The role of P38 MAPK and PKC in BLP induced TNF-alpha release, apoptosis, and NFkappaB activation in THP-1 monocyte cells.
BACKGROUND
P38 mitogen activated protein kinase (p38 MAPK) is a critical mediator of the inflammatory response, which makes it a suitable candidate as a novel therapeutic strategy for inflammatory conditions. In this study, we set out to examine the precise role of both protein kinase C (PKC) and P38 MAPK signaling kinases in bacterial lipoprotein (BLP) induced nuclear factor-kappa B (NFkappaB) activation and tumor necrosis factor-alpha (TNFalpha) release in THP-1 monocytic cell line.


METHODS
THP-1 cells were incubated with BLP(0-1000 ng/mL), phorbol myristate acetate (PMA; 0-100 microg/mL) or a combination of both for 6 and 24 h, with or without pretreatment with SB202190, a specific inhibitor of p38 MAPK and bisindolylmaleimide I, a specific inhibitor of PKC (0-200 microm). Cell supernatants were analyzed for TNF-alpha release and apoptosis. NFkappaB activity was analyzed by electromobility supershift assay.


RESULTS
BLP induced TNF-alpha release was significantly reduced by pretreatment with SB202190 at all concentrations (428.7 +/- 5.9 versus 51 +/- 0.8 rhog/mL, P < 0.05). Pretreatment with bis I significantly inhibited TNF-alpha release at higher concentrations (200 microM) (429.7 +/- 5.9 versus 194.9 +/- 42.68 rhog/mL, P < 0.05) but this was much less effective than SB202190. PMA induced TNF-alpha release was not inhibited at 6 h by either SB202190 or bis I, but was significantly so at 24 h (148.5 +/- 9.8 versus 24 +/- 1.7 and 25.1 +/- 4.4 rhog/mL, P < 0.05). BLP or lipopolysaccharide (LPS) did not result in apoptosis in THP-1 cells (P > 0.05) with PMA inducing apoptosis in a time- and dose-dependent manner. In combination with BLP (1000 ng/mL) but not LPS (1000 ng/mL), low dose PMA resulted in a significant increase in apoptosis, 6% +/- 0.5% (Control) versus 9.2% +/- 0.3% (P < 0.05) and 7% +/- 2.2% (Control) versus 7.7% +/- 0.3% (P > 0.05), respectively. This synergistic effect was inhibited by bisindolylmaleimide 100 nm, 8.9% +/- 0.9% (Control) versus 9.8% +/- 0.2% (P > 0.05). PMA and BLP induced rapid nuclear translocation of NFkappaB, which was inhibited by pretreatment with both SB-202190 and bis I, and SB202190 but not bis I, respectively.


CONCLUSIONS
P38 is a critical mediator of BLP induced TNF-alpha release and NFkappaB activation, whereas PKC is only partially responsible for its response. P38 and PKC are both critical mediators of PMA induced TNF-alpha release and NFkappaB activation.
O'Sullivan
Adrian W
AW
Department of Academic Surgery, National University of Ireland and University College Hospital, Cork, Ireland. awmos@btinternet.com
Wang
Jiang H
JH
Redmond
Henry P
HP
D017209:Apoptosis / Q000187:drug effects*; D001426:Bacterial Proteins / Q000009:adverse effects / Q000494:pharmacology; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D007249:Inflammation / Q000209:etiology / Q000378:metabolism; D008070:Lipopolysaccharides / Q000009:adverse effects / Q000494:pharmacology; D008074:Lipoproteins / Q000009:adverse effects* / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D009000:Monocytes / Q000187:drug effects / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology; D015398:Signal Transduction / Q000502:physiology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D007093:Imidazoles; D007211:Indoles; D008070:Lipopolysaccharides; D008074:Lipoproteins; D008301:Maleimides; D016328:NF-kappa B; D011725:Pyridines; D014409:Tumor Necrosis Factor-alpha; D011493:Protein Kinase C; D048051:p38 Mitogen-Activated Protein Kinases; C070515:bisindolylmaleimide I; D013755:Tetradecanoylphorbol Acetate; C090942:4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)imidazole