Palmitate induces RIP1-dependent necrosis in RAW 264.7 cells.
OBJECTIVE
The kinase receptor-interacting protein (RIP) 1, a serine/threonine protein kinase, is a key signaling molecule for necrosis. The possible involvement of RIP1 in palmitate-induced macrophage death and its underlying molecular mechanism was investigated in this study.


METHODS
Cell viability was measured by an MTT reduction assay. The type of cell death was determined by staining with annexin V, propidium iodide (PI) and the APOPercentage dye, and by examining cell morphology using transmission electron microscopy. The down-regulation of RIP1 was performed by siRNA transfection. Intracellular reactive oxygen species (ROS) were measured by staining with H(2)DCF-DA.


RESULTS
Palmitate largely induced necrosis in RAW 264.7 cells, whereas C2-ceramide induced apoptosis. Palmitate-induced necrosis was inhibited by Necrostatin-1, an inhibitor of RIP1, and by RIP1 siRNA transfection, whereas ordinary cell death was not inhibited by z-VAD-fmk. In addition, the presence of palmitate caused a significant increase in intracellular ROS levels compared to control cells. Pre-treatment with Tempol, a cell permeable ROS scavenger, and MnTBAP, an inhibitor of mitochondrial oxidative stress, protected cells from palmitate-induced cell death. Furthermore, the down-regulation of RIP1 by siRNA transfection significantly decreased palmitate-induced ROS generation compared to control cells.


CONCLUSIONS
The findings reported herein indicate that palmitate induces necrotic cell death via RIP1-dependent ROS generation in RAW 264.7 cells. These findings may provide a new mechanism that explains the link between elevated levels of free fatty acids (FFAs) and macrophage death.
Institute of Hansen's Disease, South Korea.
Seong Keun
SK
Kim
Gimoon
G
Seo
Eunhye
E
Oh
Song-Hyo
SH
Jin
Gue Tae
GT
Chae
Seong-Beom
SB
Lee
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D002460:Cell Line; D048430:Cell Shape / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D003497:Cyclic N-Oxides / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D020690:GTPase-Activating Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D007093:Imidazoles / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects* / Q000201:enzymology / Q000648:ultrastructure; D008665:Metalloporphyrins / Q000494:pharmacology; D051379:Mice; D046529:Microscopy, Electron, Transmission; D009336:Necrosis; D018384:Oxidative Stress / Q000187:drug effects; D019308:Palmitic Acid / Q000633:toxicity*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D034622:RNA Interference; D017382:Reactive Oxygen Species / Q000378:metabolism; D013110:Sphingosine / Q000031:analogs & derivatives / Q000633:toxicity; D013113:Spin Labels; D014162:Transfection
D000975:Antioxidants; D003497:Cyclic N-Oxides; D020690:GTPase-Activating Proteins; D007093:Imidazoles; D007211:Indoles; D008665:Metalloporphyrins; C064769:N-acetylsphingosine; D047428:Protein Kinase Inhibitors; C490620:Ralbp1 protein, mouse; D017382:Reactive Oxygen Species; D013113:Spin Labels; C097284:manganese(III)-tetrakis(4-benzoic acid)porphyrin; C507699:necrostatin-1; D019308:Palmitic Acid; D013110:Sphingosine; C001803:tempol