IL-1β is activated from an accessible chromatin structure by a complex array of DNA-binding proteins. A subset of these proteins constitutively associates with IL-1β regulatory elements before transcription initiation, while a second subset associates only upon monocyte stimulation. Our data indicate that one kinase, CK2, activates two processes that function in parallel to drive IL-1β transcription from the poised promoter only in stimulated cells. First, CK2 regulates p65 translocation and activity by controlling IκB-α levels and p65 phosphorylation, respectively. Second, CK2 regulates IRF-4 recruitment by modulating phosphorylation of PU.1 at Ser148. Kinetic analyses further indicate that the timing of inducible p65/IL-1β enhancer association likely primes initial IL-1β transcription in combination with constitutive IRF-8 association, while PU.1 phosphorylation and the resulting IRF-4 recruitment likely contribute to prolonging IL-1β transcription beyond early time points. This association temporally bridges the two proposed phases of IL-1β transcriptional activation. Taken together, these findings demonstrate how two pathways resulting in NF-κB and IRF association are both triggered by CK2 activity to culminate in transcriptional activation of a potent proinflammatory cytokine, IL-1β.
The roles of IRF-4 vs IRF-8 in IL-1β transcription have been highly controversial. Transient transfection experiments have shown 1) either IRF-4 or IRF-8 can activate IL-1β transcription (11
), or 2) IRF-4 plays a more important role in IL-1β activation specifically in macrophages (4
). Similarly, ChIP experiments differed on whether IRF-4 or IRF-8 association correlates with inducible IL-1β transcription (12
). Finally, experiments in mice genetically null for IRF-4 or IRF-8 demonstrated that proinflammatory cytokine production increased or decreased, respectively, indicating that IRF-8 activates IL-1β, but that IRF-4 actually inhibits cytokine production (54
). All of these findings are unified in the following model based on the demonstrated shift from a p65/PU.1/IRF-8 to a phospho-PU.1/IRF-4-dominated complex at the IL-1β enhancer. IRF-8 associates with the IL-1β enhancer in unstimulated monocytes, and this association is maintained for 30–60 min poststimulation (). We speculate that constitutive IRF-8 and PU.1 association along with rapid recruitment of NF-κB to the enhancer () contribute to the nearly instantaneous recruitment of pol II upon LPS stimulation and drive the extraordinarily rapid activation of the IL-1β gene. We propose that the demonstrated tyrosine phosphorylation of IRF-8 at 30 min poststimulation (12
) opens a window for PU.1-mediated IRF-4 recruitment to the enhancer and decreased p65 and IRF-8/enhancer association ( and ) that correlates with maximal IL-1β mRNA levels at 60–120 min poststimulation ( and ). The resolution of ChIP does not allow us to distinguish between IRF-4 and IRF-8 binding adjacent to PU.1 in the enhancer vs, for example, IRF-4 being tethered to the enhancer complex. Because IRF-4 can negatively regulate cytokine production (55
), we propose that the shift from a p65/PU.1/IRF-8 to a phospho-PU.1/IRF-4-dominated complex results in a controlled moderation of IL-1β transcription, as evidenced by decreased transcript levels at ~3h poststimulation (). The proposed mechanism of IL-1β activation by an initial IRF-8 dominance followed by a shift to IRF-4 dominance contrasts with a recently published model of sustained IFN-β activation, which is promoted by IRF-8/enhancer association (56
This detailed model of IL-1β transcriptional activation explains multiple apparent conflicts in the literature describing the roles of various sequence-specific transcription factors. However, this model does not take into account the demonstration that both IRF-4 and IRF-8 association with composite PU.1/IRF elements in other genes requires PU.1 phosphorylation at Ser148
in the rat PU.1 protein) (57
). However, in vitro transcribed/translated PU.1 and PU.1 from unstimulated macrophage nuclear extracts facilitates IRF-8/IL-1β enhancer binding (4
), indicating PU.1 phosphorylation may be dispensable for early IRF-8-mediated activation. This possibility is supported by the demonstration that expression of the nonmodifiable mPU.1, in combination with LPS stimulation (i.e., NF-κB activation), resulted in IL-1β hyper-production only at the earliest time point tested.
Synergy between NF-κB proteins and LPS-induced PU.1 and/or IRFs plays a role in activation of many immune system genes, including IL-1 receptor antagonist, IL-12p40, and RANTES (60
). Similarly, feedback loops between NF-κB and IRF induction have been identified in several experimental systems. For example, NF-κB family members regulate IRF-4 levels in lymphocytes (64
), and IRF-8 is required for activity of the IκB kinase that initiates the cascade of events leading to NF-κB nuclear translocation in dendritic cells (66
). However, the almost instantaneous activation of IL-1β transcript occurs before the times these feedback loops have been analyzed, so the contribution of direct NF-κB/PU.1/IRF cross-talk to inducible IL-1β transcription is uncertain. It is possible that serial activation of NF-κB and PU.1 phosphorylation/IRF-4 recruitment by CK2 does not require additional interaction in the earlier phases, but that feedback loops become operational at about the same time IL-1β transcription is moderated 2–3 h poststimulation. Regardless, our data support the model that CK2 is a unifying regulator of inducible IL-1β transcription, based on its ability to regulate NF-κB and IRF-4 activation.