Mechanism of UV-induced IkappaBalpha-independent activation of NF-kappaB

Abstract

Nuclear factor-kappa B (NF-kappaB) plays an important role in UV-induced skin tumorigenesis. Activation of NF-kappaB by UV-irradiation is composed of two phases. The early phase culminates with maximal levels of DNA binding ability at 4 h postirradiation and is dependent on translational inhibition. The late-phase activation of NF-kappaB occurs between 16 and 48 h post-irradiation and the mechanism is not clear due to the fact that NF-kappaB was activated in the presence of high level of IkappaBalpha. In this report, we provide evidence that without translational inhibition, the transcription of IkappaBalpha was induced by UV-irradiation. In the late-phase of UV-induced NF-kappaB activation, the IkappaBalpha depletion is the combined result of regulation at both transcriptional and translational levels. Neither ubiquitination nor proteasomal degradation have detectable attributions to IkappaBalpha breakdown. We also demonstrate that UV only induced phosphorylation of p65(S276), while tumor necrosis factor-alpha induced phosphorylation at both Ser276 and 536 sites of p65. Based upon our results, we propose a novel mechanism for translation-regulated IkappaBalpha depletion and MSK-mediated NF-kappaB activation at 24 h post-UV-irradiation.

Figure 1

Inability to block translation allows for transcriptional activation of IκBα. (A) Quantitative real-time PCR of IκBα mRNA expression in non-treated and UV-irradiated MEFs. Relative amounts of IκBα transcripts were normalized to the levels of β-actin housekeeping gene in each sample. (B) Western blot analysis of total IκBα at 24 h post UV-C irradiation (30 J m⁻²) of MEF^S/S and MEF^A/A cells. The intensities of the bands were quantified by ImageJ (v 1.31; NIH). Expression levels were normalized by the levels of β-actin and shown as a percentage of IκBα expression at 0 h after induction.

Figure 2

The ubiquitination and proteasome inhibitors failed in rescuing UV-induced IκBα depletion. Total amounts of IκB and β-actin in UV-irradiated or TNFα-treated MEF^S/S and MEF^A/A cells were determined by western blot analysis. The cells were treated or not treated with UV-irradiation (30 J m⁻², panels A and B) for 24 h or TNFα (10 ng mL⁻¹, panel C) for 1 h in the presence or absence of MG132 (5 mM, panel A) or Ro106 (5 or 10 μM, panels B and C) as indicated. The intensities of the bands were quantified by ImageJ (v 1.31; NIH). The expression levels of IκB were normalized by the expression levels of β-actin and expressed as a percentage of IκB expression at 0 h post-UV light irradiation.

Figure 3

The localization and phosphorylation of NF-κB at 24 h post-UV-irradiation. The amounts of nuclear (panels A and C) or cytosolic (panel B) p65, p65-P(536), p65-P(276) and β-actin in UV-irradiated or TNFα-treated MEF^S/S and MEF^A/A cells were determined by western blot analysis. The cells were treated or not treated with UV-irradiation (30 J m⁻², panels A and B) for 24 h or TNFα (10 ng mL⁻¹, panel C) for 1 h.

Figure 4

Proposed model for UV light-induced late-phase activation of NF-κB.