Mechanism and Role of High Density Lipoprotein-induced Activation of AMP-activated Protein Kinase in Endothelial Cells
2009; Elsevier BV; Volume: 285; Issue: 7 Linguagem: Inglês
10.1074/jbc.m109.043869
ISSN1083-351X
AutoresTakao Kimura, Hideaki Tomura, Kōichi Sato, Masaaki Ito, Isao Matsuoka, Dong‐Soon Im, Atsushi Kuwabara, Chihiro Mogi, Hiroshi Itoh, Hitoshi Kurose, Masami Murakami, Fumikazu Okajima,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoThe upstream signaling pathway leading to the activation of AMP-activated protein kinase (AMPK) by high density lipoprotein (HDL) and the role of AMPK in HDL-induced antiatherogenic actions were investigated. Experiments using genetic and pharmacological tools showed that HDL-induced activation of AMPK is dependent on both sphingosine 1-phosphate receptors and scavenger receptor class B type I through calcium/calmodulin-dependent protein kinase kinase and, for scavenger receptor class B type I system, additionally serine-threonine kinase LKB1 in human umbilical vein endothelial cells. HDL-induced activation of Akt and endothelial NO synthase, stimulation of migration, and inhibition of monocyte adhesion and adhesion molecule expression were dependent on AMPK activation. The inhibitory role of AMPK in the adhesion molecule expression and monocyte adhesion on endothelium of mouse aorta was confirmed in vivo and ex vivo. On the other hand, stimulation of ERK and proliferation were hardly affected by AMPK knockdown but completely inhibited by an N17Ras, whereas the dominant-negative Ras was ineffective for AMPK activation. In conclusion, dual HDL receptor systems differentially regulate AMPK activity through calcium/calmodulin-dependent protein kinase kinase and/or LKB1. Several HDL-induced antiatherogenic actions are regulated by AMPK, but proliferation-related actions are regulated by Ras rather than AMPK. The upstream signaling pathway leading to the activation of AMP-activated protein kinase (AMPK) by high density lipoprotein (HDL) and the role of AMPK in HDL-induced antiatherogenic actions were investigated. Experiments using genetic and pharmacological tools showed that HDL-induced activation of AMPK is dependent on both sphingosine 1-phosphate receptors and scavenger receptor class B type I through calcium/calmodulin-dependent protein kinase kinase and, for scavenger receptor class B type I system, additionally serine-threonine kinase LKB1 in human umbilical vein endothelial cells. HDL-induced activation of Akt and endothelial NO synthase, stimulation of migration, and inhibition of monocyte adhesion and adhesion molecule expression were dependent on AMPK activation. The inhibitory role of AMPK in the adhesion molecule expression and monocyte adhesion on endothelium of mouse aorta was confirmed in vivo and ex vivo. On the other hand, stimulation of ERK and proliferation were hardly affected by AMPK knockdown but completely inhibited by an N17Ras, whereas the dominant-negative Ras was ineffective for AMPK activation. In conclusion, dual HDL receptor systems differentially regulate AMPK activity through calcium/calmodulin-dependent protein kinase kinase and/or LKB1. Several HDL-induced antiatherogenic actions are regulated by AMPK, but proliferation-related actions are regulated by Ras rather than AMPK. IntroductionCirculating levels of high density lipoprotein (HDL) 2The abbreviations used are: HDLhigh density lipoproteinAMPKAMP-activated protein kinaseNOnitric oxideNOSNO synthaseeNOSendothelial NO synthaserHDLreconstituted HDLapoAapolipoprotein AS1Psphingosine 1-phosphateECendothelial cellHUVEChuman umbilical vein ECCaMKKcalcium/calmodulin-dependent protein kinase kinaseERKextracellular signal-regulated kinaseSR-BIscavenger receptor class B type IVCAM-1vascular cell adhesion moleculePI3Kphosphatidylinositol 3-kinasePBSphosphate-buffered salinePTXpertussis toxinPDZPSD-95/Dlg/ZO-1BSAbovine serum albuminAICAR5-aminoimidazole-4-carboxamide ribonucleosideZMP5-aminoimidazole-4-carboxamide 1-β-d-ribofuranosyl monophosphatesiRNAsmall interfering RNAMES4-morpholineethanesulfonic acidTNF-αtumor necrosis factor-αFBSfetal bovine serum. are inversely correlated to the risk of atherosclerosis and associated cardiovascular disease (1Assmann G. Gotto Jr., A.M. Circulation. 