FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2077366814> ?p ?o ?g. }

• W2077366814 endingPage "14890" @default.
• W2077366814 startingPage "14885" @default.
• W2077366814 abstract "We have shown that lysophosphatidylcholine (lyso-PC) increases endothelial nitric-oxide synthase (eNOS) expression at the transcriptional level (Zembowicz, A., Tang, J.-L., and Wu, K. K. (1995) J. Biol. Chem. 270, 17006–17010). To elucidate the mechanism by which lyso-PC increases the eNOS transcription, we identified Sp1 sites at −104 to −90 and PEA3 sites at −40 to −24 as being involved in lyso-PC-induced promoter activity. Site-directed mutagenesis of Sp1 sites resulted in a marked reduction of basal and lyso-PC-induced activity whereas PEA3 site mutation abrogated response to lyso-PC. Band shift assays revealed that lyso-PC augmented Sp1 binding activity. Pretreatment of cells or nuclear extracts with okadaic acid reduced the Sp1 binding activity. Furthermore, okadaic acid treatment abrogated the lyso-PC induced promoter augmentation. Lyso-PC increased the nuclear extract protein phosphatase 2A (PP2A) activity, which was suppressed by okadaic acid treatment. These results suggest that lyso-PC up-regulates eNOS transcription by a PP2A-dependent increase in Sp1 binding activity. We have shown that lysophosphatidylcholine (lyso-PC) increases endothelial nitric-oxide synthase (eNOS) expression at the transcriptional level (Zembowicz, A., Tang, J.-L., and Wu, K. K. (1995) J. Biol. Chem. 270, 17006–17010). To elucidate the mechanism by which lyso-PC increases the eNOS transcription, we identified Sp1 sites at −104 to −90 and PEA3 sites at −40 to −24 as being involved in lyso-PC-induced promoter activity. Site-directed mutagenesis of Sp1 sites resulted in a marked reduction of basal and lyso-PC-induced activity whereas PEA3 site mutation abrogated response to lyso-PC. Band shift assays revealed that lyso-PC augmented Sp1 binding activity. Pretreatment of cells or nuclear extracts with okadaic acid reduced the Sp1 binding activity. Furthermore, okadaic acid treatment abrogated the lyso-PC induced promoter augmentation. Lyso-PC increased the nuclear extract protein phosphatase 2A (PP2A) activity, which was suppressed by okadaic acid treatment. These results suggest that lyso-PC up-regulates eNOS transcription by a PP2A-dependent increase in Sp1 binding activity. Nitric oxide (NO) 1The abbreviations used are: NO, nitric oxide; lyso-PC (or LPC), lysophosphatidylcholine; eNOS or NOS-III, endothelial nitric-oxide synthase; GH, promoter construct containing −1322 to +22 fragment; HUVEC, human umbilical vein endothelial cell(s); NE, nuclear extract(s); PP, protein phosphatases; EMSA, electrophoretic mobility shift assay; OA, okadaic acid; PCR, polymerase chain reaction; FBS, fetal bovine serum; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride. 1The abbreviations used are: NO, nitric oxide; lyso-PC (or LPC), lysophosphatidylcholine; eNOS or NOS-III, endothelial nitric-oxide synthase; GH, promoter construct containing −1322 to +22 fragment; HUVEC, human umbilical vein endothelial cell(s); NE, nuclear extract(s); PP, protein phosphatases; EMSA, electrophoretic mobility shift assay; OA, okadaic acid; PCR, polymerase chain reaction; FBS, fetal bovine serum; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride. is an important mediator of diverse physiological and pathological processes, including vasodilation, cytotoxicity, and neurotransmission (1Moncada S. Palmer R.J. Higgs E.A. Pharmacol. Rev. 1991; 43: 109-142PubMed Google Scholar, 2Lowenstein C.J. Snyder S.H. Cell. 1992; 70: 705-707Abstract Full Text PDF PubMed Scopus (736) Google Scholar, 3Nathan C. FASEB J. 1992; 6: 3051-3064Crossref PubMed Scopus (4132) Google Scholar, 4Marletta M.A. J. Biol. Chem. 1993; 268: 12231-12334Abstract Full Text PDF PubMed Google Scholar). Its biosynthesis is catalyzed by nitric-oxide synthase (NOS). Three isoforms of NOS, i.e. neuronal NOS (nNOS or NOS-I), inducible-NOS (iNOS or NOS-II), and endothelial-NOS (eNOS or NOS-III) have been identified and characterized (5Bredt D.S. Synder S.H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 682-685Crossref PubMed Scopus (3114) Google Scholar, 6Stuehr D.J. Cho H.J. Kwon N.S. Weise M.F. Nathan C.F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7773-7777Crossref PubMed Scopus (727) Google Scholar, 7Sessa W.C. Harrison J.K. Barber C.M. Zeng D. Durieux M.E. D'Angelo D.D. Lynch K.R. Peach M.J. J. Biol. Chem. 1992; 267: 15274-15276Abstract Full Text PDF PubMed Google Scholar). NOS-I and -III are constitutively expressed. The inducible form is expressed by stimulation with several inflammatory and mitogenic mediators (8Nathan C. Xie Q.-W. J. Biol. Chem. 1994; 269: 13725-13728Abstract Full Text PDF PubMed Google Scholar, 9Wu K.K. Adv. Pharmacol. 