The Tyrosine Phosphatase, SHP-1, Is a Negative Regulator of Endothelial Superoxide Formation

• Published in: Journal of the American College of Cardiology Vol. 45; no. 10; pp. 1700 - 1706
• Main Authors: Krötz, Florian, Engelbrecht, Barbara, Buerkle, Martin A., Bassermann, Florian, Bridell, Hanna, Gloe, Torsten, Duyster, Justus, Pohl, Ulrich, Sohn, Hae-Young
• Format: Journal Article
• Language: English
• Published: New York, NY Elsevier Inc 17-05-2005; Elsevier Science; Elsevier Limited
• Subjects: Animals; Atherosclerosis; Biological and medical sciences; Cardiology; Cardiology. Vascular system; Cell culture; Cell Line; Cricetinae; Endothelial Cells - physiology; Endothelium, Vascular - physiology; Enzyme Activation - physiology; Enzymes; Humans; In Vitro Techniques; Intracellular Signaling Peptides and Proteins; Kinases; Medical sciences; Muscular system; NADPH Oxidases - antagonists & inhibitors; NADPH Oxidases - physiology; Phosphatidylinositol 3-Kinases - metabolism; Phosphorylation; Protein Phosphatase 1; Protein Tyrosine Phosphatase, Non-Receptor Type 6; Protein Tyrosine Phosphatases - physiology; Proteins; Signal Transduction - physiology; Smooth muscle; Superoxides - metabolism; Germany; SS; O2; AT; PI3K; HUVEC; NAD(P)H; siRNA; AS-ODN; SHP-1; PTP; RIPA; Tyrosine; Hyperoxides; Enzyme; Phosphoric monoester hydrolases; Hydrolases; Esterases; Circulatory system; Regulator; Cardiology; Formation; Phlebology
• ISSN: 0735-1097; 1558-3597
• DOI: 10.1016/j.jacc.2005.02.039

We investigated the role of SH2-domain containing phosphatase-1 (SHP-1) in endothelial reduced nicotinamide adenine dinucleotide (phosphate) (NAD[P]H)-oxidase-dependent oxidant production.
Superoxide (O2·−) generation by endothelial NAD(P)H-oxidase promotes endothelial dysfunction and atherosclerosis. Signaling pathways that regulate NAD(P)H-oxidase activity are, however, poorly understood.
SH2-domain containing phosphatase-1 was inhibited using site-directed magnetofection of antisense oligodesoxynucleotides (AS-ODN) or short interfering ribonucleic acid (siRNA) in vitro in human umbilical vein endothelial cells (HUVEC) and in isolated hamster arteries; O2·−was measured by cytochrome c reduction in vitro. Activities of NAD(P)H-oxidase activity, phosphatidyl-inositol-3-kinase (PI3K), and SHP-1 were assessed by specific assays; Rac1 activation was assessed by a pull-down assay.
Basal endothelial O2·−release was enhanced after inhibition of endothelial SHP-1 (p < 0.01), which could be prevented by specific inhibition of NAD(P)H-oxidase (p < 0.01); SHP-1 activity was high under basal conditions, further increased by vascular endothelial growth factor (10 ng/ml, p < 0.05), and abolished by SHP-1 AS-ODN treatment (p < 0.01), which also increased NAD(P)H-oxidase activity 3.3-fold (p < 0.01). Vascular endothelial growth factor also induced O2·−release (p < 0.01), which was even more enhanced when SHP-1 was knocked down (p < 0.05). The effect of SHP-1 was mediated by inhibition of PI3K/Rac1-dependent NAD(P)H-oxidase activation (p < 0.01); SHP-1 AS-ODN augmented tyrosine phosphorylation of the p85 regulatory subunit of PI3K (p < 0.05) and Rac1 activation. The latter was prevented by wortmannin, a blocker of PI3K.
In HUVEC, SHP-1 counteracts basal and stimulated NAD(P)H-oxidase activity by negative regulation of PI3K-dependent Rac1 activation; SHP-1 thus seems to be an important part of endothelial antioxidative defense controlling the activity of the O2·−-producing NAD(P)H-oxidase.