2004; 109: III8-III13Crossref PubMed Google Scholar, 2Rader D.J. J. Clin. Invest. 2006; 116: 3090-3100Crossref PubMed Scopus (469) Google Scholar). HDL promotes the process of cholesterol transport from arterial and other peripheral cells to the liver and excretes it as bile acids. The so-called reverse cholesterol transport is thought to be important for antiatherogenic properties of HDL (1Assmann G. Gotto Jr., A.M. Circulation. 2004; 109: III8-III13Crossref PubMed Google Scholar, 2Rader D.J. J. Clin. Invest. 2006; 116: 3090-3100Crossref PubMed Scopus (469) Google Scholar). HDL also exerts a variety of actions that are independent of reverse cholesterol transport; for example, HDL protects endothelium from its dysfunction, which is composed of several responses in endothelial cells (ECs), including proliferation, migration, nitric oxide (NO) production, and inhibition of adhesion molecule expression (3Nofer J.R. Walter M. Assmann G. Expert Rev. Cardiovasc. Ther. 2005; 3: 1071-1086Crossref PubMed Scopus (39) Google Scholar, 4Okajima F. Sato K. Kimura T. Endocr. J. 2009; 56: 317-334Crossref PubMed Scopus (38) Google Scholar). The adhesion of monocytes and leukocytes on endothelium is thought to be an early event of atherogenic or inflammatory responses (4Okajima F. Sato K. Kimura T. Endocr. J. 2009; 56: 317-334Crossref PubMed Scopus (38) Google Scholar). AMP-activated protein kinase (AMPK) has been shown to be involved in energy homeostasis and the regulation of a variety of cell functions (5Witters L.A. Kemp B.E. Means A.R. Trends Biochem. Sci. 2006; 31: 13-16Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 6Towler M.C. Hardie D.G. Circ. Res. 2007; 100: 328-341Crossref PubMed Scopus (1039) Google Scholar, 7Witczak C.A. Sharoff C.G. Goodyear L.J. Cell. Mol. Life Sci. 2008; 65: 3737-3755Crossref PubMed Scopus (175) Google Scholar). In ECs, AMPK has been shown to regulate the activity of endothelial NO synthase (eNOS) and NO synthesis evoked by a variety of extracellular stimuli, including HDL (8Drew B.G. Fidge N.H. Gallon-Beaumier G. Kemp B.E. Kingwell B.A. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 6999-7004Crossref PubMed Scopus (157) Google Scholar), sphingosine 1-phosphate (S1P) (9Levine Y.C. Li G.K. Michel T. J. Biol. Chem. 2007; 282: 20351-20364Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar), thrombin (10Thors B. Halldórsson H. Thorgeirsson G. FEBS Lett. 2004; 573: 175-180Crossref PubMed Scopus (77) Google Scholar), vascular endothelial growth factor (9Levine Y.C. Li G.K. Michel T. J. Biol. Chem. 2007; 282: 20351-20364Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 11Youn J.Y. Wang T. Cai H. Circ. Res. 2009; 104: 50-59Crossref PubMed Scopus (98) Google Scholar), and adiponectin (12Ouchi N. Kobayashi H. Kihara S. Kumada M. Sato K. Inoue T. Funahashi T. Walsh K. J. Biol. Chem. 2004; 279: 1304-1309Abstract Full Text Full Text PDF PubMed Scopus (657) Google Scholar). AMPK has also been suggested to be involved in the inhibition of monocyte adhesion and adhesion molecule expression in ECs, although the role of eNOS in the regulation of cell adhesion is controversial (13Hattori Y. Suzuki K. Hattori S. Kasai K. Hypertension. 2006; 47: 1183-1188Crossref PubMed Scopus (372) Google Scholar, 14Prasad R. Giri S. Nath N. Singh I. Singh A.K. J. Neurosci. Res. 2006; 84: 614-625Crossref PubMed Scopus (54) Google Scholar, 15Gaskin F.S. Kamada K. Yusof M. Korthuis R.J. Am. J. Physiol. Heart Circ. Physiol. 