Advertisement

Inhibition of Akt phosphorylation by thrombin, histamine and lysophosphatidylcholine in endothelial cells

Differential role of protein kinase C
  • Brynhildur Thors
    Affiliations
    Institute of Pharmacy, Pharmacology and Toxicology, University of Iceland, P.O. Box 8216, 128 Reykjavik, Iceland
    Search for articles by this author
  • Haraldur Halldórsson
    Affiliations
    Institute of Pharmacy, Pharmacology and Toxicology, University of Iceland, P.O. Box 8216, 128 Reykjavik, Iceland

    Department of Medicine, Landspitalinn-University Hospital, 101 Reykjavik, Iceland
    Search for articles by this author
  • Gudrun D. Clarke
    Affiliations
    Institute of Pharmacy, Pharmacology and Toxicology, University of Iceland, P.O. Box 8216, 128 Reykjavik, Iceland
    Search for articles by this author
  • Gudmundur Thorgeirsson
    Correspondence
    Corresponding author. Present address: Landspitalinn-University Hospital, 101 Reykjavik, Iceland. Tel.: +354-568-0866; fax: +354-543-6467
    Affiliations
    Institute of Pharmacy, Pharmacology and Toxicology, University of Iceland, P.O. Box 8216, 128 Reykjavik, Iceland

    Department of Medicine, Landspitalinn-University Hospital, 101 Reykjavik, Iceland
    Search for articles by this author

      Abstract

      The protein kinase Akt is involved in embryonic vascular development and neoangiogenesis as well as in several endothelial cell functions, including activation of endothelial NO-synthase (eNOS) and promotion of endothelial cell survival. We have examined the effects of G-protein activators thrombin and histamine as well as lysophosphatidylcholine (LPC) on Akt phosphorylation in cultured human umbilical vein endothelial cells (HUVEC). Akt phosphorylation was analyzed with the phosphospecific Akt (Ser473) antibody by Western blotting. While epidermal growth factor (EGF) was a potent stimulator of Akt phosphorylation histamine, thrombin and LPC blocked its activation when used in cotreatment with EGF. Following inhibition or downregulation of protein kinase C (PKC), the inhibitory effect of both histamine and thrombin on the endothelial response to EGF was prevented. Furthermore, stimulation of PKC, using short-term 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment, markedly inhibited the stimulatory effects of EGF on Akt phosphorylation. Rottlerin, an inhibitor of the PKCδ, but not Gö6976, which is an inhibitor of α, β, γ and isoforms, reversed the inhibitory effects of histamine. Conversely, inhibition or downregulation of PKC did not prevent the inhibitory effect of LPC. Akt phosphorylation was also increased by sphingosine 1-phosphate (S1P) treatment and this activity was influenced by the various cotreatments in the same way as the activation by EGF. Overall, this study demonstrated that the G-protein activators thrombin and histamine inhibited both EGF- and S1P-mediated Akt phosphorylation in HUVEC by activation of PKCδ, while the inhibitory effects of LPC were independent of PKCδ.

      Keywords

      To read this article in full you will need to make a payment

      Purchase one-time access:

      Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online access
      One-time access price info
      • For academic or personal research use, select 'Academic and Personal'
      • For corporate R&D use, select 'Corporate R&D Professionals'

      Subscribe:

      Subscribe to Atherosclerosis
      Already a print subscriber? Claim online access
      Already an online subscriber? Sign in
      Institutional Access: Sign in to ScienceDirect

