Abstract
BackgroundNuclear 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.MethodologyUsing 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.ConclusionsWe 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.
Highlights
Nuclear factor-kappa B (NF-kB) transcription factors control the expression of genes involved in a large spectrum of biological processes, including inflammation, adaptive immunity, stress responses, angiogenesis, cell proliferation and invasion [1,2]
Following cell stimulation by proinflammatory cytokines, such as tumor necrosis factor a (TNFa) and interleukin-1, inhibitor of NF-kB alpha (IkBa) is rapidly phosphorylated on serine 32 and serine 36 residues by the inhibitor of NF-kB (IkB) kinase (IKK) complex composed of three subunits: two catalytic subunits, IkB kinase a (IKKa) and IKKb, and the regulatory scaffold component Nuclear factor-kB (NF-kB) essential modulator (NEMO)
Antibodies and reagents Antibodies against NF-kB p65, IkBa, clathrin light chain a (CLCa) and IKKa were obtained from Santa Cruz Biotechnology (Santa Cruz, USA) while the clathrin heavy chain (CHC) antibody was from BD Transduction Laboratories (San Jose, USA)
Summary
Nuclear factor-kappa B (NF-kB) transcription factors control the expression of genes involved in a large spectrum of biological processes, including inflammation, adaptive immunity, stress responses, angiogenesis, cell proliferation and invasion [1,2]. There are five NF-kB isoforms in mammalian cells: p65/RelA, RelB, c-Rel, p50 (NF-kB1) and p52 (NF-kB2) All these proteins share a Rel homology domain responsible for homo- and heterodimerization as well as for sequence-specific DNA binding. Synthesized IkBa proteins bind to nuclear NF-kB dimers and dissociate them from DNA. This mechanism terminates the transcriptional activity of NF-kB and resets gene expression to basal level. Nuclear factor-kB (NF-kB) 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. We test whether clathrin heavy chain (CHC) contributes to the regulation of basal NF-kB activity in epithelial cells
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