2007; 292: H326-H332Crossref PubMed Scopus (48) Google Scholar, 16Ewart M.A. Kohlhaas C.F. Salt I.P. Arterioscler. Thromb. Vasc. Biol. 2008; 28: 2255-2257Crossref PubMed Scopus (51) Google Scholar).We and others have shown that HDL is a carrier of potent bioactive lipid mediators, including S1P, in addition to apolipoproteins (4Okajima F. Sato K. Kimura T. Endocr. J. 2009; 56: 317-334Crossref PubMed Scopus (38) Google Scholar). Recent studies have shown that HDL induced eNOS activation through lipoprotein-associated apoA-I and/or S1P, although the roles of dual receptors, i.e. scavenger receptor class B type I (SR-BI) and S1P receptors, are still controversial (4Okajima F. Sato K. Kimura T. Endocr. J. 2009; 56: 317-334Crossref PubMed Scopus (38) Google Scholar, 17Nofer J.R. van der Giet M. Tölle M. Wolinska I. von Wnuck Lipinski K. Baba H.A. Tietge U.J. Gödecke A. Ishii I. Kleuser B. Schäfers M. Fobker M. Zidek W. Assmann G. Chun J. Levkau B. J. Clin. Invest. 2004; 113: 569-581Crossref PubMed Scopus (575) Google Scholar, 18Assanasen C. Mineo C. Seetharam D. Yuhanna I.S. Marcel Y.L. Connelly M.A. Williams D.L. de la Llera-Moya M. Shaul P.W. Silver D.L. J. Clin. Invest. 2005; 115: 969-977Crossref PubMed Scopus (133) Google Scholar, 19Kimura T. Tomura H. Mogi C. Kuwabara A. Damirin A. Ishizuka T. Sekiguchi A. Ishiwara M. Im D.S. Sato K. Murakami M. Okajima F. J. Biol. Chem. 2006; 281: 37457-37467Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 20Zhu W. Saddar S. Seetharam D. Chambliss K.L. Longoria C. Silver D.L. Yuhanna I.S. Shaul P.W. Mineo C. Circ. Res. 2008; 102: 480-487Crossref PubMed Scopus (98) Google Scholar). Thus, several independent reports have shown that HDL and S1P stimulate AMPK and eNOS in ECs. However, roles of SR-BI and S1P receptors and their signaling mechanism in HDL-induced AMPK activation have not been fully characterized. Moreover, roles of AMPK in HDL-regulated functions related to the protection of endothelial dysfunctions other than eNOS activation remain poorly understood. In this study, we examined these unanswered questions in human umbilical vein endothelial cells (HUVECs) in vitro and mouse aorta in vivo.DISCUSSIONIn this study, we obtained the following important new findings with respect to the mechanism and role of HDL-induced activation of AMPK in ECs. First, HDL stimulated AMPK activation through both S1P receptors/Gi proteins and SR-BI/PDZK1. Second, CaMKK plays a role in the activation of AMPK by both receptor systems, but LKB1 may be involved in SR-BI signaling but not in S1P receptor signaling. Third, the HDL-induced activation of AMPK resulted in eNOS activation, through PI3K/Akt, and the subsequent inhibition of expression of the adhesion molecule, VCAM-1, thereby inhibiting monocyte adhesion to ECs. The role of AMPK in adhesion molecule expression was confirmed by in vivo and ex vivo experiments of mouse aorta with AICAR, an AMPK activator. Finally, HDL-induced antiatherogenic actions seem to be regulated by two independent signaling molecules, i.e. AMPK and Ras. Postulated scheme for signaling pathways for HDL-induced protection of endothelial dysfunction is shown in Fig. 10.Although previous studies have shown that HDL (8Drew B.G. Fidge N.H. Gallon-Beaumier G. Kemp B.E. Kingwell B.A. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 6999-7004Crossref PubMed Scopus (157) Google Scholar) and S1P (9Levine Y.C. Li G.K. Michel T. J. Biol. Chem. 2007; 282: 20351-20364Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar) activate AMPK and eNOS in ECs, the action mechanism of HDL is controversial. Yuhanna et al. (38Yuhanna I.S. Zhu Y. Cox B.E. Hahner L.D. Osborne-Lawrence S. Lu P. Marcel Y.L. Anderson R.G. Mendelsohn M.E. Hobbs H.H. Shaul P.W. Nat. Med. 2001; 7: 853-857Crossref PubMed Scopus (634) Google Scholar) first reported that HDL activates eNOS through SR-BI; however, they failed to detect eNOS activation by lipid-free apoA-I in ECs. Their group later reported that reconstituted apoA-I with phosphatidylcholine but without or with low concentrations of cholesterol can stimulate eNOS activation (18Assanasen C. Mineo C. Seetharam D. Yuhanna I.S. Marcel Y.L. Connelly M.A. Williams D.L. de la Llera-Moya M. Shaul P.W. Silver D.L. J. Clin. Invest. 2005; 115: 969-977Crossref PubMed Scopus (133) Google Scholar). On the other hand, Drew et al. (8Drew B.G. Fidge N.H. Gallon-Beaumier G. Kemp B.E. Kingwell B.A. