Phosphoinositide-dependent kinase 1 and p21-activated protein kinase mediate reactive oxygen species-dependent regulation of platelet-derived growth factor-induced smooth muscle cell migration.
Smooth muscle cell migration in response to platelet-derived growth factor (PDGF) is a key event in several vascular pathologies, including atherosclerosis and restenosis. PDGF increases intracellular levels of reactive oxygen species (ROS) in vascular smooth muscle cells (VSMCs), but the ROS sensitivity of migration and of the signaling pathways leading to migration are largely unknown. In VSMCs, PDGF dose-dependently increased migration compared with nonstimulated cells, with a maximum increase at 10 ng/mL. Pretreatment with the antioxidant N-acetyl-cysteine, the flavin-containing enzyme inhibitor diphenylene iodonium, or the glutathione peroxidase mimetic ebselen significantly attenuated migration (PDGF alone, 5.0+/-1.1-fold; NAC, 1.8+/-0.2-fold; diphenylene iodonium, 1.4+/-0.3-fold migration; and ebselen, 2.0+/-0.5-fold migration), as did overexpression of catalase. Pretreatment of VSMCs with the Src inhibitor PP1 or dominant-negative Rac adenovirus significantly inhibited migration, but only Src activation was attenuated by ROS inhibitors. Phosphorylation of the Src- and Rac-effector p21-activated protein kinase (PAK) 1 on Thr423 (the phosphoinositide-dependent kinase-1 [PDK1] site) was attenuated by ROS inhibition, and infection of VSMCs with dominant-negative PAK1 adenovirus attenuated migration. Moreover, kinase-inactive K111N-PDK1 inhibited PAK1 phosphorylation on Thr423, and both K111N-PDK1 and Y9F-PDK1 significantly inhibited VSMC migration. PDK1 tyrosine phosphorylation was also ROS dependent. These data indicate that PDGF-induced VSMC migration is ROS dependent and identify the Src/PDK1/PAK1 signaling pathway as an important ROS-sensitive mediator of migration. Such information is critical to understanding the role of ROS in vascular diseases in which migration of VSMCs is an important component.
Weber
David S
DS
Department of Medicine, Division of Cardiology, Emory University, Atlanta, Ga 30322, USA.
Taniyama
Yoshihiro
Y
Rocic
Petra
P
Seshiah
Puvi N
PN
Dechert
Melissa A
MA
Gerthoffer
William T
WT
Griendling
Kathy K
KK
D064413:3-Phosphoinositide-Dependent Protein Kinases; D000111:Acetylcysteine / Q000494:pharmacology; D000818:Animals; D001013:Aorta, Thoracic / Q000166:cytology; D001393:Azoles / Q000494:pharmacology; D000077214:Becaplermin; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured / Q000187:drug effects / Q000201:enzymology; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D054833:Isoindoles; D019950:Mitogen-Activated Protein Kinase 1 / Q000502:physiology; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000502:physiology; D009131:Muscle, Smooth, Vascular / Q000166:cytology*; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000201:enzymology*; D016566:Organoselenium Compounds / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology*; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000502:physiology*; D020574:Proto-Oncogene Proteins c-sis; D051381:Rats; D017382:Reactive Oxygen Species / Q000494:pharmacology*; D017479:Receptors, Platelet-Derived Growth Factor / Q000187:drug effects / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000494:pharmacology / Q000502:physiology; D015398:Signal Transduction / Q000187:drug effects; D054462:p21-Activated Kinases; D020744:rac GTP-Binding Proteins / Q000502:physiology; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*
D001393:Azoles; D054833:Isoindoles; D016566:Organoselenium Compounds; D010982:Platelet-Derived Growth Factor; D020574:Proto-Oncogene Proteins c-sis; D017382:Reactive Oxygen Species; D011993:Recombinant Fusion Proteins; D000077214:Becaplermin; C042986:ebselen; D017479:Receptors, Platelet-Derived Growth Factor; D019061:src-Family Kinases; D064413:3-Phosphoinositide-Dependent Protein Kinases; C515903:PAK1 protein, human; C578562:PDPK1 protein, human; C515906:Pak1 protein, rat; C578579:Pdpk1 protein, rat; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D020744:rac GTP-Binding Proteins; D000111:Acetylcysteine