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 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.
Preiss
Stefan
S
Faculty of Medicine, Institute of Biochemistry I, Johann Wolfgang Goethe-University, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany.
Namgaladze
Dmitry
D
Brüne
Bernhard
B
D015153:Blotting, Western / Q000379:methods; D045744:Cell Line, Tumor; D049452:Green Fluorescent Proteins / Q000235:genetics; D054513:Group IV Phospholipases A2; D006801:Humans; D008077:Lipoproteins, LDL / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D009000:Monocytes / Q000378:metabolism*; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D011493:Protein Kinase C / Q000235:genetics / Q000502:physiology*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000494:pharmacology; D015398:Signal Transduction / Q000502:physiology*; D014162:Transfection / Q000379:methods
D008077:Lipoproteins, LDL; D034741:RNA, Small Interfering; C019455:low density lipoprotein inhibitor; D049452:Green Fluorescent Proteins; D051057:Proto-Oncogene Proteins c-akt; D011493:Protein Kinase C; D010741:Phospholipases A; D054513:Group IV Phospholipases A2; D054467:Phospholipases A2