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

Reactive oxygen species (ROS) are implicated in cardiovascular diseases. ROS, such as H 2 O 2 , act as second messengers to activate diverse signaling pathways. Although H 2 O 2 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 H 2 O 2 involving a metalloprotease-dependent generation of heparin-binding EGF-like growth factor (HB-EGF) and protein kinase C (PKC)-δ activation, respectively. H 2 O 2 -induced EGF receptor tyrosine phosphorylation was inhibited by a metalloprotease inhibitor, whereas the inhibitor had no effect on H 2 O 2 -induced JAK2 tyrosine phosphorylation. HB-EGF neutralizing antibody inhibited H 2 O 2 -induced EGF receptor phosphorylation. In COS-7 cells expressing an HB-EGF construct tagged with alkaline phosphatase, H 2 O 2 stimulates HB-EGF production through metalloprotease activation. By contrast, dominant negative PKC-δ transfection inhibited H 2 O 2 -induced JAK2 phosphorylation but not EGF receptor phosphorylation. Dominant negative PYK2 inhibited H 2 O 2 -induced JAK2 activation but not EGF receptor activation, whereas dominant negative PKC-δ inhibited PYK2 activation by H 2 O 2 . These data demonstrate the presence of distinct tyrosine kinase activation pathways (PKC-δ/PYK2/JAK2 and metalloprotease/HB-EGF/EGF receptor) utilized by H 2 O 2 in VSMCs, thus providing unique therapeutic targets for cardiovascular diseases.