Redox/ROS regulation of lipopolysaccharide-induced mitogen-activated protein kinase (MAPK) activation and MAPK-mediated TNF-α biosynthesis

1. Redox and ROS regulation of MAPK-mediated TNF-α biosynthesis is not well characterized. It was hypothesized that the involvement of the MAPK pathway in regulating LPS-mediated TNF-α secretion is redox-dependent, NF-κB-sensitive and attenuated by N-acetyl-L-cysteine (NAC) and other antioxidants. 2. In alveolar epithelial cells, LPS induced a time- and dose-dependent phosphorylation of MAPKp38. This was associated with the activation of MAPK-activated protein kinase, which phosphorylated the small heat-shock protein, Hsp27. 3. MAPKp38 inhibition (SB-203580) abrogated LPS-induced TNF-α production. MAPKERK blockade (PD-98059) attenuated TNF-α secretion, an effect synergistically amplified in the presence of SB-203580. 4. Regulation of NF-κB by selective inhibitors revealed that this pathway is partially involved in regulating LPS-mediated TNF-α secretion. Whereas the proteasome inhibitor, MG-132, had no effect on LPS-mediated TNF-α production, CAPE, sulfasalazine and SN-50, a cell-permeant NF-κB inhibitor, attenuated but did not abrogate TNF-α biosynthesis. 5. LPS up-regulated ROS, an effect abrogated by 4′-hydroxy-3′-methoxy-acetophenone and NAC, which reduced TNF-α secretion, induced the accumulation of GSH, reduced the concentration of GSSG, and blockaded the phosphorylation/activation of MAPKp38 pathway. 6. ROS induced MAPKp38 phosphorylation and selective antioxidants, including the permeant GSH precursor, γ-GCE, reduced ROS-dependent MAPKp38 phosphorylation. 7. These results indicate that the MAPK pathway and MAPK-mediated regulation of TNF-α production is redox-dependent, GSH-mediated and requires, at least in part, a NF-κB/ROS-sensitive mechanism.