      References

        • Datta S.R.
        • Burnet A.
        • Greenberg M.E.
        Cellular survival: a play in three Akts.
        Genes Dev. 1999; 13: 2905-2927
        • Vanhaesebroeck B.
        • Alessi D.R.
        The PI3K–PDK1 connection: more than just a road to PKB.
        Biochem. J. 2000; 346: 551-576
        • Dimmeler S.
        • Zeiher A.M.
        Akt takes center stage in angiogenesis signaling.
        Circ. Res. 2000; 86: 4-5
        • Fulton D.
        • Gratton J.P.
        • McCabe T.J.
        • et al.
        Regulation of endothelium-derived nitric oxide production by the protein kinase Akt.
        Nature. 1999; 399: 597-601
        • Dimmeler S.
        • Fisslthaler B.
        • Flemming I.
        • Hermann C.
        • Busse R.
        • Zeiher A.M.
        Activation of nitric oxide synthase in endothelial cells via Akt-dependent phosphorylation.
        Nature. 1999; 399: 601-605
        • Gerber H.P.
        • McMurtrey A.
        • Kowalski J.
        • Yan M.
        • Keyt B.A.
        • Dixit V.
        • Ferrara N.
        Vascular endothelial growth factor regulates endothelial cell survival through the phosphatidyl-inositol 3′-kinase/Akt signal transduction pathway requirement for Flk-1/KDR activation.
        J. Biol. Chem. 1998; 273: 30336-30343
        • Murga C.
        • Fukuhara S.
        • Gutkind J.S.
        A novel role for phosphatidylinositol 3-kinase B in signaling from G-protein-coupled receptors to Akt.
        J. Biol. Chem. 2000; 275: 12069-12073
        • Tilton B.
        • Andjelkovic M.
        • Didichenko S.A.
        • Hemmings B.A.
        • Thelen M.
        G-protein-coupled receptors and Fcg-receptors mediate activation of Akt/protein kinase B in human phagocytes.
        J. Biol. Chem. 1997; 272: 28096-28101
        • Takahashi T.
        • Taniguchi T.
        • Konishi H.
        • Kikkawa U.
        • Ishikawa Y.
        • Yokoyama M.
        Activation of Akt/protein kinase B after stimulation with angiotensin II in vascular smooth muscle cells.
        Am. J. Physiol. 1999; 276: H1927-H1934
        • Pyne S.
        • Pyne N.J.
        Sphingosine 1-phosphate signaling in mammalian cells.
        Biochem. J. 2000; 349: 385-402
        • Igarashi J.
        • Bernier S.G.
        • Michel T.
        Sphingosine 1-phosphate and activation of endothelial nitric oxide synthase. Differential regulation of Akt and MAP kinase pathways by EDG and bradykinin receptors in vascular endothelial cells.
        J. Biol. Chem. 2001; 276: 12420-12426
        • Morales-Ruiz M.
        • Lee M.J.
        • Zöllner S.
        • et al.
        Sphingosine 1-phosphate activates Akt, nitric oxide production, and chemotaxis through a Gi protein/phosphoinositide 3-kinase pathway in endothelial cells.
        J. Biol. Chem. 2001; 276: 19672-19677
        • Murata N.
        • Sato K.
        • Kon J.
        • et al.
        Interaction of sphingosine 1-phosphate with plasma components, including lipoproteins, regulates the lipid receptor-mediated actions.
        Biochem. J. 2000; 352: 809-815
        • Kimura T.
