IKKα regulation of canonical NF-κB activation downstream of Nod1-mediated peptidoglycan recognition : "and" Endocytosis-independent function of clathrin heavy chain in the control of basal NF-κB activation

NF-κB is a transcription factor involved in the regulation of inflammation and innate immunity. The IκB kinase (IKK) complex contains two catalytic subunits, IKKα and IKKβ, and plays an essential role in the activation of NF-κB through the phosphorylation and degradation of the NF-κB inhibitor IκBα, thereby allowing translocation of NF-κB into the nucleus. Numerous evidences indicate that IKKβ mediates NF-κB activation in response to pro-inflammatory cytokines and microbial products, but the role of IKKα in inflammation and innate immunity is unknown.
In the first part of dissertation, we focus on understanding the previously unknown function of IKKα in the canonical NF-κB pathway, associated with inflammation and innate immunity. We show that silencing of IKKα by RNA interference (RNAi) significantly reduced phosphorylation and degradation of IκBα, and nuclear translocation of NF-κB, and secretion of the pro-inflammatory chemokine interleukin-8 (IL-8) during Shigella flexneri infection of human epithelial HeLa cells. This suggests that IKKα like IKKβ plays a pivotal role in inflammation and innate immunity by mediating NF-κB activation in response to microbial infection.
Proper control of NF-κB activation is essential for inflammation and innate immunity triggered by microbial infection, but the dysregulation of NF-κB is associated with various diseases such as chronic inflammatory diseases and cancers. Thus, the NF-κB pathway has been a target of therapeutic drug development. Although constitutive and excessive NF-κB activation has been detected in many inflammation-related diseases, the cause of the constitutive NF-κB activation in non-stimulated cells is largely unknown.
In the second part of dissertation, we focus on clathrin heavy chain (CHC), a well-known regulator of endocytosis that plays a novel endocytosis-independent function as an inhibitor of basal NF-κB activation. We show that silencing of CHC induced constitutive NF-κB nuclear translocation and high level of IL-8 secretion in resting cells. We revealed that constitutive NF-κB nuclear translocation was mediated through the constant IκBα degradation in an IKKα-dependent mechanism. We further showed that CHC depletion-induced constitutive IκBα degradation and high level of IL-8 secretion in resting cells was independent of the inhibition of clathrin-mediated endocytosis (CME) as silencing of μ2 subunit of AP2 complex (AP2M1), an adaptor protein essential for CME failed to induce the constitutive IκBα degradation and high level of IL-8 secretion. Therefore, the results presented may suggest a potential link between a defect in CHC expression and chronic inflammatory disorders and cancers.