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 6999-7004Crossref PubMed Scopus (157) Google Scholar) reported that lipid-free apoA-I stimulated eNOS by their interaction, in association with AMPK activation. The present results (Fig. 1A) and the results of the previous study (19Kimura T. Tomura H. Mogi C. Kuwabara A. Damirin A. Ishizuka T. Sekiguchi A. Ishiwara M. Im D.S. Sato K. Murakami M. Okajima F. J. Biol. Chem. 2006; 281: 37457-37467Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar) are consistent with those from Assanasen et al. (18Assanasen C. Mineo C. Seetharam D. Yuhanna I.S. Marcel Y.L. Connelly M.A. Williams D.L. de la Llera-Moya M. Shaul P.W. Silver D.L. J. Clin. Invest. 2005; 115: 969-977Crossref PubMed Scopus (133) Google Scholar), who suggested that cholesterol movement through SR-BI is critical for eNOS activation by HDL. Thus, there are many reports as to HDL-induced activation of eNOS; however, the participation of HDL receptors, i.e. SR-BI and S1P receptors, in HDL-induced AMPK activation has not yet been investigated. This study showed that the SR-BI/PZDK1 system and S1P receptor/Gi protein system contribute equally to the HDL-induced activation of AMPK in ECs. Whether internalization of apoA-I is essential, as suggested by the previous study (8Drew B.G. Fidge N.H. Gallon-Beaumier G. Kemp B.E. Kingwell B.A. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 6999-7004Crossref PubMed Scopus (157) Google Scholar), remains unknown. It should be noted, however, that the internalization of apoA-I was examined by lipid-free apoA-I but not by native HDL (8Drew B.G. Fidge N.H. Gallon-Beaumier G. Kemp B.E. Kingwell B.A. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 6999-7004Crossref PubMed Scopus (157) Google Scholar).The role of CaMKK in calcium-mobilizing G protein-coupled receptors, including S1P receptors (9Levine Y.C. Li G.K. Michel T. J. Biol. Chem. 2007; 282: 20351-20364Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar), in AMPK activation has been reported (39Hutchinson D.S. Summers R.J. Bengtsson T. Pharmacol. Ther. 2008; 119: 291-310Crossref PubMed Scopus (61) Google Scholar). Indeed, S1P has been shown to remarkably elevate the intracellular Ca2+ concentration in ECs (4Okajima F. Sato K. Kimura T. Endocr. J. 2009; 56: 317-334Crossref PubMed Scopus (38) Google Scholar). In contrast, we failed to detect a significant increase in the intracellular Ca2+ concentration by rHDL, an SR-BI ligand (data not shown). The participation of CaMKK in the SR-BI-mediated AMPK signaling, however, was suggested by the finding that knockdown of CaMKKβ and inhibition of the enzyme by STO-609 remarkably attenuated either S1P- or rHDL-induced activation of AMPK, Akt, and eNOS. It should be noted, however, that CaMKKβ is known to be substantially activated at the resting intracellular Ca2+ concentration, although the enzyme activity is further stimulated by the increase in its concentration (40Anderson K.A. Means R.L. Huang Q.H. Kemp B.E. Goldstein E.G. Selbert M.A. Edelman A.M. Fremeau R.T. Means A.R. J. Biol. Chem. 1998; 273: 31880-31889Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). The components of the SR-BI-mediated eNOS regulatory system seem to be localized in caveolae, the cholesterol-rich microdomain of the plasma membrane (36Mineo C. Shaul P.W. Cardiovasc. Res. 2006; 70: 31-41Crossref PubMed Scopus (44) Google Scholar), which are also known to be a critical platform for signaling molecules involved in a variety of signal transduction systems, including intracellular Ca2+ homeostasis (41Isshiki M. Anderson R.G. Traffic. 