title: Atrial natriuretic peptide induces mitogen-activated protein kinase phosphatase-1 in human endothelial cells via Rac1 and NAD(P)H oxidase/Nox2-activation.
abstract: The cardiovascular hormone atrial natriuretic peptide (ANP) exerts anti-inflammatory effects on tumor necrosis factor-alpha-activated endothelial cells by inducing mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1). The underlying mechanisms are as yet unknown. We aimed to elucidate the signaling pathways leading to an induction of MKP-1 by ANP in primary human endothelial cells. By using antioxidants, generation of reactive oxygen species (ROS) was shown to be crucially involved in MKP-1 upregulation. ANP was found to increase ROS formation in cultured cells as well as in the endothelium of intact rat lung vessels. We applied NAD(P)H oxidase (Nox) inhibitors (apocynin and gp91ds-tat) and revealed this enzyme complex to be crucial for superoxide generation and MKP-1 expression. Moreover, by performing Nox2/4 antisense experiments, we identified Nox2 as the critically involved Nox homologue. Pull-down assays and confocal microscopy showed that ANP activates the small Rho-GTPase Rac1. Transfection of a dominant-negative (RacN17) and constitutively active Rac1 mutant (RacV12) indicated that ANP-induced superoxide generation and MKP-1 expression are mediated via Rac1 activation. ANP-evoked production of superoxide was found to activate c-Jun N-terminal kinase (JNK). Using specific inhibitors, we linked ANP-induced JNK activation to MKP-1 expression and excluded an involvement of protein kinase C, extracellular signal-regulated kinase, and p38 MAPK. MKP-1 induction was shown to depend on activation of the transcription factor activator protein-1 (AP-1) by using electrophoretic mobility shift assay and AP-1 decoys. In summary, our work provides insights into the mechanisms by which ANP induces MKP-1 and shows that ANP is a novel endogenous activator of endothelial Rac1 and Nox/Nox2.
authors.affiliation: Department of Pharmacy, University of Munich, Germany.
authors.forename: Robert
authors.initials: R
authors.lastname: Fürst
authors.forename: Corinna
authors.initials: C
authors.lastname: Brueckl
authors.forename: Wolfgang M
authors.initials: WM
authors.lastname: Kuebler
authors.forename: Stefan
authors.initials: S
authors.lastname: Zahler
authors.forename: Florian
authors.initials: F
authors.lastname: Krötz
authors.forename: Agnes
authors.initials: A
authors.lastname: Görlach
authors.forename: Angelika M
authors.initials: AM
authors.lastname: Vollmar
authors.forename: Alexandra K
authors.initials: AK
authors.lastname: Kiemer
mesh_terms: D000098:Acetophenones / Q000494:pharmacology; D000818:Animals; D009320:Atrial Natriuretic Factor / Q000494:pharmacology / Q000502:physiology*; D002196:Capillaries; D018797:Cell Cycle Proteins / Q000096:biosynthesis* / Q000235:genetics; D002478:Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D003513:Cycloheximide / Q000494:pharmacology; D016373:DNA, Antisense / Q000494:pharmacology; D054638:Dual Specificity Phosphatase 1; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology; D004790:Enzyme Induction / Q000187:drug effects / Q000502:physiology; D006023:Glycoproteins / Q000494:pharmacology; D006162:Guanylate Cyclase / Q000187:drug effects / Q000502:physiology; D006801:Humans; D017874:Immediate-Early Proteins / Q000096:biosynthesis* / Q000235:genetics; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D008168:Lung / Q000098:blood supply; D048670:MAP Kinase Kinase 4; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D000074624:NADPH Oxidase 1; D000074662:NADPH Oxidase 2; D000074663:NADPH Oxidase 4; D000074664:NADPH Oxidase 5; D019255:NADPH Oxidases / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000096:biosynthesis* / Q000235:genetics; D054645:Protein Phosphatase 1; D017027:Protein Tyrosine Phosphatases / Q000096:biosynthesis* / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism; D017461:Receptors, Atrial Natriuretic Factor / Q000187:drug effects / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000502:physiology; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014162:Transfection; D014471:Umbilical Veins / Q000166:cytology
chemical_list: D000098:Acetophenones; D018797:Cell Cycle Proteins; D016373:DNA, Antisense; D006023:Glycoproteins; D017874:Immediate-Early Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D011993:Recombinant Fusion Proteins; D018808:Transcription Factor AP-1; C473031:gp91ds-tat protein, chimeric; C016276:8-bromocyclic GMP; D009320:Atrial Natriuretic Factor; D003513:Cycloheximide; C056165:acetovanillone; C056558:CYBB protein, human; D000074624:NADPH Oxidase 1; D000074662:NADPH Oxidase 2; D000074663:NADPH Oxidase 4; D000074664:NADPH Oxidase 5; D019255:NADPH Oxidases; C402799:NOX1 protein, human; C498910:NOX4 protein, human; C431571:NOX5 protein, human; C431570:Nox3 protein, human; C498911:Nox4 protein, rat; D048031:JNK Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4; D020929:Mitogen-Activated Protein Kinase Kinases; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; C517019:DUSP1 protein, human; D054638:Dual Specificity Phosphatase 1; C517021:Dusp1 protein, rat; D017027:Protein Tyrosine Phosphatases; D006162:Guanylate Cyclase; D017461:Receptors, Atrial Natriuretic Factor; C091715:atrial natriuretic factor receptor A; C073616:atrial natriuretic factor receptor C; D006152:Cyclic GMP