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Up-regulation of Endothelial Nitric-oxide Synthase Promoter by the Phosphatidylinositol 3-Kinase γ/Janus Kinase 2/MEK-1-dependent Pathway*

Open AccessPublished:January 12, 2001DOI:https://doi.org/10.1074/jbc.M005305200
      Our recent study indicates that lysophosphatidylcholine (LPC) enhances Sp1 binding and Sp1-dependent endothelial nitric oxide synthase (eNOS) promoter activity via the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 (MEK-1) signaling pathway (Cieslik, K., Lee, C.-M., Tang, J.-L., and Wu, K. K. (1999) J. Biol. Chem. 274, 34669–34675). To identify upstream signaling molecules, we transfected human endothelial cells with dominant negative and active mutants of Ras and evaluated their effects on eNOS promoter activity. Neither mutant altered the basal or LPC-induced eNOS promoter function. By contrast, a dominant negative mutant of phosphatidylinositol 3-kinase γ (PI-3Kγ) blocked the promoter activity induced by LPC. Wortmannin and LY 294002 had a similar effect. AG-490, a selective inhibitor of Janus kinase 2 (Jak2), also reduced the LPC-induced Sp1 binding and eNOS promoter activity to the basal level. LPC induced Jak2 phosphorylation, which was abolished by LY 294002 and the dominant negative mutant of PI-3Kγ. LY 294002 and AG-490 abrogated MEK-1 phosphorylation induced by LPC but had no effect on Raf-1. These results indicate that PI-3Kγ and Jak2 are essential for LPC-induced eNOS promoter activity. This signaling pathway was sensitive to pertussis toxin, suggesting the involvement of a Gi protein in PI-3Kγ activation. These results indicate that LPC enhances Sp1-dependent eNOS promoter activity by a pertussis toxin-sensitive, Ras-independent novel pathway, PI-3Kγ/Jak2/MEK-1/ERK1/2.
      eNOS
      endothelial nitric oxide synthase
      LPC
      lysophosphatidylcholine
      PI-3K
      phosphatidylinositol 3-kinase
      PTX
      pertussis toxin
      MEK
      mitogen-activated protein kinase/extracellular signal-regulated kinase kinase
      Jak
      Janus kinase
      PKC
      protein kinase C
      ERK
      extracellular signal-regulated kinase
      FBS
      fetal bovine serum
      TGF
      transforming growth factor
      Endothelial nitric oxide synthase (eNOS),1 a member of the NOS family, catalyzes the synthesis in blood vessels of nitric oxide, which plays a key role in maintaining blood pressure homeostasis and vascular integrity (
      • Moncada S.
      • Palmer R.J.
      • Higgs E.A.
      ,
      • Wu K.K.
      ). eNOS is constitutively expressed primarily in endothelial cells, and its level of expression has been shown to be up-regulated by exercise (
      • Sessa W.C.
      • Pritchard K.
      • Seyedi N.
      • Wang J.
      • Hintze T.H.
      ), shear stress (
      • Nadaud S.
      • Philippe M.
      • Arnal J.F.
      • Michel J.B.
      • Soubrier F.
      ), hypoxia (
      • Arnet U.A.
      • McMillan A.
      • Dinerman J.L.
      • Ballermann B.
      • Lowenstein C.J.
      ), and lysophosphatidylcholine (LPC; Ref.
      • Zembowicz A.
      • Tang J.-L.
      • Wu K.K.
      ). That LPC is capable of up-regulating the expression of vasoprotective eNOS is intriguing, because LPC has emerged as an important mediator of vascular injury, inflammation, and atherosclerosis (
      • Witztum J.
      • Steinberg D.
      ). It has been postulated that LPC-induced eNOS expression represents a crucial mechanism by which arteries exert their plastic defense against vessel wall injury (
      • Wu K.K.
      ).
      LPC is generated from oxidized low density lipoprotein (
      • Parthasarathy S.
      • Streinbrecher U.P.
      • Barnett J.
      • Witztum J.L.
      • Steinberg D.
      ) or from inflammatory cells as a result of phospholipase A2 action (
      • Asaoka Y.
      • Yoshida K.
      • Sasaki Y.
      • Nishizuka Y.
      • Murakami M.
      • Kudo I.
      • Inoue K.
      ). It possesses a variety of proinflammatory and proatherogenic properties: 1) it increases chemotactic activities of monocytes and T-lymphocytes (
      • Quinn M.T.
      • Parthasarathy S.
      • Steinberg D.
      ,
      • Asaoka Y.
      • Oka M.
      • Yoshida K.