1995; 33: 179-207Crossref PubMed Scopus (193) Google Scholar). Altered endothelial NO productions have been implicated in several important cardiovascular disorders: hypertension, atherosclerotic heart disease, and diabetes. The mechanism by which NO synthesis is deranged in these disorders remained unclear. Although NOS-III is considered to be a housekeeping gene, recent studies have provided evidence to suggest that NOS-III is induced by shear stress (10Nadaud S. Philippe M. Arnal J.F. Michel J.B. Soubrier F. Circ. Res. 1996; 79: 857-863Crossref PubMed Scopus (206) Google Scholar), physical exercise (11Sessa W.C. Prirtchard K. Seyedi N. Wang J. Hintze T.H. Circ. Res. 1994; 74: 349-353Crossref PubMed Scopus (817) Google Scholar), hypoxia (12Arnet U.A. McMillan A. Dinerman J.L. Ballermann B. Lowenstein C.J. J. Biol. Chem. 1996; 271: 15069-15073Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar), estrogen treatment (13Weiner C.P. Lizasoain I. Baylis S.A. Knowles R.G. Charles I.G. Moncada S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5212-5216Crossref PubMed Scopus (1071) Google Scholar), lysophosphatidylcholine (lyso-PC or LPC), and low levels of oxidized low density lipoprotein (14Zembowicz A. Tang J.-L. Wu K.K. J. Biol. Chem. 1995; 270: 17006-17010Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 15Hirata K. Miki N. Kuroda Y. Sakoda T. Kanashima S. Yokoyama M. Circ. Res. 1995; 76: 958-962Crossref PubMed Scopus (144) Google Scholar). These findings imply that induction of NOS-III plays an important role in protecting vascular integrity under stress. Work from our laboratory has shown that lyso-PC increases NOS-III expression at the transcriptional level (14Zembowicz A. Tang J.-L. Wu K.K. J. Biol. Chem. 1995; 270: 17006-17010Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). However, the mechanism by which lyso-PC and other inducing agents increase NOS-III gene transcription remains to be elucidated.The 5′-flanking region of human eNOS gene has been cloned and sequenced (16Marsden P.A. Heng H.H. Scherer S.W. Stewart R.L. Hall A.V. Shi X.-M. Tsui L.C. Shappert K.T. J. Biol. Chem. 1993; 268: 17478-17488Abstract Full Text PDF PubMed Google Scholar). The region adjacent to the transcription initiation sites is TATA-less and GC-rich. Functional analysis of the promoter activity conferred by the 5′-flanking region has shown that a canonical Sp1 site situated between −104 to −90 is required for the basal promoter activity (17Tang J.-L. Zembowicz A. Xu X.-M. Wu K.K. Biochem. Biophys. Res. Commun. 1995; 213: 673-680Crossref PubMed Scopus (36) Google Scholar). However, it is unclear whether this site is involved in lyso-PC-induced transcriptional activation. The purpose of this study is to identify cis-acting elements and nuclear transcriptional activators that are important in augmentation of eNOS transcription in response to lyso-PC stimulation. Our results indicate that Sp1 site at −104 to −90 and PEA3 site at −40 to −24 are required for lyso-PC-induced transcriptional activation. Lyso-PC increases the promoter function by enhancing the Sp1 binding activity mediated by the action of protein phosphatase 2A.DISCUSSIONLyso-PC is a main component of oxidized low density lipoprotein (22Parthasarathy S. Streinbrecher U.P. Barnett J. Witztum J.L. Steinberg D. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 3000-3004Crossref PubMed Scopus (306) Google Scholar). The level of lyso-PC is highly elevated in human atherosclerotic tissues (23Portman O.W. Alexander M. J. Lipid Res. 1969; 10: 158-165Abstract Full Text PDF PubMed Google Scholar). It has been shown that lyso-PC increases chemotaxis for monocytes and induces endothelial adhesive molecules such as intercellular adhesive molecule-1 and vascular cell adhesive molecule-1, which mediate monocyte and neutrophil adhesion to vascular endothelium (24Sakai M. Miyazaki A. Hakamata H. Sasaki T. Yui S. Yamazaki M. Schichiri M. Horiuchi S. J. Biol. Chem. 1994; 269: 31430-31435Abstract Full Text PDF PubMed Google Scholar). Hence, lyso-PC has been considered to play an important role in monocyte accumulation in the arterial walls. Furthermore, lyso-PC induces the expression platelet-derived growth factor A and B subunits and heparin-binding epidermal growth factor from endothelial cells (25Kume N. Gimbrone M.A. J. Clin. Invest. 1994; 93: 907-911Crossref PubMed Scopus (294) Google Scholar, 26Kume N. Cybulski M.I. Gimbrone Jr., M.A. J. Clin. Invest. 1992; 90: 1138-1144Crossref PubMed Scopus (720) Google Scholar). These growth factors promote smooth muscle cell migration and proliferation. These findings have led to the conclusion that lyso-PC is a key mediator of atherosclerosis (22Parthasarathy S. Streinbrecher U.P. Barnett J. Witztum J.L. Steinberg D. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 3000-3004Crossref PubMed Scopus (306) Google Scholar). Work from this laboratory provides evidence to indicate that the expression of NOS-III and cyclooxygenase-2 are stimulated by lyso-PC (27Zembowicz A. Jones S.L. Wu K.K. J. Clin. Invest. 