FIGURE 5.

MPO-oxidized HDL induces bovine aortic endothelial cell NF-κB activation, IKK activation, and phosphorylation of IκBα. A, BAEC were incubated with TNFα for 30 min (first through third lanes), media only (NA), HDL for 3 h, or oxHDL for the indicated times. EMSA for NF-κB activation were then performed in whole cell lysates as described under “Experimental Procedures.” Where indicated, the lysates were also incubated with anti-NF-κB p65 or isotype control IgG and supershift (SS) of the NF-κB complex monitored. Parallel immunoblots (IB) were generated using phosphoserine 32- and 36-specific IκB-α antibody (p-IκB-α), demonstrating phosphorylation of IκB-α on serines 32 and 36 in oxHDL-treated cells. Immunoblot with specific antibody to IκB-α is also shown, along with a immunoblot of lysates probed with anti-β-actin to demonstrate equal loading in each lane. B, EMSA analysis of BAEC lysates as in A except that the cells were exposed to HDL modified by the complete MPO/H₂O₂/Cl⁻ system (oxHDL) or the complete oxidant system minus the indicated components (i.e. −MPO, −H₂O₂, or −Cl⁻). Note that BAEC NF-κB activation is only observed by exposure to HDL previously incubated with the complete MPO/H₂O₂/Cl⁻ system because eliminating any one of the components of the oxidation system produces a HDL particle that fails to activate endothelial cell NF-κB. C, BAEC were incubated with TNFα (30 min) as positive control, media alone (NA) as negative control, or either HDL (3 h) or HDL previously exposed to the complete MPO/H₂O₂/Cl⁻ system (oxHDL, 2 or 3 h). IKK activity was then determined in BAEC lysates using IKK-specific immuno-pulldown coupled kinase assay (KA). IKK complex was immunoprecipitated with antibody to IKKγ, and kinase activity using recombinant GST-IκBα(1–54) and [³²P]ATP as substrate was performed as described under “Experimental Procedures.” Note that IκBα is phosphorylated in response to stimulation by TNFα and oxHDL but not HDL. Specificity of the kinase reaction was confirmed by demonstrating failure of the site-specific mutant GST-IκB-α(1–54) (32A/36A) to be phosphorylated in TNFα-stimulated extracts. Parallel immunoblots using antibodies specific to either IKKγ or β-actin are also shown. Equivalent levels of GST-IκB-α substrate addition to the IKK complexes are shown by Coomassie Blue (CB) staining. NA refers to “no addition.”