Copyright © 2006, European Society of Cardiology
Critical role for classical PKC in activating Akt by phospholipase A2-modified LDL in monocytic cells
Faculty of Medicine, Institute of Biochemistry I, Johann Wolfgang Goethe-University, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany
* Corresponding author. Tel.: +49 69 6301 7424; fax: +49 69 6301 4203. Email address: bruene{at}zbc.kgu.de
Objective: Modification of low density lipoprotein (LDL) by phospholipases confers pro-atherogenic properties, although signalling pathways of phospholipase-modified LDL (PLA-LDL) remain obscure. We questioned whether members of the protein kinase C (PKC) family are involved in PLA-LDL-induced Akt phosphorylation and survival of THP-1 monocytic cells.
Methods: Akt phosphorylation in THP-1 cells was monitored by Western analysis. To modulate PKC expression cells were transfected with dominant-negative enhanced green fluorescent protein linked PKC
(PKC-EGFP K368R) and PKCβ (PKCβ-EGFP K371M) constructs or with siRNA specific for PKC/PKCβ using nucleofection technology. Cell survival was assessed by Annexin V/propidium iodide staining or mitochondrial membrane potential measurement with 3,3'-dihexyloxacarbocyanine iodide (DiOC6) using flow cytometry.
Results: Inhibitors of phospholipase C (PLC) or classical PKCs as well as PKC depletion following phorbol ester treatments, blocked Akt phosphorylation in response to PLA-LDL. In contrast, phosphatidylinositol 3-kinase (PI3K) activation by PLA-LDL was insensitive to PKC inhibition. Using RNA interference to knockdown PKC and overexpression of dominant-negative PKC as well as PKCβ drastically lowered Akt phosphorylation after PLA-LDL. Moreover, inhibition of PKC attenuated a PLA-LDL-induced survival response towards oxidative stress in THP-1 cells.
Conclusion: We show that PKC and PKCβ are critical for PLA-LDL-induced Akt phosphorylation and survival in THP-1 monocytic cells.
KEYWORDS Atherosclerosis; Lipoprotein; Phospholipases; Protein kinase C
Abbreviations: DAG, diacylglycerol • DiOC6, 3,3'-dihexyloxacarbocyanine iodide • EGFP, enhanced green fluorescent protein • LDL, low density lipoprotein • PI3K, phosphatidylinositol 3-kinase • PKC, protein kinase C • PLA2, phospholipase A2 • PLA-LDL, phospholipase A2-modified low density lipoprotein • PLC, phospholipase C • siRNA, small interfering RNA • TPA, phorbol-12-tetradecanoate 13-acetate
Time for primary review 28 days
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