1995; 96: 1688-1692Crossref PubMed Scopus (88) Google Scholar), thereby increasing the synthesis of NO and prostacyclin, which act synergistically to block monocyte adhesion and smooth muscle cell proliferation; platelet activation and aggregation; and pathological vasoconstriction. This leads to a postulate that vascular injurious agents such as lyso-PC creates a yin-yan situation in which it induces the expression of vasoprotective genes to counteract the vascular damaging genes. It is interesting to note that lyso-PC induces the expressions of those diverse genes with two distinct types of kinetics: 1) rapid, transient as in heparin-binding epidermal growth factor and cyclooxygenase-2 gene induction and 2) delayed, sustained as in eNOS and intercellular adhesive molecule-1 gene induction. It is unclear whether these two types of genes are transcriptionally regulated by a common mechanism. In fact, the mechanism by which lyso-PC induces any of these genes had not been reported previously. This report is the first to shed light on the promoter regulation of eNOS gene by lyso-PC. Our results indicate that lyso-PC enhances the transcriptional activation of this housekeeping gene primarily by increasing the Sp1 binding activity via a PP2A-dependent reaction. Ets binding to its cognate site on the promoter region is also involved in basal and lyso-PC-induced eNOS transcription.Sp1 is a ubiquitous transcriptional activator mediating basal and regulated gene expression. Sp1 binding to its cognate motif depends not only on nuclear Sp1 levels but also on posttranslational modification of the Sp1 molecule. Recent studies indicate that Sp1 binding activity is influenced by phosphorylation: phosphorylation decreases, whereas dephosphorylation by the action of phosphatases increases Sp1 binding activity (28Daniel S. Zhang S. DePaoli-Roach A.A. Kim K.H. J. Biol. Chem. 1996; 271: 14692-14697Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 29Armstrong S.A. Barry D.A. Leggett R.W. Mueller C.R. J. Biol. Chem. 1997; 272: 13489-13495Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). A recent study on the acetyl-CoA carboxylase gene indicates that glucose induces this gene transcription by a mechanism involving dephosphorylation of Sp1 by protein phosphatase 1 and 2A (28Daniel S. Zhang S. DePaoli-Roach A.A. Kim K.H. J. Biol. Chem. 1996; 271: 14692-14697Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Our data, which show suppression of lyso-PC-induced Sp1 binding activity by okadaic acid, but not vanadate, and a selective increase in PP2A activity by lyso-PC, which was inhibited by okadaic acid, are consistent with a role of PP2A in modifying Sp1 binding activity. Results from this study led us to postulate that the Sp1 is phosphorylated at the basal cellular state, and lyso-PC activates PP2A, which in turn dephosphorylate Sp1, resulting in an increased Sp1 binding activity.Signal pathways for lyso-PC-induced transcriptional activation have not been fully established. A recent study suggests the involvement of stress-activated protein kinase/c-Jun amino-terminal kinase pathway that leads to AP1 activation and binding to its cognate site (30Fang X. Gibson S. Flowers M. Furui T. Bast Jr., R.C. Mills G.B. J. Biol. Chem. 1997; 272: 13683-13689Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). It is unclear whether JNK and/or other mitogen-activated protein kinase pathways are involved in increased Sp1 binding activity. It is possible that lyso-PC may increase Sp1 binding activity by activating protein phosphatases via the mitogen-activated protein kinase or other kinase pathways. This is being investigated in our laboratory.Our results indicate that a PEA3/Ets sequence in the promoter/enhancer region of eNOS gene is involved in basal and lyso-PC-induced promoter activity. Nuclear Ets-related proteins contain a large family of polypeptides sharing an ETS domain (31Wasylyk B. Hahn S.L. Giovane A. Eur. J. Biochem. 1993; 211: 7-18Crossref PubMed Scopus (809) Google Scholar). They bind to purine-rich sequences with a GGA core. The 5′- and 3′-flanking sequences of the GGA core confer relative specificity for Ets family proteins. The GGA-containing sequences on eNOS lack a specificity for any given class of Ets-related proteins. This is in agreement with our experimental data showing promiscuity of binding for several representatives of Ets family proteins. Ets proteins are involved in cellular response to external stresses. They generally serve as co-activators for transcriptional activators including Sp1 (32Khachigian L.A. Williams A.J. Collins T. J. Biol. Chem. 1995; 270: 27679-27686Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar, 33Gegonne A. Bosselut R. Bailly R.-A. Ghysdael J. EMBO J. 1993; 12: 1169-1178Crossref PubMed Scopus (152) Google Scholar). Several reports have suggested that PEA3 and Sp1 sequences are often colocalized on the promoter/enhancer region and binding of Ets and Sp1 to their respective sites causes a synergistic activation of a number of genes (33Gegonne A. Bosselut R. Bailly R.