Kaori Asamitsu

Inhibition of nuclear factor-κB-mediated transcription by association with the amino-terminal enhancer of split, a groucho-related protein lacking WD40 repeats.

The amino-terminal enhancer of split (AES) encodes a 197-amino acid protein that is homologous to the NH2-terminal domain of the DrosophilaGroucho protein but lacks COOH-terminal WD40 repeats. Although theDrosophila Groucho protein and its mammalian homologs, transducin-like enhancer of split proteins, are known to act as non-DNA binding corepressors, the role of the AES protein remains unclarified. Using the yeast two-hybrid system, we have identified the protein-protein interaction between AES and the p65 (RelA) subunit of the transcription factor nuclear factor κB (NF-κB), which activates various target genes involved in inflammation, apoptosis, and embryonic development. The interaction between AES and p65 was confirmed byin vitro glutathione S-transferase pull down assay and by in vivo co-immunoprecipitation study. In transient transfection assays, AES repressed p65-driven gene expression. AES also inhibited NF-κB-dependent gene expression induced by tumor necrosis factor α, interleukin-1β, and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 1, which is an upstream kinase for NF-κB activation. These data indicate that AES acts as a corepressor for NF-κB and suggest that AES may play a pivotal role in the regulation of NF-κB target genes.

NF-κB Signaling and Carcinogenesis

NF-κB is an inducible transcription factor that is controlled by the signal activation cascades. NF-κB controls a number of genes involved in immuno-inflammatory responses, cell cycle progression, inhibition of apoptosis and cell adhesion, thus promoting carcinogenesis and cancer progression. Interestingly, some proteins encoded by oncogenes and oncogenic viruses have been shown to be involved in NF-κB activation pathway. In fact, NF-κB is constitutively activated in some cancer and leukemia cells. These findings have substantiated the old concept of the link between chronic inflammation and carcinogenesis. In this review, we have attempted to overview the possible involvement of NF-κB in cancer and discuss the feasibility of anti-cancer strategy with NF-κB and its signaling cascade as novel molecular targets.

Growth Inhibition of Multiple Myeloma Cells by a Novel IκB Kinase Inhibitor

Involvement of nuclear factor-κB (NF-κB) in cell survival and proliferation of multiple myeloma has been well established. In this study we observed that NF-κB is constitutively activated in all human myeloma cell lines, thus confirming the previous studies. In addition, we found the phosphorylation of p65 subunit of NF-κB in addition to the phosphorylation of IκBα and the activation of NF-κB DNA binding and that various target genes of NF-κB including bcl-xL, XIAP, c-IAP1, cyclin D1, and IL-6 are up-regulated. We then examined the effect of a novel IκB kinase inhibitor, 2-amino-6-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-4-piperidin-4-yl nicotinonitrile (ACHP). When myeloma cells were treated with ACHP, the cell growth was efficiently inhibited with IC50 values ranging from 18 to 35 μmol/L concomitantly with inhibition of the phosphorylation of IκBα/p65 and NF-κB DNA-binding, down-regulation of the NF-κB target genes, and induction of apoptosis. In addition, we observed the treatment of ACHP augmented the cytotoxic effects of vincristine and melphalan (l-phenylalanine mustard), conventional antimyeloma drugs. These findings indicate that IκB kinase inhibitors such as ACHP can sensitize myeloma cells to the cytotoxic effects of chemotherapeutic agents by blocking the antiapoptotic nature of myeloma cells endowed by the constitutive activation of NF-κB.

RelA-Associated Inhibitor Blocks Transcription of Human Immunodeficiency Virus Type 1 by Inhibiting NF-κB and Sp1 Actions

ABSTRACT RelA-associated inhibitor (RAI) is an inhibitor of nuclear factor κB (NF-κB) newly identified by yeast two-hybrid screen as an interacting protein of the p65 (RelA) subunit. In this study, we attempted to examine the effect of RAI on transcription and replication of human immunodeficiency virus type 1 (HIV-1). We found that RAI inhibited gene expression from the HIV-1 long terminal repeat (LTR) even at the basal level. Upon in vitro DNA-binding reactions, RAI could directly block the DNA-binding of p65 subunit of NF-κB but not that of the p50 subunit or AP1. We found that RAI could also inhibit the DNA-binding of Sp1 and thus inhibit the basal HIV-1 promoter activity. We further examined the effects of RAI on Sp1 and found that RAI colocalizes with Sp1 in the nucleus and interacts with Sp1 in vitro and in vivo. Moreover, we found that RAI efficiently blocked the HIV-1 replication when cotransfected with a full-length HIV-1 clone. These findings indicate that RAI acts as an efficient inhibitor of HIV-1 gene expression in which both NF-κB and Sp1 play major roles.

