YOKODE Masayuki Kyoto University, Faculty of Medicine, Lecturer, 医学研究科, 講師 (20252447)
DOI Toshio Kyoto University, Faculty of Medicine, Lecturer, 医学研究科, 講師 (60183498)
MURAKAMI Motonobu Kyoto University, Faculty of Medicine, Associate Professor, 医学研究科, 助教授 (10157761)
ISHII Kenji Kyoto University, Faculty of Medicine, Lecturer, 医学研究科, 講師 (00212811)
WAKATUKI Yosio Kyoto University, Faculty of Medicine, Lecturer, 医学研究科, 講師 (40220826)
|Budget Amount *help
¥39,800,000 (Direct Cost: ¥39,800,000)
Fiscal Year 1998: ¥6,900,000 (Direct Cost: ¥6,900,000)
Fiscal Year 1997: ¥10,600,000 (Direct Cost: ¥10,600,000)
Fiscal Year 1996: ¥22,300,000 (Direct Cost: ¥22,300,000)
Endothelial dysfunction, or activation, elicited by oxidized low density lipoprotein (Ox-LDL) or its lipid constituent has been implicated in the pathogenesis of atherosclerosis. Lysophosphatidylcholine (lyso-PC), a phospholipid component of Ox-LDL, has been shown to induce expression of endothelial-leukocyte adhesion molecules, such as VCAM-1 and ICAM-1, and smooth muscle growth factors, including PDGF A and B chains and HB-EGF, which appear to play crucial roles in recruitment of circulating monocytes and T-lymphocytes into atherosclerotic lesions and migration and proliferation of medical smooth muscle cells into atherosclerotic intima.
One of the aims of our studies is to define transcriptional and signal transduction mechanisms involved in lyso-PC-induced gene expression. We have found that elevated levels of intracellular cyclic AMP inhibited lyso-PC-induced expression of PDGF and ICAM-1: Furthermore, lyso-PC-induced expression of PDGF was dependent upon protein tyrosine phosphory
lation. We, Therefore, explored the protein that can rapidly phosphorylated in the tyrosine residue. We have revealed that a protein with a molecular mass of 130kDa, which was designated p130, was rapidly and transiently tyrosine-phosphorylated by lyso-PC. By use of immunoblotting and immunoprecipitation, we have identified p130 as PECAM-1 which was expressed on the intercellular junction of cultured endothelial cells. Our study also have demonstrated that MAP kinases, such as ERK and JNK, were activated by lyso-PC, which appeared to depend upon protein tyrosine phosphorylation. Taken together, tyrosine phosphorylation of PECAM-1 may be the upstream of MAP kinase activation by lyso-PC.
On the other hand, vascular endothelial cells can take up Ox-LDL via receptor-dmediated endocytosis. Our recent studied have identified, for the first time, a receptor for Ox-LDL expressed in the cell-surface of vascular endothelial cells. By immunoblotting and Northern blotting, we have found that expression of LOX-1 can be upregulated by an inflammatory cytokine TNF-α and laminar flow fluid shear stress. Nuclear run-off assays have revealed that TNF-α and flow fluid shear stress can stimulate transcription of LOX-1 gene. Unregulated expression of LOX-1 by TNF-α was associated a cell line which stably expresses LOX-1 on the cell-surface. By use of this cell line, we have found that LOX-1 can bind, internalize, and degrade Ox-LDL but not significantly acetylated LDL. Binding and degradation of Ox-LDL by LOX-1 was suppressed by reagents, such as polyinosinic acid and carageenan. In contrast, fucoidin and maleylated serum albumin, which had been shown to inhibit Ox-LDL binding and degradation by class A macrophage scavenger receptors, did not significantly inhibit Ox-LDL binding or degradation by LOX-1. Delipidation of Ox-LDL did not affect the ability to bind LOX-1; therefore, protein portion of Ox-LDL particles appeared to be the ligand of LOX-1. These results, thus, indicated that ligand specificity of LOX-1 is different from that of class A or B scavenger receptors. Less