-A. Ghysdael J. EMBO J. 1993; 12: 1169-1178Crossref PubMed Scopus (152) Google Scholar). As regards the activation of eNOS gene, our data are in agreement with the proposition that Ets serves as a co-activator for Sp1. Binding of Ets alone resulted in minimal promoter activation, whereas it augmented Sp1-mediated transcriptional activation. It is possible that Ets interacts with components of the general transcription factors in the preinitiation complex through which it may enhance recruitment of the preinitiation complex to the promoter region. Nitric oxide (NO) 1The abbreviations used are: NO, nitric oxide; lyso-PC (or LPC), lysophosphatidylcholine; eNOS or NOS-III, endothelial nitric-oxide synthase; GH, promoter construct containing −1322 to +22 fragment; HUVEC, human umbilical vein endothelial cell(s); NE, nuclear extract(s); PP, protein phosphatases; EMSA, electrophoretic mobility shift assay; OA, okadaic acid; PCR, polymerase chain reaction; FBS, fetal bovine serum; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride. 1The abbreviations used are: NO, nitric oxide; lyso-PC (or LPC), lysophosphatidylcholine; eNOS or NOS-III, endothelial nitric-oxide synthase; GH, promoter construct containing −1322 to +22 fragment; HUVEC, human umbilical vein endothelial cell(s); NE, nuclear extract(s); PP, protein phosphatases; EMSA, electrophoretic mobility shift assay; OA, okadaic acid; PCR, polymerase chain reaction; FBS, fetal bovine serum; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride. is an important mediator of diverse physiological and pathological processes, including vasodilation, cytotoxicity, and neurotransmission (1Moncada S. Palmer R.J. Higgs E.A. Pharmacol. Rev. 1991; 43: 109-142PubMed Google Scholar, 2Lowenstein C.J. Snyder S.H. Cell. 1992; 70: 705-707Abstract Full Text PDF PubMed Scopus (736) Google Scholar, 3Nathan C. FASEB J. 1992; 6: 3051-3064Crossref PubMed Scopus (4132) Google Scholar, 4Marletta M.A. J. Biol. Chem. 1993; 268: 12231-12334Abstract Full Text PDF PubMed Google Scholar). Its biosynthesis is catalyzed by nitric-oxide synthase (NOS). Three isoforms of NOS, i.e. neuronal NOS (nNOS or NOS-I), inducible-NOS (iNOS or NOS-II), and endothelial-NOS (eNOS or NOS-III) have been identified and characterized (5Bredt D.S. Synder S.H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 682-685Crossref PubMed Scopus (3114) Google Scholar, 6Stuehr D.J. Cho H.J. Kwon N.S. Weise M.F. Nathan C.F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7773-7777Crossref PubMed Scopus (727) Google Scholar, 7Sessa W.C. Harrison J.K. Barber C.M. Zeng D. Durieux M.E. D'Angelo D.D. Lynch K.R. Peach M.J. J. Biol. Chem. 1992; 267: 15274-15276Abstract Full Text PDF PubMed Google Scholar). NOS-I and -III are constitutively expressed. The inducible form is expressed by stimulation with several inflammatory and mitogenic mediators (8Nathan C. Xie Q.-W. J. Biol. Chem. 1994; 269: 13725-13728Abstract Full Text PDF PubMed Google Scholar, 9Wu K.K. Adv. Pharmacol. 1995; 33: 179-207Crossref PubMed Scopus (193) Google Scholar). Altered endothelial NO productions have been implicated in several important cardiovascular disorders: hypertension, atherosclerotic heart disease, and diabetes. The mechanism by which NO synthesis is deranged in these disorders remained unclear. Although NOS-III is considered to be a housekeeping gene, recent studies have provided evidence to suggest that NOS-III is induced by shear stress (10Nadaud S. Philippe M. Arnal J.F. Michel J.B. Soubrier F. Circ. Res. 1996; 79: 857-863Crossref PubMed Scopus (206) Google Scholar), physical exercise (11Sessa W.C. Prirtchard K. Seyedi N. Wang J. Hintze T.H. Circ. Res. 1994; 74: 349-353Crossref PubMed Scopus (817) Google Scholar), hypoxia (12Arnet U.A. McMillan A. Dinerman J.L. Ballermann B. Lowenstein C.J. J. Biol. Chem. 1996; 271: 15069-15073Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar), estrogen treatment (13Weiner C.P. Lizasoain I. Baylis S.A. Knowles R.G. Charles I.G. Moncada S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5212-5216Crossref PubMed Scopus (1071) Google Scholar), lysophosphatidylcholine (lyso-PC or LPC), and low levels of oxidized low density lipoprotein (14Zembowicz A. Tang J.-L. Wu K.K. J. Biol. Chem. 1995; 270: 17006-17010Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 15Hirata K. Miki N. Kuroda Y. Sakoda T. Kanashima S. Yokoyama M. Circ. Res. 1995; 76: 958-962Crossref PubMed Scopus (144) Google Scholar). These findings imply that induction of NOS-III plays an important role in protecting vascular integrity under stress. Work from our laboratory has shown that lyso-PC increases NOS-III expression at the transcriptional level (14Zembowicz A. Tang J.-L. Wu K.K. J. Biol. Chem. 1995; 270: 17006-17010Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). However, the mechanism by which lyso-PC and other inducing agents increase NOS-III gene transcription remains to be elucidated. The 5′-flanking region of human eNOS gene has been cloned and sequenced (16Marsden P.A. Heng H.H. Scherer S.W. Stewart R.L. Hall A.V. Shi X.-M. Tsui L.C. Shappert K.T. J. Biol. Chem. 1993; 268: 17478-17488Abstract Full Text PDF PubMed Google Scholar). The region adjacent to the transcription initiation sites is TATA-less and GC-rich. Functional analysis of the promoter activity conferred by the 5′-flanking region has shown that a canonical Sp1 site situated between −104 to −90 is required for the basal promoter activity (17Tang J.-L. Zembowicz A. Xu X.-M. Wu K.K. Biochem. Biophys. Res. Commun. 