        • Sato K.
        • Kuwabara A.
        • et al.
        Sphingosine 1-phosphate may be a major component of plasma lipoproteins responsible for the cytoprotective actions in human umbilical vein endothelial cells.
        J. Biol. Chem. 2001; 276: 31780-31785
        • Chavakis E.
        • Dernbach E.
        • Hermann C.
        • Mondorf U.F.
        • Zeiher A.M.
        • Dimmeler S.
        Oxidized LDL inhibits vascular endothelial growth factor-induced endothelial cell migration by an inhibitory effect on the Akt/endothelial nitric oxide synthase pathway.
        Circulation. 2001; 103: 2102-2107
        • Gao Y.
        • Yokota R.
        • Tang S.
        • Ashton A.W.
        • Ware A.J.
        Reversal of angiogenesis in vitro, induction of apoptosis, and inhibition of Akt phosphorylation in endothelial cells by thromboxane A2.
        Circ. Res. 2000; 87: 739-745
        • Hermann C.
        • Assmus B.
        • Urbich C.
        • Zeiher A.M.
        • Dimmeler S.
        Insulin-mediated stimulation of protein kinase Akt. A potent survival-signaling cascade for endothelial cells.
        Arterioscler. Thromb. Vasc. Biol. 2000; 20: 402-409
        • Barthel A.
        • Nakatani K.
        • Dandekar A.A.
        • Roth R.A.
        Protein kinase C modulates the insulin-stimulated increase in Akt1 and Akt3 activity in 3T3-L1 adipocytes.
        Biochem. Biophys. Res. Commun. 1998; 243: 509-513
        • Zheng W.H.
        • Kar S.
        • Quirion R.
        Stimulation of protein kinase C modulates insulin-like growth factor-1-induced Akt activation in PC12 cells.
        J. Biol. Chem. 2000; 275: 13377-13385
        • Schubert K.M.
        • Scheid M.P.
        • Duronio V.
        Ceramide inhibits protein kinase B/Akt by promoting dephosphorylation of serine 473.
        J. Biol. Chem. 2000; 275: 13330-13335
        • Zhou H.L.
        • Summers S.A.
        • Birnbaum M.J.
        • Pittman R.N.
        Inhibition of Akt kinase by cell-permeable ceramide and its implications for ceramide-induced apoptosis.
        J. Biol. Chem. 1998; 273: 16568-16575
        • Guðmundsdóttir I.J.
        • Halldórsson H.
        • Magnúsdóttir K.
        • Thorgeirsson G.
        Involvement of MAP kinases in the control of cPLA2 and arachidonic acid release in endothelial cells.
        Atherosclerosis. 2001; 156: 81-90
        • Helgadóttir A.
        • Halldórsson H.
        • Magnúsdóttir K.
        • Kjeld M.
        • Thorgeirsson G.
        A role for tyrosine phosphorylation in generation of inositol phosphates and prostacyclin production in endothelial cells.
        Arterioscler. Thromb. Vasc. Biol. 1997; 17: 287-294
        • Halldórsson H.
        • Kjeld M.
        • Thorgeirsson G.
        Role of phosphoinositides in the regulation of endothelial prostacyclin production.
        Arteriosclerosis. 1988; 8: 147-154
      1. Bassa BV, Roh DO, Vaziri NO, Kirchenbaum MA, Kamanna VS. Lysophosphatidylcholine activities mesangial cell PKC and MAP kinase by PLC-1 and tyosine kinase-Ras pathways. Am J Physiol 1999;277 (Renal Physiol 46):F328–37.