AKIP1 Enhances NF-κB-dependent Gene Expression by Promoting the Nuclear Retention and Phosphorylation of p65

In this study, we have identified protein kinase A-interacting protein 1 (AKIP1) as a binding partner of NF-κB p65 subunit, and AKIP1 enhances the NF-κB-mediated gene expression. AKIP1 is a nuclear protein and known to interact with the catalytic subunit of PKA (PKAc). We identified AKIP1 by a yeast two-hybrid screen using the N terminus region of p65 as bait. The interaction between AKIP1 and p65 was confirmed by glutathione S-transferase pull-down assay in vitro and immunoprecipitation-Western blotting assay in vivo. We found that the PKAc was present in the AKIP1·p65 complex and enhanced the transcriptional activity of NF-κB by phosphorylating p65. In a transient luciferase assay, AKIP1 cotransfection efficiently increased the transcriptional activity of NF-κB induced by phorbol 12-myristate 13-acetate (PMA). When AKIP1 was knocked down by RNA interference, the PMA-mediated NF-κB-dependent gene expression was abolished, indicating a physiological role of AKIP1. We found that PKAc, which is maintained in an inactive form by binding to IκBα and NF-κB in resting cells, was activated by PMA-induced signaling and could phosphorylate p65. Overexpression of AKIP1 increased the PKAc binding to p65 and enhanced the PKAc-mediated phosphorylation of p65 at Ser-276. Interestingly, this p65 phosphorylation promoted nuclear translocation of p65 and enhanced NF-κB transcription. In fact, we observed that AKIP1 colocalized with p65 within the cells and appeared to retain p65 in nucleus. These findings indicate a positive role of AKIP1 in NF-κB signaling and suggest a novel mechanism by which AKIP1 augments the transcriptional competence of NF-κB.

Identification of Highly Selective and Potent Histone Deacetylase 3 Inhibitors Using Click Chemistry-Based Combinatorial Fragment Assembly

To find histone deacetylase 3 (HDAC3)-selective inhibitors, a series of 504 candidates was assembled using “click chemistry”, by reacting nine alkynes bearing a zinc-binding group with 56 azide building blocks in the presence of Cu(I) catalyst. Screening of the 504-member triazole library against HDAC3 and other HDAC isozymes led to the identification of potent and selective HDAC3 inhibitors T247 and T326. These compounds showed potent HDAC3 inhibition with submicromolar IC50s, whereas they did not strongly inhibit other isozymes. Compounds T247 and T326 also induced a dose-dependent selective increase of NF-κB acetylation in human colon cancer HCT116 cells, indicating selective inhibition of HDAC3 in the cells. In addition, these HDAC3-selective inhibitors induced growth inhibition of cancer cells, and activated HIV gene expression in latent HIV-infected cells. These findings indicate that HDAC3-selective inhibitors are promising candidates for anticancer drugs and antiviral agents. This work also suggests the usefulness of the click chemistry approach to find isozyme-selective HDAC inhibitors.

A-kinase-interacting Protein 1 (AKIP1) Acts as a Molecular Determinant of PKA in NF-κB Signaling

The cAMP-dependent protein kinase (PKA) signaling pathway plays a crucial role in the pathogenesis of many NF-κB-related diseases. However, there have been controversial reports with regard to the PKA actions in the regulation of NF-κB activity. In this study, we have demonstrated the effect of PKA on NF-κB activity in view of AKIP1 action; and in 293 and HeLa cells, where the endogenous AKIP1 expression is minimal, PKA-activating agents inhibited the NF-κB-dependent reporter gene expression, blocked the interaction of PKAc and p65 subunit of NF-κB, and attenuated PKA-dependent phosphorylation of p65 on Ser-276. This inhibitory function of PKAc in NF-κB signaling was reversed by overexpression of AKIP1 in 293 cells. In the breast cancer cell line, MDA-MB231 cells and MCF7 cells, where the endogenous AKIP1 is abundant, the PKA signal was found to be synergized with NF-κB activation; PKA-activating agents enhanced NF-κB-dependent transcriptional activity and the interaction between p65 and PKAc and augmented the phosphorylation of p65 on Ser-276. After RNAi knockdown of AKIP1 in these breast cancer cells, we observed that PKA-activating agents antagonized NF-κB-dependent activation. Meanwhile, PKA inhibitor suppressed NF-κB-induced breast cancer cell proliferation and multiple NF-κB-dependent anti-apoptotic gene expression. It is likely that expression of AKIP1 determines the relationship between these two signal transduction pathways. These findings explained controversial results from various independent groups regarding the action of PKA signaling on the NF-κB activation cascade and suggested a possible therapeutic potential of PKA inhibitor in developing anti-cancer strategies.

