title: Role of protein kinase C δ in apoptotic signaling of oxidized phospholipids in RAW 264.7 macrophages.
abstract: The oxidized phospholipids (oxPl) 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) and 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC) are cytotoxic components of oxidized LDL (oxLDL). Sustained exposure to oxLDL or isolated oxPl induces apoptotic signaling in vascular cells, which is a hallmark of the late phase of atherosclerosis. Activation of sphingomyelinase, the coordinate formation of ceramide and activation of caspase 3/7 as well as the activation of stress-associated kinases are causally involved in this process. Here, we provide evidence for a role of PKCδ in oxPl cytotoxicity. Silencing of the enzyme by siRNA significantly reduced caspase 3/7 activation in RAW 264.7 macrophages under the influence of oxPl. Concomitantly, PKCδ was phosphorylated as a consequence of cell exposure to PGPC or POVPC. Single molecule fluorescence microscopy provided direct evidence for oxPl-protein interaction. Both oxPl recruited an RFP-tagged PKCδ to the plasma membrane in a concentration-dependent manner. In addition, two color cross-correlation number and brightness (ccN&B) analysis of the molecular motions revealed that fluorescently labeled PGPC or POVPC analogs co-diffuse and are associated with the fluorescent protein kinase in live cells. The underlying lipid-protein interactions may be due to chemical bonding (imine formation between the phospholipid aldehyde POVPC with protein amino groups) and physical association (with POVPC or PGPC). In summary, our data supports the assumption that PKCδ acts as a proapototic kinase in oxPl-included apoptosis of RAW 264.7 macrophages. The direct association of the bioactive lipids with this enzyme seems to be an important step in the early phase of apoptotic signaling.
authors.affiliation: Institute of Biochemistry, Graz University of Technology, Graz, Austria.
authors.forename: F
authors.initials: F
authors.lastname: Vogl
authors.affiliation: J. Heyrovský Institute of Physical Chemistry, Academy of Sciences of the Czech Republic, v.v.i., Prague, Czech Republic.
authors.forename: J
authors.initials: J
authors.lastname: Humpolícková
authors.affiliation: J. Heyrovský Institute of Physical Chemistry, Academy of Sciences of the Czech Republic, v.v.i., Prague, Czech Republic.
authors.forename: M
authors.initials: M
authors.lastname: Amaro
authors.affiliation: Institute of Biochemistry, Graz University of Technology, Graz, Austria.
authors.forename: D
authors.initials: D
authors.lastname: Koller
authors.affiliation: Center for Medical Research, Core Facility for Mass Spectrometry, Medical University of Graz, Graz, Austria.
authors.forename: H
authors.initials: H
authors.lastname: Köfeler
authors.affiliation: Institute of Biochemistry, Graz University of Technology, Graz, Austria.
authors.forename: E
authors.initials: E
authors.lastname: Zenzmaier
authors.affiliation: J. Heyrovský Institute of Physical Chemistry, Academy of Sciences of the Czech Republic, v.v.i., Prague, Czech Republic.
authors.forename: M
authors.initials: M
authors.lastname: Hof
authors.affiliation: Institute of Biochemistry, Graz University of Technology, Graz, Austria. Electronic address: albin.hermetter@tugraz.at.
authors.forename: A
authors.initials: A
authors.lastname: Hermetter
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3 / Q000378:metabolism; D053179:Caspase 7 / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D015971:Gene Expression Regulation, Enzymologic; D017930:Genes, Reporter; D008264:Macrophages / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D051379:Mice; D010084:Oxidation-Reduction; D010742:Phospholipid Ethers / Q000633:toxicity*; D010766:Phosphorylation; D051745:Protein Kinase C-delta / Q000235:genetics / Q000378:metabolism*; D000067996:RAW 264.7 Cells; D034622:RNA Interference; D015398:Signal Transduction / Q000187:drug effects*; D013997:Time Factors; D014162:Transfection
chemical_list: C400184:1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphorylcholine; C400185:1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine; D010742:Phospholipid Ethers; C497259:Prkcd protein, mouse; D051745:Protein Kinase C-delta; C505409:Casp3 protein, mouse; C505524:Casp7 protein, mouse; D053148:Caspase 3; D053179:Caspase 7
keywords: Acid sphingomyelinase; Atherosclerosis; Ceramide; Fluorescent phospholipids; Lipid-protein-interactions; Oxidized LDL