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

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.

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 PKCalpha (PKCalpha-EGFP K368R) and PKCbeta (PKCbeta-EGFP K371M) constructs or with siRNA specific for PKCalpha/PKCbeta 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.

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 PKCalpha and overexpression of dominant-negative PKCalpha as well as PKCbeta 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.

We show that PKCalpha and PKCbeta are critical for PLA-LDL-induced Akt phosphorylation and survival in THP-1 monocytic cells.