1995; 213: 673-680Crossref PubMed Scopus (36) Google Scholar). However, it is unclear whether this site is involved in lyso-PC-induced transcriptional activation. The purpose of this study is to identify cis-acting elements and nuclear transcriptional activators that are important in augmentation of eNOS transcription in response to lyso-PC stimulation. Our results indicate that Sp1 site at −104 to −90 and PEA3 site at −40 to −24 are required for lyso-PC-induced transcriptional activation. Lyso-PC increases the promoter function by enhancing the Sp1 binding activity mediated by the action of protein phosphatase 2A. DISCUSSIONLyso-PC is a main component of oxidized low density lipoprotein (22Parthasarathy S. Streinbrecher U.P. Barnett J. Witztum J.L. Steinberg D. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 3000-3004Crossref PubMed Scopus (306) Google Scholar). The level of lyso-PC is highly elevated in human atherosclerotic tissues (23Portman O.W. Alexander M. J. Lipid Res. 1969; 10: 158-165Abstract Full Text PDF PubMed Google Scholar). It has been shown that lyso-PC increases chemotaxis for monocytes and induces endothelial adhesive molecules such as intercellular adhesive molecule-1 and vascular cell adhesive molecule-1, which mediate monocyte and neutrophil adhesion to vascular endothelium (24Sakai M. Miyazaki A. Hakamata H. Sasaki T. Yui S. Yamazaki M. Schichiri M. Horiuchi S. J. Biol. Chem. 1994; 269: 31430-31435Abstract Full Text PDF PubMed Google Scholar). Hence, lyso-PC has been considered to play an important role in monocyte accumulation in the arterial walls. Furthermore, lyso-PC induces the expression platelet-derived growth factor A and B subunits and heparin-binding epidermal growth factor from endothelial cells (25Kume N. Gimbrone M.A. J. Clin. Invest. 1994; 93: 907-911Crossref PubMed Scopus (294) Google Scholar, 26Kume N. Cybulski M.I. Gimbrone Jr., M.A. J. Clin. Invest. 1992; 90: 1138-1144Crossref PubMed Scopus (720) Google Scholar). These growth factors promote smooth muscle cell migration and proliferation. These findings have led to the conclusion that lyso-PC is a key mediator of atherosclerosis (22Parthasarathy S. Streinbrecher U.P. Barnett J. Witztum J.L. Steinberg D. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 3000-3004Crossref PubMed Scopus (306) Google Scholar). Work from this laboratory provides evidence to indicate that the expression of NOS-III and cyclooxygenase-2 are stimulated by lyso-PC (27Zembowicz A. Jones S.L. Wu K.K. J. Clin. Invest. 1995; 96: 1688-1692Crossref PubMed Scopus (88) Google Scholar), thereby increasing the synthesis of NO and prostacyclin, which act synergistically to block monocyte adhesion and smooth muscle cell proliferation; platelet activation and aggregation; and pathological vasoconstriction. This leads to a postulate that vascular injurious agents such as lyso-PC creates a yin-yan situation in which it induces the expression of vasoprotective genes to counteract the vascular damaging genes. It is interesting to note that lyso-PC induces the expressions of those diverse genes with two distinct types of kinetics: 1) rapid, transient as in heparin-binding epidermal growth factor and cyclooxygenase-2 gene induction and 2) delayed, sustained as in eNOS and intercellular adhesive molecule-1 gene induction. It is unclear whether these two types of genes are transcriptionally regulated by a common mechanism. In fact, the mechanism by which lyso-PC induces any of these genes had not been reported previously. This report is the first to shed light on the promoter regulation of eNOS gene by lyso-PC. Our results indicate that lyso-PC enhances the transcriptional activation of this housekeeping gene primarily by increasing the Sp1 binding activity via a PP2A-dependent reaction. Ets binding to its cognate site on the promoter region is also involved in basal and lyso-PC-induced eNOS transcription.Sp1 is a ubiquitous transcriptional activator mediating basal and regulated gene expression. Sp1 binding to its cognate motif depends not only on nuclear Sp1 levels but also on posttranslational modification of the Sp1 molecule. Recent studies indicate that Sp1 binding activity is influenced by phosphorylation: phosphorylation decreases, whereas dephosphorylation by the action of phosphatases increases Sp1 binding activity (28Daniel S. Zhang S. DePaoli-Roach A.A. Kim K.H. J. Biol. Chem. 1996; 271: 14692-14697Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 29Armstrong S.A. Barry D.A. Leggett R.W. Mueller C.R. J. Biol. Chem. 1997; 272: 13489-13495Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). A recent study on the acetyl-CoA carboxylase gene indicates that glucose induces this gene transcription by a mechanism involving dephosphorylation of Sp1 by protein phosphatase 1 and 2A (28Daniel S. Zhang S. DePaoli-Roach A.A. Kim K.H. J. Biol. Chem. 1996; 271: 14692-14697Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Our data, which show suppression of lyso-PC-induced Sp1 binding activity by okadaic acid, but not vanadate, and a selective increase in PP2A activity by lyso-PC, which was inhibited by okadaic acid, are consistent with a role of PP2A in modifying Sp1 binding activity. Results from this study led us to postulate that the Sp1 is phosphorylated at the basal cellular state, and lyso-PC activates PP2A, which in turn dephosphorylate Sp1, resulting in an increased Sp1 binding activity.Signal pathways for lyso-PC-induced transcriptional activation have not been fully established. A recent study suggests the involvement of stress-activated