title: Distinct mechanisms of receptor and nonreceptor tyrosine kinase activation by reactive oxygen species in vascular smooth muscle cells: role of metalloprotease and protein kinase C-delta.
abstract: Reactive oxygen species (ROS) are implicated in cardiovascular diseases. ROS, such as H2O2, act as second messengers to activate diverse signaling pathways. Although H2O2 activates several tyrosine kinases, including the epidermal growth factor (EGF) receptor, JAK2, and PYK2, in vascular smooth muscle cells (VSMCs), the intracellular mechanism by which ROS activate these tyrosine kinases remains unclear. Here, we identified two distinct signaling pathways required for receptor and nonreceptor tyrosine kinase activation by H2O2 involving a metalloprotease-dependent generation of heparin-binding EGF-like growth factor (HB-EGF) and protein kinase C (PKC)-delta activation, respectively. H2O2-induced EGF receptor tyrosine phosphorylation was inhibited by a metalloprotease inhibitor, whereas the inhibitor had no effect on H2O2-induced JAK2 tyrosine phosphorylation. HB-EGF neutralizing antibody inhibited H2O2-induced EGF receptor phosphorylation. In COS-7 cells expressing an HB-EGF construct tagged with alkaline phosphatase, H2O2 stimulates HB-EGF production through metalloprotease activation. By contrast, dominant negative PKC-delta transfection inhibited H2O2-induced JAK2 phosphorylation but not EGF receptor phosphorylation. Dominant negative PYK2 inhibited H2O2-induced JAK2 activation but not EGF receptor activation, whereas dominant negative PKC-delta inhibited PYK2 activation by H2O2. These data demonstrate the presence of distinct tyrosine kinase activation pathways (PKC-delta/PYK2/JAK2 and metalloprotease/HB-EGF/EGF receptor) utilized by H2O2 in VSMCs, thus providing unique therapeutic targets for cardiovascular diseases.
authors.affiliation: Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
authors.forename: Gerald D
authors.initials: GD
authors.lastname: Frank
authors.forename: Mizuo
authors.initials: M
authors.lastname: Mifune
authors.forename: Tadashi
authors.initials: T
authors.lastname: Inagami
authors.forename: Motoi
authors.initials: M
authors.lastname: Ohba
authors.forename: Terukatsu
authors.initials: T
authors.lastname: Sasaki
authors.forename: Shigeki
authors.initials: S
authors.lastname: Higashiyama
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Dempsey
authors.forename: Satoru
authors.initials: S
authors.lastname: Eguchi
mesh_terms: D000098:Acetophenones / Q000494:pharmacology; D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D001578:Benzopyrans / Q000494:pharmacology; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000378:metabolism; D005799:Genes, Dominant; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D015151:Immunoblotting; D053614:Janus Kinase 2; D008666:Metalloendopeptidases / Q000378:metabolism*; D008954:Models, Biological; D009131:Muscle, Smooth, Vascular / Q000166:cytology*; D010084:Oxidation-Reduction; D011233:Precipitin Tests; D011493:Protein Kinase C / Q000378:metabolism*; D051745:Protein Kinase C-delta; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins; D051381:Rats; D017207:Rats, Sprague-Dawley; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection
chemical_list: D000098:Acetophenones; D001578:Benzopyrans; D004791:Enzyme Inhibitors; D011518:Proto-Oncogene Proteins; D004815:Epidermal Growth Factor; D006861:Hydrogen Peroxide; C085746:rottlerin; C497260:Prkcd protein, rat; D011505:Protein-Tyrosine Kinases; D020794:Receptor Protein-Tyrosine Kinases; C507924:JAK2 protein, human; C507927:Jak2 protein, rat; D053614:Janus Kinase 2; C497258:PRKCD protein, human; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; D008666:Metalloendopeptidases