      • Saski Y.
      • Nishizuka Y.
      ); 2) it has a mitogenic effect on macrophages (
      • Sakai M.
      • Miyazaki A.
      • Hakmata H.
      • Sasaki T.
      • Yui S.
      • Yamazaki M.
      • Shichiri M.
      • Horiuchi S.
      ); 3) it activates expression of vascular adhesion molecule-1, intercellular adhesion molecule-1, platelet-derived growth factor, heparin binding epidermal growth factor, and cyclooxygenase-2 (
      • Kume N.
      • Gimbrone M.A.
      ,
      • Kume N.
      • Cybulski M.I.
      • Gimbrone M.A.
      ,
      • Nakamo T.
      • Raines E.W.
      • Abaraham J.A.
      • Klagsbrun M.
      • Ross R.
      ,
      • Zembowicz A.
      • Jones S.L.
      • Wu K.K.
      ); and 4) it increases thrombomodulin expression and reduces tissue factor and tissue factor pathway inhibitor (
      • Yuan Y.
      • Schoenwaelder S.M.
      • Salem H.H.
      • Jackson S.P.
      ,
      • Engelman B.
      • Zieseniss S.
      • Brand K.
      • Page S.
      • Lentschat A.
      • Ulmer A.J.
      • Gerlach E.
      ,
      • Sato N.
      • Kokame K.
      • Miyata T.
      • Kato H.
      ). The mechanisms by which LPC exerts the myriad cellular and molecular actions are not entirely clear. It has been suggested that LPC acts as a second messenger to activate signaling molecules such as cAMP, mitogen-activated protein kinase, protein kinase C (PKC), and phosphatidylinositol 3-kinase (PI-3K; Refs.
      • Sakai M.
      • Miyazaki A.
      • Hakmata H.
      • Sasaki T.
      • Yui S.
      • Yamazaki M.
      • Shichiri M.
      • Horiuchi S.
      ,
      • Wong J.T.
      • Tran K.
      • Pierce G.N.
      • Chan A.C., O, K.
      • Choy P.C.
      ,
      • Yamakawa T.
      • Eguchi S.
      • Yamakawa Y.
      • Motley E.D.
      • Numaguchi K.
      • Utsunomiya H.
      • Inagami T.
      ,
      • Nishioka H.
      • Horiuchi H.
      • Arai H.
      • Kita T.
      ). LPC has been shown to induce the DNA binding activity of activated protein 1 (
      • Fang X.
      • Gibson S.
      • Flowers M.
      • Furui T.
      • Bast R.C.
      • Mills G.B.
      ), cAMP response element-binding protein (
      • Ueno Y.
      • Kume N.
      • Miyamoto S.
      • Morimoto M.
      • Kataoka H.
      • Ochi H.
      • Nishi E.
      • Moriwaki H.
      • Minami M.
      • Hashimoto N.
      • Kita T.
      ), and nuclear factor κB (
      • Zhu Y.
      • Lin J.H.-C.
      • Liao H.-L.
      • Verna L.
      • Stemerman M.B.
      ). Despite these reports, the signaling pathways that lead to transcriptional up-regulation of these proinflammatory and growth factor genes remain unclear. By contrast, the transcriptional regulation of eNOS is more extensively investigated. The basal eNOS promoter activity depends on binding of Sp1 to an Sp1 cognate site (−90 to −104) on the human eNOS 5′-flanking promoter region (
      • Zhang R.
      • Min W.
      • Sessa W.C.
      ,
      • Tang J.-l.
      • Zembowicz A.
      • Xu X.-M.
      • Wu K.K.
      ). In our previous study, we have shown that LPC up-regulates eNOS promoter activity by augmenting specifically the Sp1 binding activity (
      • Cieslik K.
      • Zembowicz A.
      • Tang J.-L.
      • Wu K.K.
      ). We have further shown that LPC selectively activates extracellular signal-regulated kinase 1/2 (ERK1/2) via mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 (MEK-1), and PD98059 (2′-amino-3′-methoxyflavaone), a selective inhibitor of MEK-1, abrogated the Sp1-dependent eNOS promoter up-regulation, consistent with an essential role of MEK-1 and ERK1/2 in LPC-induced promoter activity (
      • Cieslik K.
      • Lee C.-M.
      • Tang J.-L.
      • Wu K.K.
