Pertussis toxin-sensitive G proteins as mediators of the signal transduction pathways activated by cytomegalovirus infection of smooth muscle cells.
We demonstrated recently that the arachidonic acid (AA) cascade is involved in cytomegalovirus (CMV)-induced generation of reactive oxygen species (ROS) and the activation of nuclear factor (NF)-kappaB in human smooth muscle cells (SMCs). Since AA release from neutrophils is mediated by pertussis toxin (PTx)-sensitive guanine nucleotide-binding (G) proteins, we hypothesized by analogy that CMV stimulates ROS generation in SMCs and ultimately activates NF-kappaB via a PTx-sensitive G protein-coupled pathway. Our first test of this hypothesis demonstrated that PTx blocked AA release induced by CMV infection of SMCs, as well as blocked the terminal products of this reaction, ROS generation and NF-kappaB activation. More proximal components of the pathway were then examined. CMV infection increased phosphorylation and activity of cytosolic phospholipase A2 (cPLA2), an enzyme causing AA release; these effects were inhibited by PTx. CMV infection activated mitogen-activated protein (MAP) kinase, a key enzyme for cPLA2 phosphorylation, an effect also inhibited by PTx. Finally, inhibition of MAP kinase kinase (MAPKK), which phosphorylates and thereby activates MAP kinase, inhibited CMV-induced ROS generation. These data demonstrate that a PTx-sensitive G protein-dependent signaling pathway mediates cellular effects of CMV infection of SMCs. The downstream events include phosphorylation and activation of MAP kinase by MAPKK and subsequent phosphorylation and activation of cPLA2 (with its translocation to cell membranes), followed by stimulation of the AA cascade, which generates intracellular ROS and thereby activates NF-kappaB.
Shibutani
T
T
Cardiology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892-1650, USA.
Johnson
T M
TM
Yu
Z X
ZX
Ferrans
V J
VJ
Moss
J
J
Epstein
S E
SE
D001011:Aorta / Q000166:cytology; D016718:Arachidonic Acid / Q000378:metabolism; D001692:Biological Transport; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002451:Cell Compartmentation; D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000254:growth & development*; D003593:Cytoplasm / Q000201:enzymology; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D020929:Mitogen-Activated Protein Kinase Kinases; D009131:Muscle, Smooth, Vascular / Q000821:virology*; D016328:NF-kappa B / Q000378:metabolism; D037342:Pertussis Toxin; D010741:Phospholipases A / Q000378:metabolism; D054467:Phospholipases A2; D011494:Protein Kinases / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D016328:NF-kappa B; D017382:Reactive Oxygen Species; D010566:Virulence Factors, Bordetella; D016718:Arachidonic Acid; D037342:Pertussis Toxin; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D010741:Phospholipases A; D054467:Phospholipases A2; D019204:GTP-Binding Proteins