2003; 4: 717-723Crossref PubMed Scopus (112) Google Scholar). Thus, it is not surprising that calcium/calmodulin-sensitive eNOS and CaMKK are activated without global increases in intracellular Ca2+ concentration in response to SR-BI stimulation, as is the case for the estrogen activation of eNOS (42Caulin-Glaser T. García-Cardeña G. Sarrel P. Sessa W.C. Bender J.R. Circ. Res. 1997; 81: 885-892Crossref PubMed Scopus (436) Google Scholar).In addition, LKB1 also appears to be involved in the AMPK activation through SR-BI. Similarly to CaMKK, LKB1 is also reported to be constitutively active (43Sanders M.J. Grondin P.O. Hegarty B.D. Snowden M.A. Carling D. Biochem. J. 2007; 403: 139-148Crossref PubMed Scopus (517) Google Scholar). Fogarty and Hardie (44Fogarty S. Hardie D.G. J. Biol. Chem. 2009; 284: 77-84Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) have recently shown that even though LKB1 is phosphorylated at Ser-431 (Ser-428 in human), its phosphorylation does not change the enzyme activity. Xie et al. (32Xie Z. Dong Y. Scholz R. Neumann D. Zou M.H. Circulation. 2008; 117: 952-962Crossref PubMed Scopus (224) Google Scholar) have also shown that phosphorylation of Ser-428/431 resulted in only a marginal increase in the enzyme activity. Interestingly, however, they observed that Ser-428/431 phosphorylation by metformin is necessary for LKB1 nuclear export to cytosol, where LKB1 interacts with AMPK in ECs (32Xie Z. Dong Y. Scholz R. Neumann D. Zou M.H. Circulation. 2008; 117: 952-962Crossref PubMed Scopus (224) Google Scholar). Thus, Ser-428/431 phosphorylation is essential for the LKB1 nuclear export and hence LKB1 activation of AMPK at least in ECs. The export of LKB1 into cytosol has also been shown to be associated with adiponectin activation of AMPK in muscle cells (45Zhou L. Deepa S.S. Etzler J.C. Ryu J. Mao X. Fang Q. Liu D.D. Torres J.M. Jia W. Lechleiter J.D. Liu F. Dong L.Q. J. Biol. Chem. 2009; 284: 22426-22435Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar) and breast cancer cells (46Taliaferro-Smith L. Nagalingam A. Zhong D. Zhou W. Saxena N.K. Sharma D. Oncogene. 2009; 28: 2621-2633Crossref PubMed Scopus (135) Google Scholar). Consistent with these reports, HDL, S1P, and rHDL stimulated LKB1 nuclear export in association with phosphorylation of Ser-428 in HUVECs in this study. Thus, stimulation of either S1P receptors or SR-BI causes the translocation of LKB1 and hence potentially activates AMPK.Nevertheless, LKB1 was unable to activate AMPK when CaMKK was inhibited by a specific inhibitor or down-regulated by CaMKK-siRNA. The peculiar observation may be deeply related to the regulatory mechanism of AMPK activity. It is well known that AMPK activity is regulated by the balance of phosphorylation by kinases, such as LKB1 and CaMKK, and dephosphorylation by protein phosphatase, possibly PP2Cα (7Witczak C.A. Sharoff C.G. Goodyear L.J. Cell. Mol. Life Sci. 2008; 65: 3737-3755Crossref PubMed Scopus (175) Google Scholar, 43Sanders M.J. Grondin P.O. Hegarty B.D. Snowden M.A. Carling D. Biochem. J. 2007; 403: 139-148Crossref PubMed Scopus (517) Google Scholar). Under the low CaMKK activity status, phosphorylation activity by LKB1 may be too low to overcome the dephosphorylation activity when either S1P receptors or SR-BI was stimulated. Similarly, under the low LKB1 activity status, CaMKK activity is too low to overcome the phosphatase activity in the case of SR-BI stimulation. Thus, SR-BI-mediated AMPK activation requires simultaneous activation of both CaMKK and LKB1. In the case of S1P receptor stimulation, however, strong activation of CaMKK by high Ca2+ supply may cause the AMPK activation without the aid of LKB1. In relation to this, it is interesting that AICAR can stimulate AMPK in association with LKB1 phosphorylation and nuclear export even under the low CaMKK activity status (Fig. 2C and Fig. 3, A and B). AMP binds to the γ-subunit of AMPK and thereby inhibits dephosphorylation by phosphatase. Thus, although LKB1 is not the AMP-binding site, the phosphorylation enzyme is necessary for the activation of AMPK by AMP. In the case of AICAR, the ribonucleoside is transported into cells by the adenosine transporter and metabolized by adenosine kinase into ZMP, an AMP analogue. ZMP then functions like endogenous AMP. Thus, similar to endogenous AMP, ZMP would prevent dephosphorylation of AMPK by phosphatase, such as PP2Cα (7Witczak C.A. Sharoff C.G. Goodyear L.J. Cell. Mol. Life Sci. 2008; 65: 3737-3755Crossref PubMed Scopus (175) Google Scholar, 30Corton J.M. Gillespie J.G. Hawley S.A. Hardie D.G. Eur. J. Biochem. 