Role of protein kinase C δ in apoptotic signaling of oxidized phospholipids in RAW 264.7 macrophages F. Vogl a , J. Humpolícková b , M. Amaro b , D. Koller a , H. Köfeler c , E. Zenzmaier a , M. Hof b , A. Hermetter a, ⁎ a Institute of Biochemistry, Graz University of Technology, Graz, Austria b J. Heyrovský Institute of Physical Chemistry, Academy of Sciences of the Czech Republic, v.v.i., Prague, Czech Republic c Center for Medical Research, Core Facility for Mass Spectrometry, Medical University of Graz, Graz, Austria abstract article info Article history: Received 1 September 2015 Received in revised form 26 November 2015 Accepted 17 December 2015 Available online 18 December 2015 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 cer- amide 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. Con- comitantly, PKCδ was phosphorylated as a consequence of cell exposure to PGPC or POVPC. Single molecule fluo- rescence 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 macro- phages. The direct association of the bioactive lipids with this enzyme seems to be an important step in the early phase of apoptotic signaling. © 2015 Elsevier B.V. All rights reserved. Keywords: Oxidized LDL Atherosclerosis Ceramide Acid sphingomyelinase Lipid-protein-interactions Fluorescent phospholipids 1. Introduction The oxidized phospholipids (oxPL) 1-palmitoyl-2-(5-oxovaleroyl)- sn-glycero-3-phosphocholine (POVPC) and 1-palmitoyl-2-glutaroyl- sn-glycero-3-phosphocholine (PGPC) are generated from 1-palmitoyl- 2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC) under conditions of oxidative stress. They are biologically active components of oxidized low-density lipoprotein (oxLDL) and contribute to the toxicity of this particle in vascular cells [1–4]. We have previously shown that PGPC and POVPC induce apoptotic cell death in cultured vascular smooth muscle cells [4,5], in RAW 264.7 macrophages and in murine bone marrow-derived macrophages [6]. In vascular smooth muscle cells, apoptotic signaling was associated with activation of acid sphingomyelinase (aSMase), phosphorylation of p38 MAPK and JNK and activation of caspase-3. In macrophages, activation of ceramide synthases (CerS), mainly CerS 2, catalyzing a specific subset of ceramides, was identified as a delayed response to the oxPL [7]. All these features of oxPL-induced cell death can also be observed when vascular cells are exposed to oxLDL [7,8] and therefore it was concluded that POVPC and PGPC are main toxic components of this particle in the vascular wall. Acid sphingomyelinase (aSMase) is a key upstream signaling com- ponent of oxPL and causally related to apoptotic cell death induced by PGPC or POVPC. Chemical inhibition of this enzyme abolishes activation of downstream signaling enzymes such as p38 MAPK, JNK and caspase- 3. As a consequence, apoptotic cell death is decreased [4,9]. It has been shown by Hannun et al. that PKCδ is involved in the activation of aSMase under the influence of external stimuli. If cancer cells were stimulated with phorbol ester [10] PKCδ was recruited to the cytoplasmic side of the plasma membrane followed by autophosphorylation and transfer of the kinase to the lysosomes where it phosphorylates aSMase. Phos- phorylated aSMase moves to the plasma membrane where it generates ceramide. Larroque-Cardoso et al. [11] have published information that supports the assumption of a key signaling role of PKCδ in vascular smooth muscle cells, too. They found that PKCδ was activated under the influence of oxLDL and triggers apoptosis in these cells, which is as- sociated to activation of stress-induced kinases and ER stress. In J774A.1 Biochimica et Biophysica Acta 1861 (2016) 320–330 ⁎ Corresponding author at: Institute of Biochemistry, Graz University of Technology, 8010 Graz, Austria. E-mail address: albin.hermetter@tugraz.at (A. Hermetter). http://dx.doi.org/10.1016/j.bbalip.2015.12.009 1388-1981/© 2015 Elsevier B.V. All rights reserved. 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