Atrial Natriuretic Peptide Induces Mitogen-Activated Protein Kinase Phosphatase-1 in Human Endothelial Cells via Rac1 and NAD(P)H Oxidase/Nox2-Activation

• Published in: Circulation research 2005-01, Vol.96 (1), p.43-53
• Main Authors: Fürst, Robert, Brueckl, Corinna, Kuebler, Wolfgang M, Zahler, Stefan, Krötz, Florian, Görlach, Agnes, Vollmar, Angelika M, Kiemer, Alexandra K
• Format: Article
• Language: English
• Subjects: Acetophenones - pharmacology; Animals; Atrial Natriuretic Factor - pharmacology; Atrial Natriuretic Factor - physiology; Biological and medical sciences; Capillaries; Cell Cycle Proteins - biosynthesis; Cell Cycle Proteins - genetics; Cells, Cultured - drug effects; Cells, Cultured - metabolism; Cyclic GMP - analogs & derivatives; Cyclic GMP - metabolism; Cyclic GMP - pharmacology; Cycloheximide - pharmacology; DNA, Antisense - pharmacology; Dual Specificity Phosphatase 1; Endothelial Cells - drug effects; Endothelial Cells - metabolism; Endothelium, Vascular - cytology; Enzyme Induction - drug effects; Enzyme Induction - physiology; Fundamental and applied biological sciences. Psychology; Glycoproteins - pharmacology; Guanylate Cyclase - drug effects; Guanylate Cyclase - physiology; Humans; Immediate-Early Proteins - biosynthesis; Immediate-Early Proteins - genetics; JNK Mitogen-Activated Protein Kinases - metabolism; Lung - blood supply; MAP Kinase Kinase 4; MAP Kinase Signaling System - drug effects; MAP Kinase Signaling System - physiology; Membrane Glycoproteins - biosynthesis; Membrane Glycoproteins - genetics; Membrane Glycoproteins - physiology; Membrane Proteins - biosynthesis; Membrane Proteins - genetics; Mitogen-Activated Protein Kinase Kinases - metabolism; NADPH Oxidase 1; NADPH Oxidase 2; NADPH Oxidase 4; NADPH Oxidase 5; NADPH Oxidases - biosynthesis; NADPH Oxidases - genetics; NADPH Oxidases - physiology; Oligonucleotides, Antisense - pharmacology; Phosphoprotein Phosphatases - biosynthesis; Phosphoprotein Phosphatases - genetics; Protein Phosphatase 1; Protein Tyrosine Phosphatases - biosynthesis; Protein Tyrosine Phosphatases - genetics; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species - metabolism; Receptors, Atrial Natriuretic Factor - drug effects; Receptors, Atrial Natriuretic Factor - physiology; Recombinant Fusion Proteins - physiology; RNA, Messenger - biosynthesis; RNA, Messenger - genetics; Transcription Factor AP-1 - metabolism; Transfection; Umbilical Veins - cytology; Vertebrates: cardiovascular system
• ISSN: 0009-7330; 1524-4571
• DOI: 10.1161/01.RES.0000151983.01148.06

The cardiovascular hormone atrial natriuretic peptide (ANP) exerts anti-inflammatory effects on tumor necrosis factor-α–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.