title: Palmitate induces RIP1/RIP3-dependent necrosis via MLKL-mediated pore formation in the plasma membrane of RAW 264.7 cells.
abstract: We previously reported that palmitate induces receptor-interacting protein (RIP)1-dependent necrosis in RAW 264.7 macrophage cells. In response to death receptor stimuli, RIP1 is reported to activate RIP3, which causes the phosphorylation and translocation of mixed-lineage kinase domain-like (MLKL) protein to the plasma membrane, subsequent pore formation in the plasma membrane, and necrotic cell death. In the current study, we investigated the role of MLKL in palmitate-induced, RIP1/RIP3-dependent necrotic cell death in RAW 264.7 cells. The down-regulation of RIP1 or RIP3 by siRNA transfection protected the cells from palmitate-induced cell death. In addition, MLKL was phosphorylated at the serine residue and translocated to the plasma membrane in palmitate-treated cells. In these cells, MLKL was observed as aggregate dots on the plasma membrane. The findings also show that palmitate induced the formation of pores with varied shapes and sizes, and an increase in propidium iodide (PI) uptake and lactate dehydrogenase (LDH) release. Furthermore, the down-regulation of MLKL by siRNA transfection significantly decreased palmitate-induced PI uptake and LDH release, resulting in protection against palmitate-induced necrotic cell death. The findings reported here indicate that palmitate induces RIP1/RIP3-dependent necrosis via MLKL-mediated pore formation of RAW 264.7 cells in the plasma membrane, which could provide a new mechanism to explain the link between elevated levels of free fatty acids (FFAs), palmitate in particular, and macrophage death.
authors.affiliation: Institute of Hansen's Disease, Department of Pathology, College of Medicine, The Catholic University of Korea, Seoul 06591, South Korea.
authors.forename: Seong Keun
authors.initials: SK
authors.lastname: Kim
authors.affiliation: Institute of Hansen's Disease, Department of Pathology, College of Medicine, The Catholic University of Korea, Seoul 06591, South Korea.
authors.forename: Mihee
authors.initials: M
authors.lastname: Yun
authors.affiliation: Institute of Hansen's Disease, Department of Pathology, College of Medicine, The Catholic University of Korea, Seoul 06591, South Korea.
authors.forename: Gimoon
authors.initials: G
authors.lastname: Seo
authors.affiliation: Institute of Hansen's Disease, Department of Pathology, College of Medicine, The Catholic University of Korea, Seoul 06591, South Korea.
authors.forename: Ji-Young
authors.initials: JY
authors.lastname: Lee
authors.affiliation: Institute of Hansen's Disease, Department of Pathology, College of Medicine, The Catholic University of Korea, Seoul 06591, South Korea. Electronic address: sblee@catholic.ac.kr.
authors.forename: Seong-Beom
authors.initials: SB
authors.lastname: Lee
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000378:metabolism* / Q000473:pathology; D002463:Cell Membrane Permeability; D020690:GTPase-Activating Proteins / Q000378:metabolism*; D051379:Mice; D009336:Necrosis / Q000139:chemically induced / Q000378:metabolism / Q000473:pathology; D019308:Palmitic Acid; D011494:Protein Kinases / Q000378:metabolism*; D000067996:RAW 264.7 Cells; D053422:Receptor-Interacting Protein Serine-Threonine Kinases / Q000378:metabolism*
chemical_list: D020690:GTPase-Activating Proteins; C490620:Ralbp1 protein, mouse; D019308:Palmitic Acid; C570694:MLKL protein, mouse; D011494:Protein Kinases; D053422:Receptor-Interacting Protein Serine-Threonine Kinases; C485615:Ripk3 protein, mouse
keywords: MLKL; Necrosis; Palmitate; RAW 264.7 cells; RIP1; RIP3