HDL interrupt a signaling cascade—the SphK pathway that is involved in regulation of endothelial cell activation, a key event in atherogenesis. This provides a mechanistic explanation for the well-documented ability of HDL to protect against atherosclerosis and might ultimately lead to the development of novel strategies for the prevention and treatment of this disease.

      Acknowledgments

      We thank M. Berndt, M. F. Shannon, A. Ullrich, and B. Wattenberg for helpful comments on the manuscript; L. J. Wang and J. Drew for technical assistance and cell cultures; D. Ashby, P. Baker, and M. Clay for the isolation of lipoproteins; staff at the delivery ward of the Women's and Children's Hospital, Adelaide, and Burnside War Memorial Hospital, for collection of umbilical cords, and M. Walker for secretarial assistance.

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