        • Sugiyama S.
        • Kugiyama K.
        • Ogata N.
        • et al.
        Biphasic regulation of transcription factor nuclear factor-κB activity in human endothelial cells by lysophosphatidylcholine through protein kinase C-mediated pathway.
        Arterioscler. Thromb. Vasc. Biol. 1998; 18: 568-576
        • Motley E.D.
        • Kabir S.M.
        • Gardner C.D.
        • et al.
        Lysophosphatidylcholine inhibits insulin-induced Akt activation through protein kinase C-alpha in vascular smooth muscle cells.
        Hypertension. 2002; 39: 508-512
        • Cazzolli R.
        • Carpenter L.
        • Biden T.J.
        • Schmitz-Pfeiffer C.
        A role for protein phosphatase 2A-like activity, but not atypical protein kinase Cζ, in the inhibition of protein kinase B/Akt and glycogen synthesis by palmitate.
        Diabetes. 2001; 50: 2210-2218
        • Bourbon N.A.
        • Sandirasegarane L.
        • Kester M.
        Ceramide-induced inhibition of Akt is mediated through protein kinase Cζ: implications for growth arrest.
        J. Biol. Chem. 2002; 277: 3286-3292
        • Stratford S.
        • Dewald D.B.
        • Summers S.
        Ceramide dissociates 3′-phosphoinositide production from pleckstrin homology domain translocation.
        Biochem. J. 2001; 354: 359-368
        • Kureishi Y.
        • Luo Z.
        • Shiojima I.
        • et al.
        The HMG-CoA reductase inhibitor simvastatin activates the protein kinase Akt and promotes angiogenesis in normocholesterolemic animals.
        Nat. Med. 2000; 6: 1004-1010
        • Tanimoto R.
        • Jin Z.
        • Berk B.C.
        Transactivation of vascular endothelial growth factor (VEGF) receptor Flk-1/KDR is involved in sphingosine 1-phosphate-stimulated phosphorylation of Akt and endothelial nitric-oxide synthase (eNOS).
        J. Biol. Chem. 2002; 277: 42997-43001
        • McIntyre T.M.
        • Zimmerman G.A.
        • Prescott S.M.
        Biologically active oxidized phospholipids.
        J. Biol. Chem. 1999; 274: 25189-25192
        • Chisolm III, G.M.
        • Hazen S.L.
        • Fox P.L.
        • Cathcart M.K.
        The oxidation of lipoproteins by monocytes–macrophages: biochemical and biological mechanisms.
        J. Biol. Chem. 1999; 274: 25959-25962
        • Yokoyama M.
        • Hirata K.
        • Miyake R.
        • Akita H.
        • Ishikawa Y.
        • Fukuzaki H.
        Lysophosphatidylcholine: essential role in the inhibition of endothelium-dependent vasorelaxation by oxidized low-density lipoprotein.
        Biochem. Biophys. Res. Commun. 1990; 168: 301-308
        • Kugiyama K.
        • Kerns S.A.
        • Morrisett J.D.
        • Roberts R.
        • Henry P.D.
        Impairment of endothelium-dependent arterial relaxation by lysoledithin in modified low-density lipoproteins.
        Nature. 1990; 344: 160-162
        • Ross R.
        Atherosclerosis—an inflammatory disease.
        N. Engl. J. Med. 1999; 340: 115-126
        • Steinberg D.
        Low-density lipoprotein oxidation and its pathobiological significance.
        J. Biol. Chem. 1997; 272: 20963-20966
        • Tesfamariam B.
        • Brown M.L.
        • Cohen R.A.
        Elevated glucose impairs endothelium-dependent relaxation by activating protein kinase C.
        J. Clin. Invest. 1991; 87: 1643-1648
        • Kugiyama K.
        • Ohgushi M.
        • Sugiyama S.
        • Murohara T.
        • Fununaga K.
        • Miyamoto E.
        • Yasue H.
        Lysophosphatidylcholine inhibits surface receptor-mediated intracellular signals in endothelial cells by a pathway involving protein kinase C activation.
        Circ. Res. 1992; 71: 1422-1428
        • Chen L.
        • Liang B.
        • Froese D.E.
        • et al.
        Oxidative modification of low-density lipoprotein in normal and hyperlipidemic patients: effect of lysophosphatidylcholine composition on vascular relaxation.
        J. Lipid Res. 1997; 38: 546-553
        • Bommakanti R.K.
        • Vinayak S.
        • Simonds W.F.
        Dual regulation of Akt/protein kinase B by heterotrimeric G-protein subunits.
        J. Biol. Chem. 2000; 275: 38870-38876
        • Exton J.H.