      ). The signaling pathway upstream of MEK-1 has not been reported. In this study, we searched for upstream signaling kinases by using dominant negative mutants or selective pharmacological inhibitors, or both. Here, we report identification of PI-3Kγ as an essential signaling molecule in MEK-1 activation with subsequent Sp1-dependent eNOS promoter up-regulation by LPC. Our results further show that PI-3Kγ activates a downstream Janus kinase 2 (Jak2), which in turn activates MEK-1. Neither Ras nor Raf-1 activation is required for eNOS promoter activation by LPC.

      DISCUSSION

      Several laboratories including ours have reported that LPC activates MEK-1, which in turn activates ERK1/2 (
      • Wong J.T.
      • Tran K.
      • Pierce G.N.
      • Chan A.C., O, K.
      • Choy P.C.
      ,
      • Cieslik K.
      • Lee C.-M.
      • Tang J.-L.
      • Wu K.K.
      ). Results from our previous studies further indicate that the MEK-1/ERK1/2 signaling pathway is essential for Sp1-dependent eNOS promoter activity induced by LPC (
      • Cieslik K.
      • Lee C.-M.
      • Tang J.-L.
      • Wu K.K.
      ). In the present study, we provide information for the first time that activation of the MEK-1 signaling pathway leading to eNOS promoter up-regulation depends on activation of upstream PI-3Kγ and Jak2. Several pieces of evidence from the present study indicate that PI-3Kγ and Jak2 activations are essential for eNOS promoter up-regulation by LPC: 1) wortmannin and LY 294002 block LPC-induced MEK-1 phosphorylation (Fig. 8 B), Sp1 binding (Fig. 2, A and B), and eNOS promoter activity (Fig. 2 C); 2) the dominant negative mutant of p110γ selectively abrogated eNOS promoter activity increased by LPC (Fig. 3); and 3) AG-490, a selective inhibitor of Jak2, reduced LPC-induced Sp1 binding (Fig. 6, A and B), eNOS promoter activity (Fig. 6 C), and MEK-1 phosphorylation (Fig. 8 B) to the basal level. Importantly, our results indicate that Jak2 acts downstream of PI-3Kγ. Compelling evidence to support this conclusion includes the abrogation of Jak2 autophosphorylation by Δ110γ (Fig.7 C) as well as by LY 294002 (Fig. 7 A). By contrast, the conventional upstream signaling molecules of MEK-1,i.e. Ras and Raf-1, do not play a significant role in signaling LPC-induced eNOS promoter activity. Neither dominant negative nor dominant active Ras mutants exerted any effect on basal or LPC-induced Sp1 binding and eNOS promoter activity (Fig. 1). Furthermore, LPC did not increase Raf-1 phosphorylation (Fig.8 A). Taken together, these findings indicate that LPC induces eNOS promoter function in endothelial cells by a novel signaling pathway involving PI-3Kγ→Jak2→MEK-1→ERK1/2.
      PI-3K catalyzes the synthesis of phosphatidylinositol 3,4,5-trisphosphate, which is an important second messenger for diverse cellular responses (
      • Toker A.
      • Gantley L.C.
      ). At least four PI-3K isoforms have been characterized (
      • Vanhaesebroeck B.
      • Leevers S.J.
      • Panayotou G.
      • Waterfield M.D.
      ). Heterodimeric PI-3Kα, PI-3Kβ, and PI-3Kδ constitute a p110 catalytic subunit and a p85 or p55 regulatory subunit. The heterodimeric PI-3Kγ constitutes a p110γ catalytic subunit, which does not interact with p85 adaptors but forms a dimer with a p101 regulatory subunit. The dominant negative deletion mutants Δp110γ and Δp85 specifically inhibit PI-3Kγ and PI-3Kα, -β, or -δ, respectively. Our results clearly show that Δp110γ but not Δp85 blocked LPC-induced eNOS promoter activity signaled via the MEK-1/ERK1/2 pathway. Both PI-3Kγ and PI-3Kα have been shown to transmit signals to activate MEK-1 in different cell types via a common pathway involving Shc, Grb2, and SOS (son ofsevenless) with subsequent activation of Ras and Raf-1 (
      • Leopoldt D.
      • Hanck T.
      • Exner T.
      • Maier U.
      • Wetzker R.
      • Nurnberg B.
      ,
      • Van Biesen T.
      • Hawes B.E.
      • Luttrell D.K.
      • Krueger K.M.
      • Touhara K.
      • Porfiri E.
      • Sakaue M.
      • Luttrell L.M.
      • Lefkowitz R.J.
      ,
      • Hawes B.E.
      • Luttrell L.M.
      • Van Biesen T.
      • Lefkowitz R.J.
      ). eNOS promoter activation by LPC does not signal through this pathway, because neither Ras nor Raf-1 activation was required for LPC-induced eNOS promoter activity. Alternatively, Gγ-stimulated PI-3Kγ has recently been reported to activate PKCξ (
      • Wang Y.-X.
