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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 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.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 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.CONCLUSIONS
We show that PKCalpha and PKCbeta are critical for PLA-LDL-induced Akt phosphorylation and survival in THP-1 monocytic cells.
Akt ⊣ PLA-LDL: " 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 "
Akt → phospholipase C (PLC): " 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 "
phosphatidylinositol 3-kinase (PI3K) → PLA-LDL: " In contrast, phosphatidylinositol 3-kinase (PI3K) activation by PLA-LDL was insensitive to PKC inhibition "
Akt → PLA-LDL: " We show that PKCalpha and PKCbeta are critical for PLA-LDL induced Akt phosphorylation and survival in THP-1 monocytic cells "
Akt → PKCalpha: " We show that PKCalpha and PKCbeta are critical for PLA-LDL induced Akt phosphorylation and survival in THP-1 monocytic cells "
Akt → PKCbeta: " We show that PKCalpha and PKCbeta are critical for PLA-LDL induced Akt phosphorylation and survival in THP-1 monocytic cells "