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-δ

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)-δ 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-δ 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-δ inhibited PYK2 activation by H₂O₂. These data demonstrate the presence of distinct tyrosine kinase activation pathways (PKC-δ/PYK2/JAK2 and metalloprotease/HB-EGF/EGF receptor) utilized by H₂O₂ in VSMCs, thus providing unique therapeutic targets for cardiovascular diseases.

ACKNOWLEDGMENTS

We thank Kunie Eguchi and Trinita Fitzgerald for their excellent technical assistance.

This work was supported by National Institute of Health training grant HL07323 and a United Negro College Fund/Merck postdoctoral science research fellowship (G. Frank), by an American Heart Association Scientist Development Grant and a Vanderbilt University Diabetes Center Pilot & Feasibility Proposal (S. Eguchi), and in part by the research grants HL58205, DK20593, and CA68485 from the National Institute of Health (T. Inagami).