Thrombin-mediated ET-1 gene regulation involves CaM kinases and calcineurin in human endothelial cells.
The preproendothelin-1 (preproET-1) gene is induced by thrombin after phosphorylation of nonreceptor protein tyrosine kinase pathways. This study investigated the contribution of Ca2+/calmodulin-dependent intracellular signaling cascades to this pathway and measured ET-1 mRNA levels by Northern blot analysis in human endothelial cells. Increased intracellular Ca2+ levels in response to Ca2+ ionophore or Ca2+ ATPase inhibitors tert-butylhydroquinone and thapsigargin mimicked thrombin actions on ET-1 mRNA induction. Thrombin-mediated activation of ET-1 mRNA was reduced by specific calmodulin antagonists W7 or calmidazolium and after inhibition of CaM kinase II by KN-62. Inhibition of calcium/calmodulin-dependent phosphatase calcineurin by cyclosporin A, however, stimulated ET-1 mRNA in human endothelial cells. Phosphotyrosine immunoblot assays show that calcium/calmodulin-dependent signaling pathways precede thrombin-induced tyrosine phosphorylation, and that the calcium/calmodulin-dependent phosphatase calcineurin also exerts its effects via activation of protein tyrosine kinases. These observations demonstrate that thrombin stimulates the preproET-1 gene in human endothelial cells through calcium-dependent activation of CaM kinase and protein tyrosine kinases, and that calcineurin may also participate in regulation of the prepro ET-1 gene.
Marsen
T A
TA
Department of Medicine, School of Medicine, Case Western Reserve University, Cleveland, Ohio, USA.
Simonson
M S
MS
Dunn
M J
MJ
D019703:Calcineurin; D002118:Calcium / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000502:physiology*; D002148:Calmodulin-Binding Proteins / Q000502:physiology*; D002478:Cells, Cultured; D016232:Endothelins / Q000235:genetics*; D004730:Endothelium, Vascular / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D010749:Phosphoprotein Phosphatases / Q000502:physiology*; D013917:Thrombin / Q000494:pharmacology*
D002148:Calmodulin-Binding Proteins; D016232:Endothelins; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D013917:Thrombin; D002118:Calcium