      • Dhulipala P.D.K.
      • Li L.
      • Benovic J.L.
      • Kotlikoff M.I.
      ,
      • Takeda H.
      • Matozaki T.
      • Takada T.
      • Noguchi T.
      • Yamao T.
      • Tsuda M.
      • Ochi F.
      • Fukunaga K.
      • Inagaki K.
      • Kasuga M.
      ), and PKCξ activates MEK-1 independently of Ras and Raf-1 (
      • Hawes B.E.
      • Luttrell L.M.
      • Van Biesen T.
      • Lefkowitz R.J.
      ). The LPC-induced eNOS promoter up-regulation may be signaled through this pathway. In our preliminary work, we found that several PKC inhibitors blocked ERK1/2 activation and eNOS promoter activity,
      C.-M. Lee, K. Cieslik, and K. K. Wu, unpublished data.
      supporting the involvement of a PKC in this pathway. Furthermore, Jak2 activation in thrombin-stimulated platelets (G protein-coupled receptor) is downstream of PKC activation (
      • Rodriguez B.
      • Watson S.P.
      ). It is possible that a PKC isoform, such as atypical PKCξ, serves as an intermediate signal between PI-3Kγ and Jak2 in the LPC-induced MEK-1 activation. Further studies are required to evaluate this possibility. It was reported that PI-3Kγ possesses a protein kinase activity that signals to ERK1/2 in addition to the well characterized lipid kinase activity, which signals to protein kinase B/Akt (
      • Bondeva T.
      • Pirola L.
      • Bulgarelli-Leva G.
      • Pubio I.
      • Reinhard W.
      • Wymann M.P.
      ). It is unclear whether signaling of PI-3Kγ to Jak2 and MEK-1 is mediated by its protein kinase activity or via lipid kinase activity, nor is it clear whether the Akt pathway is involved in ERK1/2-mediated promoter up-regulation by LPC. Nevertheless, it should be noted that Akt activation leads to phosphorylation of a serine residue located at the C-terminal region of eNOS, and the phosphorylated eNOS exhibits enhanced catalytic activity (
      • Fulton D.
      • Gratton J.P.
      • McCabe T.J.
      • Fontana J.
      • Fujio Y.
      • Walsh K.
      • Franke T.F.
      • Papapetropoulos A.
      • Sessa W.C.
      ). Thus, the PI-3K pathway(s) occupies a central position in regulating eNOS activity.
      Jak2 is a member of the Janus kinase family of nonreceptor protein tyrosine kinases, which consists of three additional members: Jak1, Jak3, and Tyk2. Each member has a conserved C-terminal kinase domain. Jak2 was reported to activate PI-3Kα, but the exact mechanism by which these two molecules interact is unclear (
      • Al-Shami A.
      • Naccache P.H.
      ). This Jak2-dependent PI-3Kα pathway is unlikely to signal LPC-induced eNOS promoter function, because transfection of Δp85, which blocks PI-3Kα activity, had no effect of eNOS promoter activity. Jak2 has been reported to be directly associated with and stimulated by the angiotensin II receptor (
      • Marrero M.B.
      • Schleffer B.
      • Paxton W.G.
      • Heerdt L.
      • Berk B.C.
      • Delafontaine P.
      • Bernstein K.E.
      ,
      • Ali M.S.
      • Sayeski P.P.
      • Dirksen L.B.
      • Hayzer D.J.
      • Marrero M.B.
      • Bernstein K.E.
      ). The relevance of these reports to LPC-induced signaling Jak2 is unclear, because it is unknown whether the action of LPC is receptor-mediated. However, on the basis of our experimental results, it is unlikely that Jak2 is directly activated by LPC receptor, even if such a receptor exists. Our results clearly show that Jak2 activation is downstream of PI-3Kγ. A previous study has shown that PI-3Kγ activates Bruton's tyrosine kinase in lymphocytes (
      • Li Z.
      • Wahl M.I.
      • Equinoa A.
      • Stephens L.R.
      • Hawkins P.T.
      • Witte D.N.
      ). Further studies are likely to identify additional nonreceptor protein-tyrosine kinases that are activated by PI-3Kγ. The mechanism by which PI-3Kγ activates Jak2 is unclear. It is possible that phosphatidylinositol 3,4,5-trisphosphate generated by PI-3Kγ may bind to the JH motif (Jakhomology domain) located in the catalytic domain of Jak2, leading to Jak2 activation. The JH motif is structurally similar to the Src homology 2 domain (SH2), and phosphatidylinositol 3,4,5-trisphosphate is known to bind the Src homology 2 domain for activation of kinases. Alternatively, PI-3Kγ may activate Jak2 through its protein kinase activity (
      • Bondeva T.
