Endothelin-mediated vascular growth requires p42/p44 mitogen-activated protein kinase and p70 S6 kinase cascades via transactivation of epidermal growth factor receptor.
Endothelin-1 (ET-1), a potent endothelium-derived vasoconstrictor peptide, exerts a growth-promoting effect on vascular smooth muscle cells, implicating its pathogenic role in vascular remodeling. To gain insight into the cellular and molecular mechanism whereby ET-1 induces vascular growth, we studied whether transactivation of receptor tyrosine kinases, such as epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor, are required for activation of p42/p44 mitogen-activated protein (MAP) kinase and p70 S6 kinase (p70S6K), and subsequent growth-promotion by ET-1 in cultured rat vascular smooth muscle cells. Immunoblotting with antiphosphotyrosine antibody revealed that ET-1 rapidly (within 2 min) and transiently induced tyrosine phosphorylation of several proteins, among which 180-kDa protein was shown to be EGFR. ET-1 rapidly increased association of EGFR and Shc with glutathione-S-transferase-Grb2 fusion protein. The ET-1-induced activation of MAP kinase was reduced by an EGFR kinase inhibitor (AG1478) but not by a platelet-derived growth factor receptor kinase inhibitor (AG1296). AG1478 dose-dependently decreased ET-1-stimulated MAP kinase activity as well as [3H]leucine and [3H]thymidine uptake. The ET-1-induced tyrosine phosphorylation of EGFR, as well as MAP kinase activation, was inhibited by an ETA receptor antagonist and intracellular Ca2+ antagonists but not by an ETB receptor antagonist, pertussis toxin, or protein kinase C inhibitors. In addition, dominant negative mutant of H-Ras and a MAP kinase kinase (MEK-1) inhibitor (PD98059) completely blocked ET-1-induced MAP kinase activation as well as [3H]leucine and [3H]thymidine uptake. Both AG1478 and PD98059 inhibited ET-1-induced phosphorylation and activation of p70S6K. Furthermore, rapamycin, a selective inhibitor of mammalian target of rapamycin, completely blocked ET-1-stimulated [3H]leucine and [3H]thymidine uptake. These results suggest that ETA receptor-mediated vascular growth by ET-1 requires both MAP kinase and p70S6K cascades mediated partly via Ca2+-dependent EGFR transactivation.
Iwasaki
H
H
Second Department of Internal Medicine, Tokyo Medical and Dental University, Japan.
Eguchi
S
S
Ueno
H
H
Marumo
F
F
Hirata
Y
Y
D000818:Animals; D002118:Calcium / Q000502:physiology; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D019332:Endothelin-1 / Q000494:pharmacology; D016232:Endothelins / Q000502:physiology*; D004789:Enzyme Activation / Q000502:physiology; D066246:ErbB Receptors / Q000235:genetics*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D024510:Muscle Development; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000201:enzymology / Q000254:growth & development*; D015689:Oncogene Protein p21(ras) / Q000378:metabolism; D010766:Phosphorylation; D051381:Rats; D044022:Receptor, Endothelin A; D017466:Receptors, Endothelin / Q000502:physiology; D019893:Ribosomal Protein S6 Kinases / Q000378:metabolism* / Q000502:physiology; D015533:Transcriptional Activation / Q000235:genetics*; D014443:Tyrosine / Q000378:metabolism
D019332:Endothelin-1; D016232:Endothelins; D044022:Receptor, Endothelin A; D017466:Receptors, Endothelin; D014443:Tyrosine; D066246:ErbB Receptors; D019893:Ribosomal Protein S6 Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D015689:Oncogene Protein p21(ras); D002118:Calcium