protein kinase/c-Jun amino-terminal kinase pathway that leads to AP1 activation and binding to its cognate site (30Fang X. Gibson S. Flowers M. Furui T. Bast Jr., R.C. Mills G.B. J. Biol. Chem. 1997; 272: 13683-13689Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). It is unclear whether JNK and/or other mitogen-activated protein kinase pathways are involved in increased Sp1 binding activity. It is possible that lyso-PC may increase Sp1 binding activity by activating protein phosphatases via the mitogen-activated protein kinase or other kinase pathways. This is being investigated in our laboratory.Our results indicate that a PEA3/Ets sequence in the promoter/enhancer region of eNOS gene is involved in basal and lyso-PC-induced promoter activity. Nuclear Ets-related proteins contain a large family of polypeptides sharing an ETS domain (31Wasylyk B. Hahn S.L. Giovane A. Eur. J. Biochem. 1993; 211: 7-18Crossref PubMed Scopus (809) Google Scholar). They bind to purine-rich sequences with a GGA core. The 5′- and 3′-flanking sequences of the GGA core confer relative specificity for Ets family proteins. The GGA-containing sequences on eNOS lack a specificity for any given class of Ets-related proteins. This is in agreement with our experimental data showing promiscuity of binding for several representatives of Ets family proteins. Ets proteins are involved in cellular response to external stresses. They generally serve as co-activators for transcriptional activators including Sp1 (32Khachigian L.A. Williams A.J. Collins T. J. Biol. Chem. 1995; 270: 27679-27686Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar, 33Gegonne A. Bosselut R. Bailly R.-A. Ghysdael J. EMBO J. 1993; 12: 1169-1178Crossref PubMed Scopus (152) Google Scholar). Several reports have suggested that PEA3 and Sp1 sequences are often colocalized on the promoter/enhancer region and binding of Ets and Sp1 to their respective sites causes a synergistic activation of a number of genes (33Gegonne A. Bosselut R. Bailly R.-A. Ghysdael J. EMBO J. 1993; 12: 1169-1178Crossref PubMed Scopus (152) Google Scholar). As regards the activation of eNOS gene, our data are in agreement with the proposition that Ets serves as a co-activator for Sp1. Binding of Ets alone resulted in minimal promoter activation, whereas it augmented Sp1-mediated transcriptional activation. It is possible that Ets interacts with components of the general transcription factors in the preinitiation complex through which it may enhance recruitment of the preinitiation complex to the promoter region. Lyso-PC is a main component of oxidized low density lipoprotein (22Parthasarathy S. Streinbrecher U.P. Barnett J. Witztum J.L. Steinberg D. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 3000-3004Crossref PubMed Scopus (306) Google Scholar). The level of lyso-PC is highly elevated in human atherosclerotic tissues (23Portman O.W. Alexander M. J. Lipid Res. 1969; 10: 158-165Abstract Full Text PDF PubMed Google Scholar). It has been shown that lyso-PC increases chemotaxis for monocytes and induces endothelial adhesive molecules such as intercellular adhesive molecule-1 and vascular cell adhesive molecule-1, which mediate monocyte and neutrophil adhesion to vascular endothelium (24Sakai M. Miyazaki A. Hakamata H. Sasaki T. Yui S. Yamazaki M. Schichiri M. Horiuchi S. J. Biol. Chem. 1994; 269: 31430-31435Abstract Full Text PDF PubMed Google Scholar). Hence, lyso-PC has been considered to play an important role in monocyte accumulation in the arterial walls. Furthermore, lyso-PC induces the expression platelet-derived growth factor A and B subunits and heparin-binding epidermal growth factor from endothelial cells (25Kume N. Gimbrone M.A. J. Clin. Invest. 1994; 93: 907-911Crossref PubMed Scopus (294) Google Scholar, 26Kume N. Cybulski M.I. Gimbrone Jr., M.A. J. Clin. Invest. 1992; 90: 1138-1144Crossref PubMed Scopus (720) Google Scholar). These growth factors promote smooth muscle cell migration and proliferation. These findings have led to the conclusion that lyso-PC is a key mediator of atherosclerosis (22Parthasarathy S. Streinbrecher U.P. Barnett J. Witztum J.L. Steinberg D. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 3000-3004Crossref PubMed Scopus (306) Google Scholar). Work from this laboratory provides evidence to indicate that the expression of NOS-III and cyclooxygenase-2 are stimulated by lyso-PC (27Zembowicz A. Jones S.L. Wu K.K. J. Clin. Invest. 