Thrombin stimulation of vascular adhesion molecule-1 in endothelial cells is mediated by protein kinase C (PKC)-delta-NF-kappa B and PKC-zeta-GATA signaling pathways.
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.
Minami
Takashi
T
Department of Molecular Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA.
Abid
Md Ruhul
MR
Zhang
Jie
J
King
George
G
Kodama
Tatsuhiko
T
Aird
William C
WC
D000098:Acetophenones / Q000494:pharmacology; D020816:Amino Acid Motifs; D001578:Benzopyrans / Q000494:pharmacology; D015152:Blotting, Northern; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000378:metabolism; D019281:Dimerization; D004730:Endothelium, Vascular / Q000166:cytology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D050989:GATA2 Transcription Factor; D005799:Genes, Dominant; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008954:Models, Biological; D009025:Morpholines / Q000494:pharmacology; D009130:Muscle, Smooth / Q000166:cytology; D016328:NF-kappa B / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism*; D051745:Protein Kinase C-delta; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism; D015398:Signal Transduction; D013917:Thrombin / Q000378:metabolism / Q000494:pharmacology*; D013997:Time Factors; D051996:Transcription Factor RelA; D014157:Transcription Factors / Q000378:metabolism; D015533:Transcriptional Activation; D014162:Transfection; D019010:Vascular Cell Adhesion Molecule-1 / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases; D000099221:Protein Kinase C zeta
D000098:Acetophenones; D001578:Benzopyrans; D002867:Chromones; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D050989:GATA2 Transcription Factor; D008156:Luciferases; D020928:Mitogen-Activated Protein Kinases; D009025:Morpholines; D016328:NF-kappa B; D010455:Peptides; D019869:Phosphatidylinositol 3-Kinases; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; D012313:RNA; D012333:RNA, Messenger; D012260:Ribonucleases; D013917:Thrombin; D051996:Transcription Factor RelA; D014157:Transcription Factors; D019010:Vascular Cell Adhesion Molecule-1; D048051:p38 Mitogen-Activated Protein Kinases; D000099221:Protein Kinase C zeta; C494711:GATA2 protein, human; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C085746:rottlerin; C497258:PRKCD protein, human