Abstract
We recently demonstrated that thrombin induces the expression of vascular adhesion molecule-1 (VCAM-1) in endothelial cells by an NF-kappaB- and GATA-dependent mechanism. In the present study, we describe the signaling pathways that mediate this response. Thrombin stimulation of the VCAM-1 gene and promoter in human umbilical vein endothelial cells was inhibited by preincubation with the phosphatidylinositol 3-kinase inhibitor, LY294002, the protein kinase C (PKC)-delta inhibitor, rottlerin, a PKC-zeta peptide inhibitor, or by overexpression of dominant negative (DN)-PKC-zeta. In electrophoretic mobility shift assays, thrombin-mediated induction of NF-kappaB p65 binding to two NF-kappaB motifs in the upstream promoter region of VCAM-1 was blocked by LY294002 and rottlerin, whereas the inducible binding of GATA-2 to a tandem GATA motif was inhibited by LY294002 and the PKC-zeta peptide inhibitor. In co-transfection assays, thrombin stimulation of a minimal promoter containing multimerized VCAM-1 NF-kappaB sites was inhibited by DN-PKC-delta but not DN-PKC-zeta. In contrast, thrombin-mediated transactivation of a minimal promoter containing tandem VCAM-1 GATA motifs was inhibited by DN-PKC-zeta but not DN-PKC-delta. Finally, thrombin failed to induce VCAM-1 expression in vascular smooth muscle cells. Taken together, these data suggest that the endothelial cell-specific effect of thrombin on VCAM-1 expression involves the coordinate activity of PKC-delta-NF-kappaB and PKC-zeta-GATA signaling pathways.
Publication types
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Acetophenones / pharmacology
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Amino Acid Motifs
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Benzopyrans / pharmacology
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Blotting, Northern
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Cell Nucleus / metabolism
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Cells, Cultured
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Chromones / pharmacology
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DNA-Binding Proteins / metabolism
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Dimerization
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Endothelium, Vascular / cytology
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Enzyme Inhibitors / pharmacology
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GATA2 Transcription Factor
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Genes, Dominant
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Humans
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Luciferases / metabolism
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Mitogen-Activated Protein Kinases / metabolism
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Models, Biological
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Morpholines / pharmacology
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Muscle, Smooth / cytology
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NF-kappa B / metabolism*
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Peptides / metabolism
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Phosphatidylinositol 3-Kinases / metabolism
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Promoter Regions, Genetic
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Protein Binding
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Protein Kinase C / metabolism*
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Protein Kinase C-delta
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RNA / metabolism
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RNA, Messenger / metabolism
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Ribonucleases / metabolism
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Signal Transduction
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Thrombin / metabolism
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Thrombin / pharmacology*
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Time Factors
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Transcription Factor RelA
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Transcription Factors / metabolism
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Transcriptional Activation
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Transfection
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Vascular Cell Adhesion Molecule-1 / metabolism*
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p38 Mitogen-Activated Protein Kinases
Substances
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Acetophenones
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Benzopyrans
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Chromones
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DNA-Binding Proteins
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Enzyme Inhibitors
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GATA2 Transcription Factor
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GATA2 protein, human
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Morpholines
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NF-kappa B
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Peptides
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RNA, Messenger
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Transcription Factor RelA
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Transcription Factors
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Vascular Cell Adhesion Molecule-1
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2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one
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RNA
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rottlerin
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Luciferases
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Phosphatidylinositol 3-Kinases
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protein kinase C zeta
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PRKCD protein, human
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Protein Kinase C
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Protein Kinase C-delta
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Mitogen-Activated Protein Kinases
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p38 Mitogen-Activated Protein Kinases
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Ribonucleases
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Thrombin