Dominant-negative lox-1 blocks homodimerization of wild-type lox-1-induced cell proliferation through extracellular signal regulated kinase 1/2 activation.
C-type lectin-like oxidized low-density lipoprotein (Ox-LDL) receptor-1 (Lox-1) belongs to the same family as natural killer cell receptors Ly49A and CD94 and functionally undergoes dimerization. Although Lys262 and Lys263 in the C terminus of bovine (b)Lox-1 play an important role in the uptake of Ox-LDL, mutation of these residues has not been suggested to be a potential source of the dominant-negative property. We hypothesize that dominant-negative human (h)Lox-1 forms a heterodimer with Lox-1-wild-type (WT) and blocks Lox-1-WT-induced cell signaling. Based on the use of molecular imaging techniques with laser scanning confocal microscopy and immunoprecipitation in an hLox-1-expressing Chinese hamster ovary cell system, homodimerization of hLox-1-WT was localized in the cell membrane, and Ox-LDL activated extracellular signal regulated kinase (ERK)1/2 without the translocation of hLox-1-WT. Lys266 and Lys267 of hLox-1, corresponding with Lys262 and Lys263 of bLox-1, were mutated (hLox1-K266A/K267A), and the mutant receptor inhibited hLox-1-WT-induced thymidine incorporation and ERK1/2 activation. Although Ox-LDL binds to the dominant-negative mutant receptor and is taken up by cytoplasm, ERK1/2 activation was blocked by heterodimerization with the mutant receptor and hLox-1-WT in the cell membrane. In addition, in human coronary artery smooth muscle cells, which express hLox-1-WT, we confirmed that the activation of ERK1/2 and [3H]-thymidine incorporation was caused by the addition of Ox-LDL, and these actions were blocked by hLox1-K266A/K267A. In conclusion, the present findings constitute the first evidence that strategies aimed at blocking cell-proliferative pathways at the receptor level could be useful for impairing Lox-1-induced cell proliferation.
Tanigawa
Hiroyuki
H
Department of Cardiology, Fukuoka University School of Medicine, 7-45-1 Nanakuma, Fukuoka, 814-0180, Japan.
Miura
Shin-Ichiro
S
Matsuo
Yoshino
Y
Fujino
Masahiro
M
Sawamura
Tatsuya
T
Saku
Keijiro
K
D000818:Animals; D001667:Binding, Competitive / Q000235:genetics; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D049109:Cell Proliferation; D002478:Cells, Cultured; D003331:Coronary Vessels / Q000166:cytology; D006224:Cricetinae; D019281:Dimerization; D004789:Enzyme Activation / Q000187:drug effects; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D005456:Fluorescent Dyes; D018014:Gene Transfer Techniques; D005799:Genes, Dominant; D006801:Humans; D008077:Lipoproteins, LDL / Q000378:metabolism / Q000494:pharmacology; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D051127:Scavenger Receptors, Class E / Q000235:genetics* / Q000378:metabolism*; D013936:Thymidine / Q000493:pharmacokinetics; D014316:Tritium
D005456:Fluorescent Dyes; D008077:Lipoproteins, LDL; C495137:OLR1 protein, human; D051127:Scavenger Receptors, Class E; C099252:oxidized low density lipoprotein; D014316:Tritium; D048049:Extracellular Signal-Regulated MAP Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D013936:Thymidine