1995; 229: 558-565Crossref PubMed Scopus (1018) Google Scholar). Under the condition of inhibiting dephosphorylation of AMPK, LKB1 may be able to phosphorylate and activate AMPK without the aid of other phosphorylation enzymes. It remains unknown, however, how AICAR stimulates LKB1 phosphorylation and nuclear export.This study showed that wortmannin, a PI3K inhibitor, inhibited HDL- and S1P-induced phosphorylation of Akt and eNOS without any significant change in AMPK phosphorylation. Knockdown of AMPK almost completely inhibited the phosphorylation of Akt and eNOS, suggesting that AMPK is an upstream signaling molecule of PI3K/Akt, which regulates the eNOS activity. The finding that an AMPK activator, AICAR, stimulated Akt and eNOS further supports this conclusion. The signaling pathway of AMPK/PI3K/Akt/eNOS is consistent with those suggested for adiponectin-induced eNOS activation in HUVECs (12Ouchi N. Kobayashi H. Kihara S. Kumada M. Sato K. Inoue T. Funahashi T. Walsh K. J. Biol. Chem. 2004; 279: 1304-1309Abstract Full Text Full Text PDF PubMed Scopus (657) Google Scholar) and S1P- and vascular endothelial growth factor-induced eNOS activation in bovine aortic ECs (9Levine Y.C. Li G.K. Michel T. J. Biol. Chem. 2007; 282: 20351-20364Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). However, the eNOS activation mechanism in ECs is complex. Thors et al. (10Thors B. Halldórsson H. Thorgeirsson G. FEBS Lett. 2004; 573: 175-180Crossref PubMed Scopus (77) Google Scholar) showed that thrombin and histamine stimulate eNOS via the AMPK-mediated pathway independent of PI3K/Akt in HUVECs. On the other hand, it was reported that AMPK is not involved in eNOS activation by insulin (33Morrow V.A. Foufelle F. Connell J.M. Petrie J.R. Gould G.W. Salt I.P. J. Biol. Chem. 2003; 278: 31629-31639Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar) and vascular endothelial growth factor (34Nagata D. Mogi M. Walsh K. J. Biol. Chem. 2003; 278: 31000-31006Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar). Further complicating the issue, PI3K works as an upstream regulator of AMPK as indicated by the finding that PI3K inhibitors inactivated AMPK (11Youn J.Y. Wang T. Cai H. Circ. Res. 2009; 104: 50-59Crossref PubMed Scopus (98) Google Scholar). Thus, the signaling pathways of AMPK, PI3K/Akt, and eNOS seem to be differentially regulated depending on the differences in sources (or sites) or species, even in vascular ECs. The differences in time and stimuli employed may also partly explain the different regulatory mechanism of eNOS activation (35Hu Z. Chen J. Wei Q. Xia Y. J. Biol. Chem. 2008; 283: 25256-25263Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Further experiments are required to determine the relationship of the regulation of these important signaling enzyme activities.ATP-consuming proliferation is expected to be inhibited by AMPK activation (5Witters L.A. Kemp B.E. Means A.R. Trends Biochem. Sci. 2006; 31: 13-16Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 6Towler M.C. Hardie D.G. Circ. Res. 2007; 100: 328-341Crossref PubMed Scopus (1039) Google Scholar, 7Witczak C.A. Sharoff C.G. Goodyear L.J. Cell. Mol. Life Sci. 2008; 65: 3737-3755Crossref PubMed Scopus (175) Google Scholar). On the other hand, recent studies have shown that AMPK (34Nagata D. Mogi M. Walsh K. J. Biol. Chem. 2003; 278: 31000-31006Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar) and LKB1, an upstream regulator of AMPK (47Londesborough A. Vaahtomeri K. Tiainen M. Katajisto P. Ekman N. Vallenius T. Mäkelä T.P. Development. 2008; 135: 2331-2338Crossref PubMed Scopus (33) Google Scholar), are essential for angiogenesis, which must be associated with migration and proliferation of ECs. Moreover, AICAR, an AMPK stimulator, has been shown to activate ERK in osteoblasts (48Kim J.E. Ahn M.W. Baek S.H. Lee I.K. Kim Y.W. Kim J.Y. Dan J.M. Park S.Y. Bone. 2008; 43: 394-404Crossref PubMed Scopus (72) Google Scholar, 49Kanazawa I. Yamaguchi T. Yano S. Yamauchi M. Sugimoto T. Am. J. Physiol. Endocrinol. Metab. 