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Role of protein kinase C δ in apoptotic signaling of oxidized phospholipids in RAW 264.7 macrophages
F. Vogl, J. Humpolícková, M. Amaro, D. Koller, H. Köfeler, E. Zenzmaier, M. Hof, A. Hermetter,
Language English Country Netherlands
Document type Journal Article, Research Support, Non-U.S. Gov't
- MeSH
- Enzyme Activation MeSH
- Apoptosis drug effects MeSH
- Time Factors MeSH
- Phospholipid Ethers toxicity MeSH
- Phosphorylation MeSH
- Caspase 3 metabolism MeSH
- Caspase 7 metabolism MeSH
- Macrophages drug effects enzymology pathology MeSH
- Mice MeSH
- Oxidation-Reduction MeSH
- Protein Kinase C-delta genetics metabolism MeSH
- RAW 264.7 Cells MeSH
- Gene Expression Regulation, Enzymologic MeSH
- Genes, Reporter MeSH
- RNA Interference MeSH
- Signal Transduction drug effects MeSH
- Transfection MeSH
- Dose-Response Relationship, Drug MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
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.
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