A-kinase interacting protein 1 (AKIP1) acts as a molecular determinant of the role of PKA in NF-κB signaling

The cAMP-dependent protein kinase (PKA) signaling pathway plays a crucial role in the pathogenesis of many NF-κB-related diseases. However, there have been controversial reports with regard to the PKA actions in the regulation of NF-κB activity. In this study, we have demonstrated the effect of PKA on NF-κB activity in view of AKIP1 action; and in 293 and HeLa cells, where the endogenous AKIP1 expression is minimal, PKA-activating agents inhibited the NF-κB-dependent reporter gene expression, blocked the interaction of PKAc and p65 subunit of NF-κB, and attenuated PKA-dependent phosphorylation of p65 on Ser-276. This inhibitory function of PKAc in NF-κB signaling was reversed by overexpression of AKIP1 in 293 cells. In the breast cancer cell line, MDA-MB231 cells and MCF7 cells, where the endogenous AKIP1 is abundant, the PKA signal was found to be synergized with NF-κB activation; PKA-activating agents enhanced NF-κB-dependent transcriptional activity and the interaction between p65 and PKAc and augmented the phosphorylation of p65 on Ser-276. After RNAi knockdown of AKIP1 in these breast cancer cells, we observed that PKA-activating agents antagonized NF-κB-dependent activation. Meanwhile, PKA inhibitor suppressed NF-κB-induced breast cancer cell proliferation and multiple NF-κB-dependent anti-apoptotic gene expression. It is likely that expression of AKIP1 determines the relationship between these two signal transduction pathways. These findings explained controversial results from various independent groups regarding the action of PKA signaling on the NF-κB activation cascade and suggested a possible therapeutic potential of PKA inhibitor in developing anti-cancer strategies.

Inhibition of Human Immunodeficiency Virus Type 1 Replication in Latently Infected Cells by a Novel IκB Kinase Inhibitor

ABSTRACT In human immunodeficiency virus type 1 (HIV-1) latently infected cells, NF-κB plays a major role in the transcriptional induction of HIV-1 replication. Hence, downregulation of NF-κB activation has long been sought for effective anti-HIV therapy. Tumor necrosis factor alpha (TNF-α) stimulates IκB kinase (IKK) complex, a critical regulator in the NF-κB signaling pathway. A novel IKK inhibitor, ACHP {2-amino-6-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-4-piperidin-4-yl-nicotinonitrile}, was developed and evaluated as a potent and specific inhibitor for IKK-α and IKK-β. In this study, we examined the ability of this compound to inhibit HIV-1 replication in OM10.1 cells latently infected with HIV. When these cells were pretreated with ACHP, TNF-α-induced HIV-1 replication was dramatically inhibited, as measured by the HIV p24 antigen levels in the culture supernatants. Its 50% effective concentration was approximately 0.56 μM, whereas its 50% cytotoxic concentration was about 15 μM. Western blot analysis revealed inhibition of IκBα phosphorylation, IκBα degradation, p65 nuclear translocation, and p65 phosphorylation. ACHP was also found to suppress HIV-1 long terminal repeat (LTR)-driven gene expression through the inhibition of NF-κB activation. Furthermore, ACHP inhibited TNF-α-induced NF-κB (p65) recruitment to the HIV-1 LTR, as assessed by chromatin immunoprecipitation assay. These findings suggest that ACHP acts as a potent suppressor of TNF-α-induced HIV replication in latently infected cells and that this inhibition is mediated through suppression of IKK activity.

Novel histone deacetylase inhibitor NCH-51 activates latent HIV-1 gene expression.

Pharmacological manipulations to purge human immunodeficiency virus (HIV) from latent reservoirs have been considered as an adjuvant therapeutic approach to highly‐active antiretroviral therapy for the eradication of HIV. Our novel histone deacetylase inhibitor NCH‐51 induced expression of latent HIV‐1 with minimal cytotoxicity. Using chromatin immunoprecipitation assays, we observed a reduction of HDAC1 occupancy, histone hyperacetylation and the recruitment of positive transcription factors at the HIV‐1 promoter in latently infected‐cells under the treatment with NCH‐51. Mutation studies of the long terminal repeat (LTR) revealed NCH‐51 mediated gene expression through the Sp1 sites. When Sp1 expression was knocked‐down by small interfering RNA, the NCH‐51‐mediated activation of a stably integrated HIV‐1 LTR was attenuated. Moreover, the Sp1 inhibitor mithramycin A abolished the effects of NCH‐51.