Thrombin induces the preproendothelin-1 gene in endothelial cells by a protein tyrosine kinase-linked mechanism.
Thrombin stimulates synthesis and secretion of endothelin-1 (ET-1), a vasoactive peptide that triggers responses in the vascular endothelium and smooth muscle. We investigated the signal transduction pathways by which thrombin stimulates preproET-1 gene expression and ET-1 peptide secretion in macrovascular cells (human umbilical vein endothelial cells [HUVECs] and bovine pulmonary artery endothelial cells [BPAECs]) and microvascular cells (human microvascular endothelial cell line [HMEC-1]). Thrombin (4 U/mL) stimulated maximal induction of ET-1 peptide secretion and preproET-1 mRNA after 2 hours in HUVECs and BPAECs and after 1 hour in HMEC-1. A synthetic thrombin receptor activator peptide confirmed ligand-specific receptor actions to induce preproET-1 mRNA. Protein kinase C (PKC) activation by phorbol ester transiently induced preproET-1 mRNA but had no effect on ET-1 peptide synthesis. PKC inhibitors sangivamycin and calphostin C and PKC depletion failed to suppress thrombin-stimulated preproET-1 mRNA. Adenylate cyclase and cAMP-dependent protein kinase did not participate in thrombin-induced preproET-1 gene activation. Thrombin stimulated a rapid increase in phosphotyrosine-containing proteins, suggesting a role for tyrosine phosphorylation in thrombin signaling. These data demonstrate that thrombin induces the preproET-1 gene and ET-1 peptide synthesis by a PKC-independent PTK-dependent pathway in macrovascular and microvascular endothelial cells. Protein tyrosine kinase inhibitors herbimycin A and genistein blocked thrombin-stimulated preproET-1 mRNA and peptide secretion, whereas daidzein, which lacks inhibitory activity, did not suppress thrombin-induced ET-1.
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
D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D019332:Endothelin-1; D016232:Endothelins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology* / Q000378:metabolism; D004789:Enzyme Activation; D005786:Gene Expression Regulation; D006801:Humans; D015151:Immunoblotting; D011493:Protein Kinase C / Q000378:metabolism; D011498:Protein Precursors / Q000235:genetics*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011651:Pulmonary Artery; D012333:RNA, Messenger / Q000235:genetics; D011863:Radioimmunoassay; D018179:Receptors, Thrombin / Q000502:physiology; D015398:Signal Transduction; D013917:Thrombin / Q000502:physiology*; D015533:Transcriptional Activation; D014471:Umbilical Veins
D019332:Endothelin-1; D016232:Endothelins; D011498:Protein Precursors; D012333:RNA, Messenger; D018179:Receptors, Thrombin; D011505:Protein-Tyrosine Kinases; D011493:Protein Kinase C; D013917:Thrombin