Critical role for classical PKC in activating Akt by phospholipase A2-modified LDL in monocytic cells

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 alpha (PKC alpha-EGFP K368R) and PK beta (PKC beta-EGFP K371M) constructs or with siRNA specific for PKC alpha/PK beta using nucleofection technology. Cell survival was assessed by Annexin V/propidium iodide staining or mitochondrial membrane potential measurement with 3,3'-dihexyloxacarbocyanine iodide (DiOC(6)) 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 PKCa and overexpression of dominant-negative PKC alpha as well as PKC 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 alpha and PKC beta are critical for PLA-LDL-induced Akt phosphorylation and survival in THP-1 monocytic cells. (c) 2006 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.