The Role of P38 MAPK and PKC in BLP Induced TNF-α Release, Apoptosis, and NFκB 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 (NFκB) activation and tumor necrosis factor-alpha (TNFα) release in THP-1 monocytic cell line. Materials and methods: THP-1 cells were incubated with BLP(0-1000 ng/mL), phorbol myristate acetate (PMA; 0-100 μg/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 μm). Cell supernatants were analyzed for TNF-α release and apoptosis. NFκB activity was analyzed by electromobility supershift assay. Results: BLP induced TNF-α release was significantly reduced by pretreatment with SB202190 at all concentrations (428.7 ± 5.9 versus 51 ± 0.8 ρg/mL, P < 0.05). Pretreatment with bis I significantly inhibited TNF-α release at higher concentrations (200 μM) (429.7 ± 5.9 versus 194.9 ± 42.68 ρg/mL, P < 0.05) but this was much less effective than SB202190. PMA induced TNF-α 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 ρg/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 ngmL) 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 NFκB, 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-α release and NFκB activation, whereas PKC is only partially responsible for its response. P38 and PKC are both critical mediators of PMA induced TNF-α release and NFκB activation.