      • Pirola L.
      • Bulgarelli-Leva G.
      • Pubio I.
      • Reinhard W.
      • Wymann M.P.
      ).
      It has been reported in HeLa and 3T3 cells that LPC can also activate the c-Jun N-terminal kinase pathway and thereby increase AP1-dependent gene transcription (
      • Fang X.
      • Gibson S.
      • Flowers M.
      • Furui T.
      • Bast R.C.
      • Mills G.B.
      ). We did not detect c-Jun N-terminal kinase (JNK) activation by LPC in endothelial cells (
      • Cieslik K.
      • Lee C.-M.
      • Tang J.-L.
      • Wu K.K.
      ). Our results are consistent with other reports, which show that in vascular endothelial cells or smooth muscle cells, LPC predominantly activates the ERK1/2 pathway (
      • Wong J.T.
      • Tran K.
      • Pierce G.N.
      • Chan A.C., O, K.
      • Choy P.C.
      ,
      • Cieslik K.
      • Lee C.-M.
      • Tang J.-L.
      • Wu K.K.
      ,
      • Rikitake Y.
      • Kawashima S.
      • Yamashita T.
      • Ueyama T.
      • Ishido S.
      • Hotta H.
      • Hirata K.
      • Yokoyama M.
      ). It is possible that PI-3Kγ→Jak2→MEK-1→ERK1/2 may represent a common signaling pathway through which LPC exerts its transcriptional responses in vascular cells. LPC has been shown to increase mRNA or protein expression, or both, of a myriad of endothelial genes, including thrombomodulin, tissue plasminogen activator, plasminogen activator inhibitor 1, intercellular adhesion molecule 1, vascular cell adhesion molecule 1, platelet-derived growth factor, and heparin binding epidermal growth factor-like growth factor (
      • Sato N.
      • Kokame K.
      • Shimokado K.
      • Kato H.
      • Miyata T.
      ,
      • Kume N.
      • Gimbrone M.A.
      ,
      • Kume N.
      • Cybulski M.I.
      • Gimbrone M.A.
      ,
      • Nakamo T.
      • Raines E.W.
      • Abaraham J.A.
      • Klagsbrun M.
      • Ross R.
      ). How LPC increases the expression of these biologically active genes is generally unknown. It is unclear whether LPC augments the promoter activities of these genes, nor is it known which transactivators are stimulated to account for the increased expression of such a diverse group of genes. Results from our study suggest that LPC may stimulate the promoter activities of certain genes, such as thrombomodulin, vascular cell adhesion molecule 1 and plasminogen activator inhibitor 1, that depend in part on Sp1 binding for their transcriptional activation (
      • Shingu T.
      • Bornstein P.
      ,
      • Neish A.S.
      • Khachigian L.M.
      • Park A.
      • Baichwal V.R.
      • Collins T.
      ,
      • Chen Y.Q.
      • Su M.
      • Walia R.R.
      • Hao Q.
      • Covington J.W.
      • Vaughan D.E.
      ) by a similar PI-3k→ Jak2 signaling pathway, resulting in increased Sp1 binding and promoter activity. Further studies are needed to test this hypothesis.
      Previous studies suggest that LPC activates G proteins. A more recent study shows in platelets and megakaryocytic cell lines that LPC stimulates Gs, thereby increasing adenylyl cyclase and cellular cAMP levels (
      • Yuan Y.
      • Schoenwaelder S.M.
      • Salem H.H.
      • Jackson S.P.
      ). Our results indicate that LPC stimulates PTX-sensitive Gi in endothelial cells. The mechanism by which LPC activates G proteins is unclear. It has been assumed that LPC activates G proteins by membrane perturbation. LPC is an amphipathic molecule and has been shown to transit through plasma membrane and to enter into cells at a rapid rate (
      • Mohandas N.
      • Wyatt J.
      • Mel S.F.
      • Rossi M.E.
      • Shohet S.B.
      ). It is unlikely that it will exert a specific effect on activation of G proteins. Alternatively, its action may be mediated by Gi-coupled receptor activation. In view of the important roles that LPC plays in diverse pathophysiological processes, it should be valuable to determine whether its action is mediated by a specific receptor. Identification of such a specific receptor should have important therapeutic implications.

      Acknowledgments

      We thank Susan Mitterling for editorial assistance.

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