Promoter-dependent effect of IKKalpha on NF-kappaB/p65 DNA binding

IKKalpha regulates many chromatin events in the nuclear phase of the NF-kappaB program, including phosphorylation of histone H3 and removal of co-repressors from NF-kappaB-dependent promoters. However, all of the nuclear functions of IKKalpha are not understood. In this study, using mouse embryonic fibroblasts IKKalpha knock-out and reexpressing IKKalpha after retroviral transduction, we demonstrate that IKKalpha contributes to NF-kappaB/p65 DNA binding activity on an exogenous kappaB element and on some, but not all, endogenous NF-kappaB-target promoters. Indeed, p65 chromatin immunoprecipitation assays revealed that IKKalpha is crucial for p65 binding on kappaB sites of icam-1 and mcp-1 promoters but not on ikappabalpha promoter. The mutation of IKKalpha putative nuclear localization sequence, which prevents its nuclear translocation, or of crucial serines in the IKKalpha activation loop completely inhibits p65 binding on icam-1 and mcp-1 promoters and rather enhances p65 binding on the ikappabalpha promoter. Further molecular studies demonstrated that the removal of chromatin-bound HDAC3, a histone deacetylase inhibiting p65 DNA binding, is differentially regulated by IKKalpha in a promoter-specific manner. Indeed, whereas the absence of IKKalpha induces HDAC3 recruitment and repression on the icam-1 promoter, it has an opposite effect on the ikappabalpha promoter, where a better p65 binding occurs. We conclude that nuclear IKKalpha is required for p65 DNA binding in a gene-specific manner.     

NIK is involved in nucleosomal regulation by enhancing histone H3 phosphorylation by IKKalpha

The exact physiological role of NF-kappaB-inducing kinase (NIK) in the NF-kappaB activation pathway has not been defined, although it is an upstream kinase of IKKalpha. Recent studies have indicated that IKKalpha is a nucleosomal modifier of NF-kappaB signaling. We hypothesized that NIK generates a proximal signal that contributes to IKKalpha modification of nucleosomal structure through phosphorylation of histone H3 and enhancement of target gene expression. By using a chromatin immunoprecipitation assay, our data show that endogenous IKKalpha is recruited to the promoter site of several NF-kappaB-dependent genes in macrophages. Our data show that immunoreactive NIK is rapidly recruited to nuclear compartment in macrophages in response to treatment with endotoxin where it augments phosphorylation of histone H3 by inducing phosphorylation and kinase activity of IKKalpha. A small interfering RNA knockdown of NIK markedly reduces phosphorylation of histone H3 in endotoxin treated macrophages. These data, together, demonstrate a novel role for NIK as a histone H3 modifier, through an accessory pathway from NIK to IKKalpha, that could play an important role in the endotoxin response through modification of nucleosomal structure.     

HIV-1 Tat protein induces IL-10 production in monocytes by classical and alternative NF-kappaB pathways

The human immunodeficiency virus (HIV) transactivating Tat protein is not only critical for viral replication but also affects the host immune system by inducing the production of cytokines such as IL-10. This anti-inflammatory cytokine is upregulated during the course of HIV infection, representing an important pathway by which HIV may induce immunodeficiency. Here, we show that, by acting at the membrane, Tat induces IL-10 expression in primary monocytes and promonocytic U937 cells by NF-kappaB-dependent pathways. The trans-dominant negative mutants of NF-kappaB-inducing kinase (NIK), IKKalpha and IKKbeta expressed in our transactivation model, in accordance with the nuclear binding of p65 and p52 NF-kappaB subunits to the IL-10 promoter, suggest the involvement of both classical and alternative NF-kappaB pathways. In inactivated cells, IKKalpha is localized predominantly in the cytoplasm. Interestingly, Tat stimulates IKKalpha translocation from the cytoplasm to the nucleus in monocytes. Chromatin immunoprecipitation (ChIP) assay experiments, after Tat treatment, revealed IKKalpha and CBP/p300 recruitment to the IL-10 promoter and histone H3 phosphorylation (Ser 10) and acetylation (Lys 14) in this region, presumably leading to chromatin remodeling. We demonstrate that, upstream of NF-kappaB, PKC, ERK1/2 and p38 MAP kinases are involved in Tat-induced IKKalpha nuclear translocation and histone H3 modifications on the IL-10 promoter in accordance with the role of these three kinases in IL-10 production. As a whole, the study demonstrates that Tat activates at least three signaling pathways concurrently, including the classical, alternative and IKKalpha pathways, to promote production of IL-10.     

A cell cycle regulatory network controlling NF-kappaB subunit activity and function

Aberrantly active NF-kappaB complexes can contribute to tumorigenesis by regulating genes that promote the growth and survival of cancer cells. We have investigated NF-kappaB during the cell cycle and find that its ability to regulate the G1-phase expression of key proto-oncogenes is subject to regulation by the integrated activity of IkappaB kinase (IKK)alpha, IKKbeta, Akt and Chk1. The coordinated binding of NF-kappaB subunits to the Cyclin D1, c-Myc and Skp2 promoters is dynamic with distinct changes in promoter occupancy and RelA(p65) phosphorylation occurring through G1, S and G2 phases, concomitant with a switch from coactivator to corepressor recruitment. Akt activity is required for IKK-dependent phosphorylation of NF-kappaB subunits in G1 and G2 phases, where Chk1 is inactive. However, in S-phase, Akt is inactivated, while Chk1 phosphorylates RelA and associates with IKKalpha, inhibiting the processing of the p100 (NF-kappaB2) subunit, which also plays a critical role in the regulation of these genes. These data reveal a complex regulatory network integrating NF-kappaB with the DNA-replication checkpoint and the expression of critical regulators of cell proliferation.     

Aberrant Cytoplasmic Localization of N-CoR in Colorectal Tumors

We have previously shown that IKKs are aberrantly activated in colon cancer cells leading to SMRT phosphorylation and its release from the chromatin. We now show that IKKα phosphorylates the homologous N-CoR corepressor in serines 2345 and 2348 creating a functional 14-3-3 binding domain (RKpS2348KSP). Moreover, we have analyzed the subcellular localization of N-CoR in 43 colorectal cancer samples and we have found that aberrant cytoplasmic distribution of N-CoR is a general trait of these tumors.