2009; 296: E139-E146Crossref PubMed Scopus (78) Google Scholar). Thus, the activation of AMPK potentially acts on ERK and proliferation in an inhibitory or stimulatory manner. Our results showed that HDL-induced ERK activation and proliferation were independent of AMPK, whereas migration response to HDL was depending on AMPK, in ECs. Instead, HDL utilizes the Ras system, which is an independent signaling molecule from AMPK, to induce proliferation. This signaling cascade resembles the insulin signaling pathways; insulin stimulates the PI3K/Akt pathway, which usually regulates a variety of differentiated functions of the cells, and the Ras/ERK pathway, which regulates proliferation (50Saltiel A.R. Pessin J.E. Trends Cell Biol. 2002; 12: 65-71Abstract Full Text Full Text PDF PubMed Scopus (499) Google Scholar). In conclusion, HDL activates AMPK through the apoA-I/SR-BI/PDZK1 and the S1P/S1P receptor/Gi protein systems, thereby stimulating PI3K/Akt and the subsequent eNOS activation, cell migration, and the inhibition of adhesion molecule expression. Both systems seem to be regulated by CaMKK and, for the SR-BI system, additionally by LKB1. HDL also stimulates ERK and proliferation, which are mediated by Ras but not by AMPK. Thus, HDL exerts antiatherogenic actions through dual systems involving AMPK and Ras. IntroductionCirculating levels of high density lipoprotein (HDL) 2The abbreviations used are: HDLhigh density lipoproteinAMPKAMP-activated protein kinaseNOnitric oxideNOSNO synthaseeNOSendothelial NO synthaserHDLreconstituted HDLapoAapolipoprotein AS1Psphingosine 1-phosphateECendothelial cellHUVEChuman umbilical vein ECCaMKKcalcium/calmodulin-dependent protein kinase kinaseERKextracellular signal-regulated kinaseSR-BIscavenger receptor class B type IVCAM-1vascular cell adhesion moleculePI3Kphosphatidylinositol 3-kinasePBSphosphate-buffered salinePTXpertussis toxinPDZPSD-95/Dlg/ZO-1BSAbovine serum albuminAICAR5-aminoimidazole-4-carboxamide ribonucleosideZMP5-aminoimidazole-4-carboxamide 1-β-d-ribofuranosyl monophosphatesiRNAsmall interfering RNAMES4-morpholineethanesulfonic acidTNF-αtumor necrosis factor-αFBSfetal bovine serum. are inversely correlated to the risk of atherosclerosis and associated cardiovascular disease (1Assmann G. Gotto Jr., A.M. Circulation. 2004; 109: III8-III13Crossref PubMed Google Scholar, 2Rader D.J. J. Clin. Invest. 2006; 116: 3090-3100Crossref PubMed Scopus (469) Google Scholar). HDL promotes the process of cholesterol transport from arterial and other peripheral cells to the liver and excretes it as bile acids. The so-called reverse cholesterol transport is thought to be important for antiatherogenic properties of HDL (1Assmann G. Gotto Jr., A.M. Circulation. 2004; 109: III8-III13Crossref PubMed Google Scholar, 2Rader D.J. J. Clin. Invest. 2006; 116: 3090-3100Crossref PubMed Scopus (469) Google Scholar). HDL also exerts a variety of actions that are independent of reverse cholesterol transport; for example, HDL protects endothelium from its dysfunction, which is composed of several responses in endothelial cells (ECs), including proliferation, migration, nitric oxide (NO) production, and inhibition of adhesion molecule expression (3Nofer J.R. Walter M. Assmann G. Expert Rev. Cardiovasc. Ther. 2005; 3: 1071-1086Crossref PubMed Scopus (39) Google Scholar, 4Okajima F. Sato K. Kimura T. Endocr. J. 2009; 56: 317-334Crossref PubMed Scopus (38) Google Scholar). The adhesion of monocytes and leukocytes on endothelium is thought to be an early event of atherogenic or inflammatory responses (4Okajima F. Sato K. Kimura T. Endocr. J. 2009; 56: 317-334Crossref PubMed Scopus (38) Google Scholar). AMP-activated protein kinase (AMPK) has been shown to be involved in energy homeostasis and the regulation of a variety of cell functions (5Witters L.A. Kemp B.E. Means A.R. Trends Biochem. Sci. 2006; 31: 13-16Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 6Towler M.C. Hardie D.G. Circ. Res. 2007; 100: 328-341Crossref PubMed Scopus (1039) Google Scholar, 7Witczak C.A. Sharoff C.G. Goodyear L.J. Cell. Mol. Life Sci. 2008; 65: 3737-3755Crossref PubMed Scopus (175) Google Scholar). In ECs, AMPK has been shown to regulate the activity of endothelial NO synthase (eNOS) and NO synthesis evoked by a variety of extracellular stimuli, including HDL (8Drew B.G. Fidge N.H. Gallon-Beaumier G. Kemp B.E. Kingwell B.A. