Lipid raft clustering and redox signaling platform formation in coronary arterial endothelial cells.
Recent studies have indicated that lipid rafts (LRs) in the cell membrane are clustered in response to different stimuli to form signaling platforms for transmembrane transduction. It remains unknown whether this LR clustering participates in redox signaling in endothelial cells. The present study tested a hypothesis that clustering of LRs on the membrane of coronary endothelial cells produces aggregation and activation of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase, thereby forming a redox signaling platform. By confocal microscopic analysis of agonist-stimulated rafts patch formation, we found that several death receptor ligands or apoptotic factors, including tumor necrosis factor alpha, Fas ligand, or endostatin, stimulated the clustering and trafficking of individual LRs on the plasma membrane of coronary endothelial cells. Interestingly, double labeling of a membrane-bound NADPH oxidase subunit, gp91phox, and LRs showed that gp91phox colocalized within the LR patches when endothelial cells were stimulated by Fas ligand. In isolated LR fractions from Fas-stimulated endothelial cells, gp91phox, p47phox (a crucial cytosolic regulatory subunit of NADPH oxidase), and Rac GTPase were markedly increased and blocked by nystatin, a compound that disrupts LRs. These clustered LRs contained high NADPH oxidase activity, which increased in response to Fas stimulation. Functionally, Fas ligand-induced inhibition of endothelium-dependent vasorelaxation was reduced if LRs were disrupted or NADPH oxidase was inhibited. These results suggest that LR clustering occurs in coronary endothelial cells. The formation of redox signaling platforms on the cell membrane mediates transmembrane signaling of death receptors, resulting in endothelial dysfunction.
Zhang
Andrew Y
AY
Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, USA.
Yi
Fan
F
Zhang
Guo
G
Gulbins
Erich
E
Li
Pin-Lan
PL
D000818:Animals; D001920:Bradykinin / Q000494:pharmacology; D002417:Cattle; D002478:Cells, Cultured; D003331:Coronary Vessels / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D042783:Endothelial Cells / Q000502:physiology*; D008024:Ligands; D008562:Membrane Glycoproteins / Q000378:metabolism; D021962:Membrane Microdomains / Q000235:genetics / Q000502:physiology*; D009097:Multienzyme Complexes / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D019255:NADPH Oxidases / Q000378:metabolism; D010084:Oxidation-Reduction; D010750:Phosphoproteins / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000502:physiology; D018124:Receptors, Tumor Necrosis Factor / Q000819:agonists / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D014018:Tissue Distribution; D014665:Vasodilator Agents / Q000494:pharmacology; D020744:rac GTP-Binding Proteins / Q000378:metabolism
D008024:Ligands; D008562:Membrane Glycoproteins; D009097:Multienzyme Complexes; D010750:Phosphoproteins; D018124:Receptors, Tumor Necrosis Factor; D014665:Vasodilator Agents; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; D020744:rac GTP-Binding Proteins; D001920:Bradykinin