Alpha-lipoic acid inhibits TNF-α induced NF-κB activation through blocking of MEKK1–MKK4–IKK signaling cascades

The therapeutic effects of α-lipoic acid (α-LA) via NF-κB down regulation were demonstrated on joint inflammation and erosion in an animal model. In this study, we investigated how α-LA inhibits the pathway of NF-κB activation by TNF-α via the mitogen-activated protein kinase (MAPK) pathway in rheumatoid arthritis (RA) fibroblast-like synovial cells (FLS). FLS were stimulated with TNF-α following pre-treatment with or without α-LA. Electrophoretic mobility shift assays (EMSA) revealed that TNF-α activates NF-κB in FLS. This was inhibited by α-LA at concentrations of 1 mM. TNF-α induced IKK mediated phosphorylation of GST-IκB and pre-treatment with α-LA inhibited this pathway. FLS constitutively express MEKK1, MEKK2, MEKK3, and TAK1 and that their levels are unaffected by TNF-α or α-LA. Immunoprecipitation using anti-MEKK1 antibody phosphorylated GST-IκB and pre-treating the cells with α-LA could abolish the reaction. FLS were immunoprecipitated using an antibody to MEKK1, and MKK4 was coprecipitated with MEKK1. In addition, immune complexes precipitated with anti-MKK4 antibody phosphorylated GST-IκB, and pre-treatment with α-LA inhibited the phosphorylation. Immunoprecipitation assay showed that MEKK1, MKK4, IKK-α, IKK-β, IκB, and NF-κB comprised immunocomplex. It can be concluded that TNF-α activates NF-κB in FLS through MEKK1–MKK4–IKK signaling complex, and α-LA inhibits this signaling at the level of or upstream of IKK-α and IKK-β.