1995; 96: 1688-1692Crossref PubMed Scopus (88) Google Scholar), thereby increasing the synthesis of NO and prostacyclin, which act synergistically to block monocyte adhesion and smooth muscle cell proliferation; platelet activation and aggregation; and pathological vasoconstriction. This leads to a postulate that vascular injurious agents such as lyso-PC creates a yin-yan situation in which it induces the expression of vasoprotective genes to counteract the vascular damaging genes. It is interesting to note that lyso-PC induces the expressions of those diverse genes with two distinct types of kinetics: 1) rapid, transient as in heparin-binding epidermal growth factor and cyclooxygenase-2 gene induction and 2) delayed, sustained as in eNOS and intercellular adhesive molecule-1 gene induction. It is unclear whether these two types of genes are transcriptionally regulated by a common mechanism. In fact, the mechanism by which lyso-PC induces any of these genes had not been reported previously. This report is the first to shed light on the promoter regulation of eNOS gene by lyso-PC. Our results indicate that lyso-PC enhances the transcriptional activation of this housekeeping gene primarily by increasing the Sp1 binding activity via a PP2A-dependent reaction. Ets binding to its cognate site on the promoter region is also involved in basal and lyso-PC-induced eNOS transcription. Sp1 is a ubiquitous transcriptional activator mediating basal and regulated gene expression. Sp1 binding to its cognate motif depends not only on nuclear Sp1 levels but also on posttranslational modification of the Sp1 molecule. Recent studies indicate that Sp1 binding activity is influenced by phosphorylation: phosphorylation decreases, whereas dephosphorylation by the action of phosphatases increases Sp1 binding activity (28Daniel S. Zhang S. DePaoli-Roach A.A. Kim K.H. J. Biol. Chem. 1996; 271: 14692-14697Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar, 29Armstrong S.A. Barry D.A. Leggett R.W. Mueller C.R. J. Biol. Chem. 1997; 272: 13489-13495Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). A recent study on the acetyl-CoA carboxylase gene indicates that glucose induces this gene transcription by a mechanism involving dephosphorylation of Sp1 by protein phosphatase 1 and 2A (28Daniel S. Zhang S. DePaoli-Roach A.A. Kim K.H. J. Biol. Chem. 1996; 271: 14692-14697Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Our data, which show suppression of lyso-PC-induced Sp1 binding activity by okadaic acid, but not vanadate, and a selective increase in PP2A activity by lyso-PC, which was inhibited by okadaic acid, are consistent with a role of PP2A in modifying Sp1 binding activity. Results from this study led us to postulate that the Sp1 is phosphorylated at the basal cellular state, and lyso-PC activates PP2A, which in turn dephosphorylate Sp1, resulting in an increased Sp1 binding activity. Signal pathways for lyso-PC-induced transcriptional activation have not been fully established. A recent study suggests the involvement of stress-activated protein kinase/c-Jun amino-terminal kinase pathway that leads to AP1 activation and binding to its cognate site (30Fang X. Gibson S. Flowers M. Furui T. Bast Jr., R.C. Mills G.B. J. Biol. Chem. 1997; 272: 13683-13689Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). It is unclear whether JNK and/or other mitogen-activated protein kinase pathways are involved in increased Sp1 binding activity. It is possible that lyso-PC may increase Sp1 binding activity by activating protein phosphatases via the mitogen-activated protein kinase or other kinase pathways. This is being investigated in our laboratory. Our results indicate that a PEA3/Ets sequence in the promoter/enhancer region of eNOS gene is involved in basal and lyso-PC-induced promoter activity. Nuclear Ets-related proteins contain a large family of polypeptides sharing an ETS domain (31Wasylyk B. Hahn S.L. Giovane A. Eur. J. Biochem. 1993; 211: 7-18Crossref PubMed Scopus (809) Google Scholar). They bind to purine-rich sequences with a GGA core. The 5′- and 3′-flanking sequences of the GGA core confer relative specificity for Ets family proteins. The GGA-containing sequences on eNOS lack a specificity for any given class of Ets-related proteins. This is in agreement with our experimental data showing promiscuity of binding for several representatives of Ets family proteins. Ets proteins are involved in cellular response to external stresses. They generally serve as co-activators for transcriptional activators including Sp1 (32Khachigian L.A. Williams A.J. Collins T. J. Biol. Chem. 1995; 270: 27679-27686Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar, 33Gegonne A. Bosselut R. Bailly R.-A. Ghysdael J. EMBO J. 1993; 12: 1169-1178Crossref PubMed Scopus (152) Google Scholar). Several reports have suggested that PEA3 and Sp1 sequences are often colocalized on the promoter/enhancer region and binding of Ets and Sp1 to their respective sites causes a synergistic activation of a number of genes (33Gegonne A. Bosselut R. Bailly R.-A. Ghysdael J. EMBO J. 1993; 12: 1169-1178Crossref PubMed Scopus (152) Google Scholar). As regards the activation of eNOS gene, our data are in agreement with the proposition that Ets serves as a co-activator for Sp1. Binding of Ets alone resulted in minimal promoter activation, whereas it augmented Sp1-mediated transcriptional activation. It is possible that Ets interacts with components of the general transcription factors in the preinitiation complex through which it may enhance recruitment of the preinitiation complex to the promoter region. We thank Zhifei Zu for technical assistance and Susan Mitterling for excellent assistance in preparing this manuscript." @default.
• W2077366814 created "2016-06-24" @default.
• W2077366814 creator A5014157914 @default.
• W2077366814 creator A5035975194 @default.
• W2077366814 creator A5050785570 @default.
• W2077366814 creator A5052376231 @default.
