title: The tyrosine phosphatase, SHP-1, is a negative regulator of endothelial superoxide formation.
abstract: OBJECTIVE
  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.
  
  
  BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Institute of Physiology, Ludwig-Maximilians University, Munich, Germany. fkroetz@lmu.de
authors.forename: Florian
authors.initials: F
authors.lastname: Krötz
authors.forename: Barbara
authors.initials: B
authors.lastname: Engelbrecht
authors.forename: Martin A
authors.initials: MA
authors.lastname: Buerkle
authors.forename: Florian
authors.initials: F
authors.lastname: Bassermann
authors.forename: Hanna
authors.initials: H
authors.lastname: Bridell
authors.forename: Torsten
authors.initials: T
authors.lastname: Gloe
authors.forename: Justus
authors.initials: J
authors.lastname: Duyster
authors.forename: Ulrich
authors.initials: U
authors.lastname: Pohl
authors.forename: Hae-Young
authors.initials: HY
authors.lastname: Sohn
mesh_terms: D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D042783:Endothelial Cells / Q000502:physiology; D004730:Endothelium, Vascular / Q000502:physiology*; D004789:Enzyme Activation / Q000502:physiology; D006801:Humans; D066298:In Vitro Techniques; D047908:Intracellular Signaling Peptides and Proteins; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors* / Q000502:physiology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D054645:Protein Phosphatase 1; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology; D013481:Superoxides / Q000378:metabolism*
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D013481:Superoxides; D019255:NADPH Oxidases; D019869:Phosphatidylinositol 3-Kinases; D054645:Protein Phosphatase 1; C509249:PTPN6 protein, human; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases