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.
Publication types
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Comparative Study
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Research Support, Non-U.S. Gov't
MeSH terms
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Acetophenones / pharmacology
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Animals
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Atrial Natriuretic Factor / pharmacology
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Atrial Natriuretic Factor / physiology*
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Capillaries
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Cell Cycle Proteins / biosynthesis*
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Cell Cycle Proteins / genetics
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Cells, Cultured / drug effects
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Cells, Cultured / metabolism
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Cyclic GMP / analogs & derivatives*
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Cyclic GMP / metabolism
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Cyclic GMP / pharmacology
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Cycloheximide / pharmacology
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DNA, Antisense / pharmacology
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Dual Specificity Phosphatase 1
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Endothelial Cells / drug effects*
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Endothelial Cells / metabolism
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Endothelium, Vascular / cytology
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Enzyme Induction / drug effects
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Enzyme Induction / physiology
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Glycoproteins / pharmacology
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Guanylate Cyclase / drug effects
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Guanylate Cyclase / physiology
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Humans
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Immediate-Early Proteins / biosynthesis*
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Immediate-Early Proteins / genetics
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JNK Mitogen-Activated Protein Kinases / metabolism
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Lung / blood supply
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MAP Kinase Kinase 4
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MAP Kinase Signaling System / drug effects
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MAP Kinase Signaling System / physiology
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Membrane Glycoproteins / biosynthesis
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Membrane Glycoproteins / genetics
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Membrane Glycoproteins / physiology*
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Membrane Proteins / biosynthesis
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Membrane Proteins / genetics
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Mitogen-Activated Protein Kinase Kinases / metabolism
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NADPH Oxidase 1
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NADPH Oxidase 2
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NADPH Oxidase 4
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NADPH Oxidase 5
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NADPH Oxidases / biosynthesis
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NADPH Oxidases / genetics
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NADPH Oxidases / physiology*
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Oligonucleotides, Antisense / pharmacology
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Phosphoprotein Phosphatases / biosynthesis*
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Phosphoprotein Phosphatases / genetics
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Protein Phosphatase 1
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Protein Tyrosine Phosphatases / biosynthesis*
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Protein Tyrosine Phosphatases / genetics
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RNA, Messenger / biosynthesis
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RNA, Messenger / genetics
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Rats
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Rats, Sprague-Dawley
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Reactive Oxygen Species / metabolism
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Receptors, Atrial Natriuretic Factor / drug effects
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Receptors, Atrial Natriuretic Factor / physiology
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Recombinant Fusion Proteins / physiology
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Transcription Factor AP-1 / metabolism
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Transfection
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Umbilical Veins / cytology
Substances
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Acetophenones
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Cell Cycle Proteins
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DNA, Antisense
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Glycoproteins
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Immediate-Early Proteins
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Membrane Glycoproteins
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Membrane Proteins
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Oligonucleotides, Antisense
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RNA, Messenger
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Reactive Oxygen Species
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Recombinant Fusion Proteins
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Transcription Factor AP-1
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gp91ds-tat protein, chimeric
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8-bromocyclic GMP
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Atrial Natriuretic Factor
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Cycloheximide
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acetovanillone
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CYBB protein, human
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NADPH Oxidase 1
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NADPH Oxidase 2
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NADPH Oxidase 4
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NADPH Oxidase 5
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NADPH Oxidases
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NOX1 protein, human
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NOX4 protein, human
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NOX5 protein, human
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Nox3 protein, human
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Nox4 protein, rat
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JNK Mitogen-Activated Protein Kinases
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MAP Kinase Kinase 4
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Mitogen-Activated Protein Kinase Kinases
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Phosphoprotein Phosphatases
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Protein Phosphatase 1
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DUSP1 protein, human
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Dual Specificity Phosphatase 1
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Dusp1 protein, rat
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Protein Tyrosine Phosphatases
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Guanylate Cyclase
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Receptors, Atrial Natriuretic Factor
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atrial natriuretic factor receptor A
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atrial natriuretic factor receptor C
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Cyclic GMP