Potentiation of Tumor Necrosis Factor-Induced NF-κB Activation by Deacetylase Inhibitors Is Associated with a Delayed Cytoplasmic Reappearance of IκBα

Abstract

Previous studies have implicated acetylases and deacetylases in regulating the transcriptional activity of NF-B. Here, we show that inhibitors of deacetylases such as trichostatin A (TSA) and sodium butyrate (NaBut) potentiated TNF-induced expression of several natural NF-B-driven promoters. This transcriptional synergism observed between TNF and TSA (or NaBut) required intact B sites in all promoters tested and was biologically relevant as demonstrated by RNase protection on two instances of endogenous NF-B-regulated gene transcription. Importantly, TSA prolonged both TNF-induced DNA-binding activity and the presence of NF-B in the nucleus. We showed that the p65 subunit of NF-B was acetylated in vivo. However, this acetylation was weak, suggesting that other mechanisms could be implicated in the potentiated binding and transactivation activities of NF-B after TNF plus TSA versus TNF treatment. Western blot and immunofluorescence confocal microscopy experiments revealed a delay in the cytoplasmic reappearance of the IB␣ inhibitor that correlated temporally with the prolonged intranuclear binding and presence of NF-B. This delay was due neither to a defect in IB␣ mRNA production nor to a nuclear retention of IB␣ but was rather due to a persistent proteasome-mediated degradation of IB␣. A prolongation of IB kinase activity could explain, at least partially, the delayed IB␣ cytoplasmic reappearance observed in presence of TNF plus TSA.NF-B is a ubiquitously expressed family of transcription factors controlling the expression of numerous genes involved in inflammatory and immune responses and cellular proliferation (reviewed in references 4, 5, 19, and 26). There are five known members of the mammalian NF-B/Rel family: p50 (NF-B1), p52 (NF-B2), p65 (RelA), c-Rel, and RelB. The most abundant form of NF-B is a heterodimer of p50 and p65. In unstimulated cells, NF-B is sequestered in the cytoplasm in an inactive form through interaction with the IB inhibitory proteins (including IB␣, IB␤, and IBε, of which the best studied is IB␣). Upon stimulation of cells by specific inducers, such as the proinflammatory cytokine tumor necrosis factor SF2 (referred to as TNF hereinafter), IB␣ is phosphorylated on two specific serine residues by a large cytoplasmic IB kinase (IKK) complex that consists of the kinase catalytic subunits IKK␣ and IKK␤ and the regulatory subunit NEMO/ IKK␥ (reviewed in references 23 and 26). This phosphorylation marks IB␣ for polyubiquitination by the E3-SCF ␤-TrCP ubiquitin ligase complex, a specific ubiquitin ligase belonging to the SCF (i.e., Skp-1/Cul/Fbox) family, and for degradation by the 26S proteasome (reviewed in reference 5). Degradation of IB␣ allows a rapid and transient translocation of NF-B to the nucleus, where it activates transcription from a wide variety of promoters-including that of its own inhibitor, IB␣. The newly synthesized IB␣ enters the nucleus and removes NF-B from its DNA binding sites and transports it back to the cytoplasm, thereby terminating NF-B-dependent transcription (...

Inhibition of histone deacetylases prevents cytokine-induced toxicity in beta cells

“…However, the underlying mechanisms seem to depend on the cell type investigated and include on the one hand an HDAC-inhibitor-mediated continual degradation of de novo synthesised IκBα protein (possibly by prolonged activation of the upstream IκB-kinase) causing prolonged presence and DNA-binding of NFκB in the nucleus and enhancing NFκB-dependent transactivation, or on the other hand a reduction in proteasomal degradation of IκBα, preventing NFκB nuclear translocation and DNA binding [4,8,[46][47][48][49].…”

Identification of PP2A as a crucial regulator of the NF-κB feedback loop: its inhibition by UVB turns NF-κB into a pro-apoptotic factor

“…30 Enhanced proteasomal degradation of IkBa in our setting also appeared to be linked to signals upstream of its phosphorylation, because it could be counteracted by inhibition of IKKb. Correspondingly, resynthesized IkBa captured by proteasome inhibition after IL-1 þ UVB treatment displayed phosphorylation of Ser32/36 residues.…”

Deacetylase Inhibitors and the Viral Transactivator TaxBLV Synergistically Activate Bovine Leukemia Virus Gene Expression via a cAMP-responsive Element- and cAMP-responsive Element-binding Protein-dependent Mechanism

“…Our laboratory has previously demonstrated that histone hyperacetylation induces HIV-1 expression by specifically disrupting a single nucleosome positioned immediately downstream of the transcription start site (73)(74)(75). Moreover, HIV-1 transcription can be synergistically activated either by the viral transactivator Tat HIV-1 and HDACi through direct acetylation of Tat HIV-1 (50, 76 -78) or by NF-B and HDACi through a persistent degradation of the NF-B inhibitor I B-␣ (38,43).…”

“…In Vivo Acetylation Assays-In vivo acetylation assays were performed as described previously (43). In brief, COS-7 cells were transfected, using FuGENE-6 (Roche Molecular Biochemicals) according to the manufacturer's protocol, with expression vectors for WT-Tax BLV , Tax BLV -mut3K, HA-Tax BLV , bovine CREB2, or p53 (500 ng).…”