IKKalpha and IKKbeta each function to regulate NF-kappaB activation in the TNF-induced/canonical pathway

Abstract

Background: Activation of the transcription factor NF-kappaB by cytokines is rapid, mediated through the activation of the IKK complex with subsequent phosphorylation and degradation of the inhibitory IkappaB proteins. The IKK complex is comprised of two catalytic subunits, IKKalpha and IKKbeta, and a regulatory protein known as NEMO. Using cells from mice that are genetically deficient in IKKbeta or IKKalpha, or using a kinase inactive mutant of IKKbeta, it has been proposed that IKKbeta is critical for TNF-induced IkappaB phosphorylation/degradation through the canonical pathway while IKKalpha has been shown to be involved in the non-canonical pathway for NF-kappaB activation. These conclusions have led to a focus on development of IKKbeta inhibitors for potential use in inflammatory disorders and cancer.

Methodology: Analysis of NF-kappaB activation in response to TNF in MEFs reveals that IKKbeta is essential for efficient phosphorylation and subsequent degradation of IkappaB alpha, yet IKKalpha contributes to the NF-kappaB activation response in these cells as measured via DNA binding assays. In HeLa cells, both IKKalpha and IKKbeta contribute to IkappaB alpha phosphorylation and NF-kappaB activation. A kinase inactive mutant of IKKbeta, which has been used as evidence for the critical importance of IKKbeta in TNF-induced signaling, blocks activation of NF-kappaB induced by IKKalpha, even in cells that are deficient in IKKbeta.

Conclusions: These results demonstrate the importance of IKKalpha in canonical NF-kappaB activation, downstream of cytokine treatment of cells. The experiments suggest that IKKalpha will be a therapeutic target in inflammatory disorders.

Figure 1. The role of IKKα and IKKβ in p65 phosphorylation and IκBα degradation in response to TNFα.

MEF cells that are deficient for IKKα, IKKβ, or both IKKα and IKKβ (DKO) were treated with TNFα for the indicated times. NF-κB activity, as measured by IκBα degradation and p65 phosphorylation, is diminished in IKKα and IKKβ deficient MEF cells. IKKα and IKKβ DKO cells show no detectable p65 phosphorylation. Tubulin levels are shown as a loading control.

Figure 2. NF-κB DNA binding activity is reduced in IKKα and in IKKβ deficient cells.

DNA binding activity of NF-κB was measured by gel shift assay. Indicated MEF cells were treated with TNFα for indicated times. Nuclear proteins were subject to gel shift assay for DNA binding analysis.

Figure 3. IKKα and IKKβ knock-down in Hela cells leads to diminished IκB degradation and p65 phosphorylation.

HeLa cells were grown in 6-well plates and transfected with the indicated siRNA for 3 days. Western blots were performed on total cell extracts after treatment with TNF for the indicated times. Tubulin levels are shown as a loading control.

Figure 4. IKKα and IKKβ each contribute to TNF-induced NF-κB activity in HeLa cells.

HeLa cells, seeded in 24-well plates were transiently transfected with indicated siRNA constructs for 48 hr. Then media was replaced and cells were further transfected with NF-κB responsive 3x-κB luciferase and a control Renilla luciferase contructs. TNF was added (as indicated) and 24 hr later cells were lysed and dual lucifearse assay was performed. Luciferase readings in untreated and control vector transfected cells were normalized as 1.

Figure 5. Knockdown of IKKα or IKKβ blocks basal and TNF-α induced NF-κB luciferase activity in breast cancer cells.

SKBR3 cells stably expressing 4x-κB firefly luciferase reporter gene were transfected with 100 nM siRNA against IKKα, IKKβ or both. Cells were treated with PBS (black) or 10 ng/ml TNF (gray striped) for 12 hr. Luciferase activity was measured and normalized to total protein levels.

Figure 6. IKKβ kinase mutant inhibits TNF and IKKα-induced NF-κB-dependent reporter gene activity.

WT and IKKβ null MEFS were transfected with the indicated vector construct and with the NF-κB-dependent luciferase reporter. Luciferase luciferase activity was measured 48 hr after transfected. Where indicated, cells were treated with TNF for 4 hrs. Relative luciferase values were calculated using a renilla control expression vector for normalization. Relative luciferase values are normalized to vector control samples.