        Regulation of phosphoinositide phospholipases by hormones, neurotransmitters, and other agonists linked to G-proteins.
        Annu. Rev. Pharmacol. Toxicol. 1996; 36: 481-509
        • Nishizuka Y.
        Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C.
        Science. 1992; 258: 607-614
        • Haller H.
        • Ziegler W.
        • Lindschau C.
        • Luft F.C.
        Endothelial cell tyrosine kinase receptor and G-protein-coupled receptor activation involves distinct protein kinase C isoforms.
        Arterioscler. Thromb. Vasc. Biol. 1996; 16: 678-686
        • Mattila P.
        • Majuri M.L.
        • Tiisala S.
        • Renkonen R.
        Expression of six protein kinase C isotypes in endothelial cells.
        Life Sci. 1994; 55: 1253-1260
        • Davies S.P.
        • Reddy H.
        • Caivano M.
        • Cohen P.
        Specificity and mechanism of action of some commonly used protein kinase inhibitors.
        Biochem. J. 2000; 351: 95-105
        • Le Good J.A.
        • Ziegler W.H.
        • Parekh D.B.
        • Alessi D.R.
        • Cohen P.
        • Parker P.J.
        Protein kinase C isotypes controlled by phophoinositide 3-kinase through the protein kinase PDK1.
        Science. 1998; 281: 2042-2045
        • Watanabe T.
        • Ono Y.
        • Taniyama Y.
        • et al.
        Cell division arrest induced by phorbol ester in CHO cells overexpressing protein kinase C-δ subspecies.
        Proc. Natl. Acad. Sci. USA. 1992; 89: 10159-10163
        • Mischak H.
        • Goodnight J.A.
        • Kolch W.
        • et al.
        Overexpression of protein kinase C-δ and -ε in NIH 3T3 cells induces opposite effects on growth, morphology, anchorage dependence, and tumorigenicity.
        J. Biol. Chem. 1993; 268: 6090-6096
        • Emoto Y.
        • Manome Y.
        • Meinhardt G.
        • et al.
        Proteolytic activation of protein kinase C-δ by an ICE-like protease in apoptotic cells.
        EMBO J. 1995; 14: 6148-6156
        • Li L.
        • Lorenzo P.S.
        • Bogi K.
        • Blumberg P.M.
        • Yuspa S.H.
        Protein kinase C-δ targets mitochondria, alters mitochondrial membrane potential, and induces apoptosis in normal and neoplastic keratinocytes when overexpressed by an adenoviral vector.
        Mol. Cell. Biol. 1999; 19: 8547-8558
        • Takahashi T.
        • Shibuya M.
        The overexpression of PKCδ is involved in vascular endothelial growth factor-resistant apoptosis in cultured primary sinusoidal endothelial cells.
        Biochem. Biophys. Res. Commun. 2001; 280: 415-420
        • Steinberg D.
        • Parthasarathy S.
        • Carew T.E.
        • Khoo J.C.
        Beyond cholesterol. Modification of low-density lipoprotein that increase its atherogenicity.
        N. Engl. J. Med. 1989; 320: 915-924
        • Witztum J.L.
        • Steinberg D.
        Role of oxidized low-density lipoprotein in atherogenesis.
        J. Clin. Invest. 1991; 88: 1785-1792
        • Kume N.
        • Cybulsky M.I.
        • Gimbrone Jr., M.A.
        Lysophosphatidylcholine, a component of atherogenic lipoproteins, induces mononuclear leukocyte adhesion molecules in cultured human and rabbit arterial endothelial cells.
        J. Clin. Invest. 1992; 90: 1138-1144
        • Kume N.
        • Gimbrone Jr., M.A.
        Lysophosphatidylcholine transcriptionally induces growth factor gene expression in cultured human endothelial cells.
        J. Clin. Invest. 1994; 93: 907-911
        • Mangin Jr., E.L.
        • Kugiyama K.
        • Nguy J.H.
        • Kerns S.A.
        • Henry P.D.
        Effects of lysolipids and oxidatively modified low-density lipoprotein on endothelium-dependent relaxation of rabbit aorta.
        Circ. Res. 1993; 72: 161-166
        • Fulton D.
        • Gratton J.
        • Sessa W.C.
        Post-translational control of endothelial nitric-oxide synthase: why isn't calcium/calmodulin enough?.
        JPET. 2001; 299: 818-824
        • Chen Z.P.
        • Mitchelhill K.I.
        • Michell B.J.
        • et al.
        AMP-activated protein kinase phosphorylation of endothelial NO-synthase.
        FEBS Lett. 1999; 443: 285-289