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 6999-7004Crossref PubMed Scopus (157) Google Scholar), sphingosine 1-phosphate (S1P) (9Levine Y.C. Li G.K. Michel T. J. Biol. Chem. 2007; 282: 20351-20364Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar), thrombin (10Thors B. Halldórsson H. Thorgeirsson G. FEBS Lett. 2004; 573: 175-180Crossref PubMed Scopus (77) Google Scholar), vascular endothelial growth factor (9Levine Y.C. Li G.K. Michel T. J. Biol. Chem. 2007; 282: 20351-20364Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 11Youn J.Y. Wang T. Cai H. Circ. Res. 2009; 104: 50-59Crossref PubMed Scopus (98) Google Scholar), and adiponectin (12Ouchi N. Kobayashi H. Kihara S. Kumada M. Sato K. Inoue T. Funahashi T. Walsh K. J. Biol. Chem. 2004; 279: 1304-1309Abstract Full Text Full Text PDF PubMed Scopus (657) Google Scholar). AMPK has also been suggested to be involved in the inhibition of monocyte adhesion and adhesion molecule expression in ECs, although the role of eNOS in the regulation of cell adhesion is controversial (13Hattori Y. Suzuki K. Hattori S. Kasai K. Hypertension. 2006; 47: 1183-1188Crossref PubMed Scopus (372) Google Scholar, 14Prasad R. Giri S. Nath N. Singh I. Singh A.K. J. Neurosci. Res. 2006; 84: 614-625Crossref PubMed Scopus (54) Google Scholar, 15Gaskin F.S. Kamada K. Yusof M. Korthuis R.J. Am. J. Physiol. Heart Circ. Physiol. 2007; 292: H326-H332Crossref PubMed Scopus (48) Google Scholar, 16Ewart M.A. Kohlhaas C.F. Salt I.P. Arterioscler. Thromb. Vasc. Biol. 2008; 28: 2255-2257Crossref PubMed Scopus (51) Google Scholar).We and others have shown that HDL is a carrier of potent bioactive lipid mediators, including S1P, in addition to apolipoproteins (4Okajima F. Sato K. Kimura T. Endocr. J. 2009; 56: 317-334Crossref PubMed Scopus (38) Google Scholar). Recent studies have shown that HDL induced eNOS activation through lipoprotein-associated apoA-I and/or S1P, although the roles of dual receptors, i.e. scavenger receptor class B type I (SR-BI) and S1P receptors, are still controversial (4Okajima F. Sato K. Kimura T. Endocr. J. 2009; 56: 317-334Crossref PubMed Scopus (38) Google Scholar, 17Nofer J.R. van der Giet M. Tölle M. Wolinska I. von Wnuck Lipinski K. Baba H.A. Tietge U.J. Gödecke A. Ishii I. Kleuser B. Schäfers M. Fobker M. Zidek W. Assmann G. Chun J. Levkau B. J. Clin. Invest. 2004; 113: 569-581Crossref PubMed Scopus (575) Google Scholar, 18Assanasen C. Mineo C. Seetharam D. Yuhanna I.S. Marcel Y.L. Connelly M.A. Williams D.L. de la Llera-Moya M. Shaul P.W. Silver D.L. J. Clin. Invest. 2005; 115: 969-977Crossref PubMed Scopus (133) Google Scholar, 19Kimura T. Tomura H. Mogi C. Kuwabara A. Damirin A. Ishizuka T. Sekiguchi A. Ishiwara M. Im D.S. Sato K. Murakami M. Okajima F. J. Biol. Chem. 2006; 281: 37457-37467Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 20Zhu W. Saddar S. Seetharam D. Chambliss K.L. Longoria C. Silver D.L. Yuhanna I.S. Shaul P.W. Mineo C. Circ. Res. 2008; 102: 480-487Crossref PubMed Scopus (98) Google Scholar). Thus, several independent reports have shown that HDL and S1P stimulate AMPK and eNOS in ECs. However, roles of SR-BI and S1P receptors and their signaling mechanism in HDL-induced AMPK activation have not been fully characterized. Moreover, roles of AMPK in HDL-regulated functions related to the protection of endothelial dysfunctions other than eNOS activation remain poorly understood. In this study, we examined these unanswered questions in human umbilical vein endothelial cells (HUVECs) in vitro and mouse aorta in vivo.
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