Endocytosis-independent function of clathrin heavy chain in the control of basal NF-κB activation.

Abstract

Nuclear factor-κB (NF-κB) is a transcription factor that regulates the transcription of genes involved in a variety of biological processes, including innate and adaptive immunity, stress responses and cell proliferation. Constitutive or excessive NF-κB activity has been associated with inflammatory disorders and higher risk of cancer. In contrast to the mechanisms controlling inducible activation, the regulation of basal NF-κB activation is not well understood. Here we test whether clathrin heavy chain (CHC) contributes to the regulation of basal NF-κB activity in epithelial cells. Using RNA interference to reduce endogenous CHC expression, we found that CHC is required to prevent constitutive activation of NF-κB and gene expression. Immunofluorescence staining showed constitutive nuclear localization of the NF-κB subunit p65 in absence of stimulation after CHC knockdown. Elevated basal p65 nuclear localization is caused by constitutive phosphorylation and degradation of inhibitor of NF-κB alpha (IκBα) through an IκB kinase α (IKKα)-dependent mechanism. The role of CHC in NF-κB signaling is functionally relevant as constitutive expression of the proinflammatory chemokine interleukin-8 (IL-8), whose expression is regulated by NF-κB, was found after CHC knockdown. Disruption of clathrin-mediated endocytosis by chemical inhibition or depletion of the μ2-subunit of the endocytosis adaptor protein AP-2, and knockdown of clathrin light chain a (CHLa), failed to induce constitutive NF-κB activation and IL-8 expression, showing that CHC acts on NF-κB independently of endocytosis and CLCa. We conclude that CHC functions as a built-in molecular brake that ensures a tight control of basal NF-κB activation and gene expression in unstimulated cells. Furthermore, our data suggest a potential link between a defect in CHC expression and chronic inflammation disorder and cancer.