• W2077366814 date "1998-06-01" @default.
• W2077366814 modified "2023-10-16" @default.
• W2077366814 title "Transcriptional Regulation of Endothelial Nitric-oxide Synthase by Lysophosphatidylcholine" @default.
• W2077366814 cites W1511098597 @default.
• W2077366814 cites W1519102962 @default.
• W2077366814 cites W1519147845 @default.
• W2077366814 cites W1529376098 @default.
• W2077366814 cites W1553107571 @default.
• W2077366814 cites W1599900714 @default.
• W2077366814 cites W1834041853 @default.
• W2077366814 cites W1965077361 @default.
• W2077366814 cites W1966883946 @default.
• W2077366814 cites W2001381510 @default.
• W2077366814 cites W2005476698 @default.
• W2077366814 cites W2011574071 @default.
• W2077366814 cites W2029551349 @default.
• W2077366814 cites W2042277367 @default.
• W2077366814 cites W2059039694 @default.
• W2077366814 cites W2059379142 @default.
• W2077366814 cites W2076332509 @default.
• W2077366814 cites W2080512950 @default.
• W2077366814 cites W2091548699 @default.
• W2077366814 cites W2091732701 @default.
• W2077366814 cites W2097794242 @default.
• W2077366814 cites W2099992849 @default.
• W2077366814 cites W2122824674 @default.
• W2077366814 cites W2125823894 @default.
• W2077366814 cites W2128154787 @default.
• W2077366814 cites W2135038548 @default.
• W2077366814 cites W2164319707 @default.
• W2077366814 cites W2167120310 @default.
• W2077366814 cites W2418871452 @default.
• W2077366814 cites W99079292 @default.
• W2077366814 doi "https://doi.org/10.1074/jbc.273.24.14885" @default.
• W2077366814 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9614091" @default.
• W2077366814 hasPublicationYear "1998" @default.
• W2077366814 type Work @default.
• W2077366814 sameAs 2077366814 @default.
• W2077366814 citedByCount "77" @default.
• W2077366814 countsByYear W20773668142012 @default.
• W2077366814 countsByYear W20773668142014 @default.
• W2077366814 countsByYear W20773668142015 @default.
• W2077366814 countsByYear W20773668142016 @default.
• W2077366814 countsByYear W20773668142017 @default.
• W2077366814 countsByYear W20773668142020 @default.
• W2077366814 countsByYear W20773668142021 @default.
• W2077366814 countsByYear W20773668142023 @default.
• W2077366814 crossrefType "journal-article" @default.
• W2077366814 hasAuthorship W2077366814A5014157914 @default.
• W2077366814 hasAuthorship W2077366814A5035975194 @default.
• W2077366814 hasAuthorship W2077366814A5050785570 @default.
• W2077366814 hasAuthorship W2077366814A5052376231 @default.
• W2077366814 hasBestOaLocation W20773668141 @default.
• W2077366814 hasConcept C112243037 @default.
• W2077366814 hasConcept C178790620 @default.
• W2077366814 hasConcept C181199279 @default.
• W2077366814 hasConcept C185592680 @default.
• W2077366814 hasConcept C2776330855 @default.
• W2077366814 hasConcept C2777622882 @default.
• W2077366814 hasConcept C2778326061 @default.
• W2077366814 hasConcept C2778596009 @default.
• W2077366814 hasConcept C2778918659 @default.
• W2077366814 hasConcept C2908688039 @default.
• W2077366814 hasConcept C2992676626 @default.
• W2077366814 hasConcept C41625074 @default.
• W2077366814 hasConcept C519581460 @default.
• W2077366814 hasConcept C55493867 @default.
• W2077366814 hasConcept C86803240 @default.
• W2077366814 hasConcept C95444343 @default.
• W2077366814 hasConceptScore W2077366814C112243037 @default.
• W2077366814 hasConceptScore W2077366814C178790620 @default.
• W2077366814 hasConceptScore W2077366814C181199279 @default.
• W2077366814 hasConceptScore W2077366814C185592680 @default.
• W2077366814 hasConceptScore W2077366814C2776330855 @default.
• W2077366814 hasConceptScore W2077366814C2777622882 @default.
• W2077366814 hasConceptScore W2077366814C2778326061 @default.
• W2077366814 hasConceptScore W2077366814C2778596009 @default.
• W2077366814 hasConceptScore W2077366814C2778918659 @default.
• W2077366814 hasConceptScore W2077366814C2908688039 @default.
• W2077366814 hasConceptScore W2077366814C2992676626 @default.
• W2077366814 hasConceptScore W2077366814C41625074 @default.
• W2077366814 hasConceptScore W2077366814C519581460 @default.
• W2077366814 hasConceptScore W2077366814C55493867 @default.
• W2077366814 hasConceptScore W2077366814C86803240 @default.
• W2077366814 hasConceptScore W2077366814C95444343 @default.
• W2077366814 hasIssue "24" @default.
• W2077366814 hasLocation W20773668141 @default.
• W2077366814 hasOpenAccess W2077366814 @default.
• W2077366814 hasPrimaryLocation W20773668141 @default.
• W2077366814 hasRelatedWork W1977080241 @default.
• W2077366814 hasRelatedWork W2016244203 @default.
• W2077366814 hasRelatedWork W2152012885 @default.

• next