Clathrin heavy chain is required for TNF-induced inflammatory signaling

BackgroundTumor necrosis factor receptor I recruits tumor necrosis factor receptor-associated death domain (TRADD) and multiple kinases that ultimately phosphorylate inhibitor kappa B (IKBα). Degradation of phospho-IKBα (p-IKBα) frees nuclear factor kappa B (NFKB) to be active and phosphorylated. Many receptors require clathrin-mediated endocytosis to provide the scaffolds necessary for signaling. Therefore, we investigated the role of clathrin heavy chain (CHC) in tumor necrosis factor α (TNF-α)–induced IKBα phosphorylation and NFKB activation. We hypothesized that CHC was required for TNF-α–induced inflammatory signaling.MethodsWe treated human pulmonary epithelial cells with small interfering RNA to knock down intracellular CHC (CHCsil). TRADD and scrambled (noncoding) small interfering RNA sequences were used as positive and negative controls, respectively. Treatment groups were exposed to 10 ng/mL of TNF-α. Total IκBα, p-IκBα, and phosphorylated P65 (a subunit of NFKB) were determined by immunoblot staining. Densitometry was normalized to controls for the analysis of the stains. TNF-α–induced release of monocyte chemoattractant protein 1 (MCP-1) was determined by enzyme-linked immunosorbent assay. Statistical analyses were determined by analysis of variance or paired t test as appropriate.ResultsTNF-α−induced IκBα phosphorylation and degradation at 5 and 30 minutes, respectively, and induced P65 phosphorylation. CHCsil diminished p-IκBα by 91% (P < .03); however, IκBα degradation was not affected. CHC knockdown caused a 66% decrease in P65 phosphorylation after 3 minutes of TNF-α. CHCsil decreased TNF-α–induced MCP-1 by 46% (P < .05), compared with control.ConclusionsCHCsil significantly impairs phosphorylation of both IκBα and P65. CHCsil also significantly decreased MCP-1 production. These data suggest that CHC is required for certain TNF-α–induced, inflammatory signaling pathways.