Thrombin activates nuclear factor-kappaB and potentiates endothelial cell activation by TNF.
Thrombin is the central bioregulatory enzyme in hemostasis and is generated in vascular beds in which inflammatory responses are ongoing. In this study, we examined the effect of thrombin, both alone and in combination with TNF, on gene expression in porcine aortic endothelial cells (EC). Thrombin (1-10 U/ml) induced increased mRNA levels of E-selectin, monocyte chemoattractant protein-1, IL-8, plasminogen activator inhibitor-1, and IkappaB-alpha. These effects were mimicked by a thrombin receptor-activating peptide; preincubation of thrombin with hirudin blocked the induction of mRNA, suggesting that the increased gene expression was due to thrombin-specific activity. Because these genes are known to contain nuclear-factor-kappaB (NF-kappaB)-binding elements in their promoter region, we next examined the ability of thrombin to activate this transcription factor. As detected by electrophoretic mobility shift assay, thrombin (10 U/ml) or thrombin receptor-activating peptide (100 microM) stimulated increased NF-kappaB-binding activity. Supershift analysis revealed that these complexes were comprised principally of the RelA (p65) and NF-kappaB1 (p50) Rel family members. Thrombin alone did not substantively increase protein levels of E-selectin despite the increase in E-selectin mRNA levels. However, thrombin (3-10 U/ml) stimulated a 10-fold enhancement in the ability of TNF (0.3-1.0 ng/ml) to induce E-selectin surface expression. Similar potentiation of TNF-induced NF-kappaB activity and E-selectin transcription by thrombin was observed in experiments utilizing luciferase reporter constructs expressed in bovine aortic EC. The ability of thrombin to potentiate TNF-induced EC activation thus provides an important mechanism by which products of the coagulation cascade may enhance cytokine-mediated inflammatory responses.
Anrather
D
D
Sandoz Center for Immunobiology, Beth Israel Deaconess Medical Center, Boston, MA 02115, USA.
Millan
M T
MT
Palmetshofer
A
A
Robson
S C
SC
Geczy
C
C
Ritchie
A J
AJ
Bach
F H
FH
Ewenstein
B M
BM
D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D004357:Drug Synergism; D019040:E-Selectin / Q000378:metabolism; D004730:Endothelium, Vascular; D006801:Humans; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D018179:Receptors, Thrombin / Q000378:metabolism; D013552:Swine; D013917:Thrombin / Q000494:pharmacology*; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D015854:Up-Regulation
D019040:E-Selectin; D016328:NF-kappa B; D018179:Receptors, Thrombin; D014409:Tumor Necrosis Factor-alpha; D013917:Thrombin