IkappaB Kinase Complex Research Articles

Abstract B022: The adaptor protein FRS2beta fashions a cytokine-rich inflammatory microenvironment that is associated with DCIS and promotes breast cancer carcinogenesis

Abstract Although it is commonly held that pro-inflammatory changes precede the onset of breast cancer, the underlying mechanisms remain obscure. Here, we report that FRS2beta, an adaptor protein with a highly restricted epithelial expression pattern, mediates the pro-inflammatory changes that alter the stroma in premalignant mammary tissues. We posit that these changes, which include lesions of ductal carcinoma in situ (DCIS), precede disease onset. In the MMTV-ErbB2 mouse model of mammary carcinoma, FRS2beta-deficiency markedly attenuated tumorigenesis. Importantly, tumor cells derived from MMTV-ErbB2 mice failed to generate tumors when grafted in the FRS2beta-deficient premalignant tissues. We found that the co-localization of FRS2beta and the NEMO subunit of the IkappaB kinase (IKK) complex in early endosomes activated nuclear factor-kappaB (NFkappaB), a master regulator of inflammation. Moreover, the inhibition of NFkappaB-induced cytokines CXC chemokine ligand (CXCL)12 and insulin-like growth factor (IGF1) abrogated tumorigenesis. In line with this, human breast cancer tissues that express higher levels of FRS2beta contain more stroma. The elucidation of the FRS2beta-NFkappaB axis uncovers a hitherto unknown molecular link between the pro-inflammatory changes in the DCIS and the disease onset. Citation Format: Noriko Gotoh. The adaptor protein FRS2beta fashions a cytokine-rich inflammatory microenvironment that is associated with DCIS and promotes breast cancer carcinogenesis [abstract]. In: Proceedings of the AACR Special Conference on Rethinking DCIS: An Opportunity for Prevention?; 2022 Sep 8-11; Philadelphia, PA. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_1): Abstract nr B022.

Mas-related G protein-coupled receptor D participates in inflammatory pain by promoting NF-κB activation through interaction with TAK1 and IKK complex

Mas-related G protein-coupled receptor D (MrgprD) is mainly expressed in small-diameter sensory neurons of the dorsal root ganglion (DRG). Results from previous studies suggest that MrgprD participates in mechanical hyperalgesia and nerve injury-induced neuropathic pain. However, it remains elusive whether and how MrgprD is involved in inflammatory pain. Here, we used a mouse model of chronic inflammatory pain established by intraperitoneal administration of lipopolysaccharide (LPS). The LPS injection induced an evident peripheral neuroinflammation and mechanical hyperalgesia in the mice and increased MrgprD expression in the DRG. The LPS administration also augmented the proportion of MrgprD-expressing neurons in the lumbar 4 DRG. Behaviorally, the LPS-induced hypersensitivities to mechanical and cold stimuli, but not to a heat stimulus, were substantially attenuated in Mrgprd-knockout mice compared with wildtype littermates. Mrgprd deletion in DRGs suppressed the LPS-triggered activation of the NF-κB signaling pathway and attenuated LPS-induced up-regulation of pro-inflammatory factors. Moreover, ectopic overexpression of MrgprD in HEK293 cells stably expressing mouse toll-like receptor 4 (TLR4) markedly promoted the LPS-induced NF-κB activation and enhanced NF-κB's DNA-binding activity. Furthermore, MrgprD physically interacted with TGF-β-activated kinase 1 (TAK1) and I-kappa-B-kinase (IKK) complexes, but not with mitogen-activated protein kinases (MAPKs) in mouse DRGs. In macrophage-like RAW 264.7 cells, MrgprD also interacted with TAK1 and IKK complex, and the treatment of MrgprD agonist elicited the activation of NF-κB signaling, but not of mitogen-activated protein kinases (MAPKs) signaling pathway. Our findings indicate that MrgprD facilitates the development of LPS-triggered persistent inflammatory hyperalgesia by promoting canonical NF-κB activation, highlighting the important roles of MrgprD in NF-κB-mediated inflammation and chronic pain.

Cytokine signalling in haematopoietic cells

Cytokine receptor signalling is essential for cell survival, proliferation and subsequent differentiation of haematopoietic stem cells (HSCs). Cytokines control development of haematopoietic progenitors into cells of the myeloid, lymphoid and erythroid lineages by stimulating cell cycle progression, proliferation and differentiation as well as by inhibiting apoptosis. My work focusses on Interleukin-3 (IL-3) and Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF), two essential cytokines in haematopoiesis. Binding of a cytokine to its specific receptor leads to the activation of multiple kinase signalling pathways, including the JAK/STAT, Ras-MAP kinase (MAPK) and PI3-kinase/AKT pathways. In this signalling network, the IkappaB Kinase (IKK) complex plays an important role as a downstream signalling hub. My research investigates how IL-3 mediated IKK activation promotes the survival of myeloid cells and what role this process may play in the development of related diseases, such as myeloproliferative disorders. Using immortalised growth factor (IL-3 or GM-CSF) dependent myeloid progenitor cells (FDMs), as well as employing various in vivo mouse models of haematopoietic development, I was able to show that 1) IKK is a major signalling hub linking IL-3, TNFR1 and p53 signalling to control the survival in haematopoietic cells by describing for the first time a role for the E3 ubiquitin ligase MDM2 downstream of IL-3- or TNFalpha-mediated IKK2 activation, suggesting crosstalk between the NF-kB signalling and p53 signalling pathways; 2) IKK regulates cellular metabolism through activation of NF-kB- and p53-dependent metabolic target genes by showing that deletion of IKK2 but not IKK1 in hematopoietic cells significantly alters cellular metabolism, impairing oxidative phosphorylation and upregulating glycolysis due to altered expression of p53-dependent metabolic target genes; 3) IKK plays a crucial role during haematopoietic development, regulating myeloid cell proliferation, lineage commitment and survival, showing that deletion of IKK2 but not IKK1 in haematopoietic progenitor cells severely affects haematopoietic development by skewing lineage commitment in vivo, resulting in neutrophilia, elevated circulating interleukin-6 and lethality due to severe gastrointestinal inflammation. The work presented in this thesis provides new important insights into the role of IKK in haematopoietic cells and haematopoietic development and clearly demonstrate that IKK1 and IKK2, the two catalytic subunits of the IKK complex, have distinct functions depending on the context of activation. In the future, this fact could be exploited to develop novel targeted therapies to specifically target a subunit in disease settings such as haematopoietic malignancies where aberrant NF-kappaB activity is frequently observed.

Hsa‐mir‐3199‐2 and hsa‐mir‐1293 as Novel Prognostic Biomarkers of Papillary Renal Cell Carcinoma by COX Ratio Risk Regression Model Screening

Papillary renal cell carcinoma(PRCC) is the second most common and aggressive renal cell carcinoma. Identification of novel microRNA biomarkers could be beneficial for the diagnosis and prognosis of PRCC patients. We aimed to screen differentially expressed miRNAs that can act as prognostic factors and to predict the survival of PRCC patients. High-throughput data of miRNAs of 274 PRCC samples were downloaded from TCGA (The Cancer Genome Atlas) dataset and interested miRNAs were identified. Hierarchical clustering and principal component analysis (PCA) were performed on these miRNAs. Critical genes that can act as prognostic factors were screened by LASSO. What's more, Kaplan-Meier survival analysis and ROC (Receiver Operating Characteristic) growth curve were used to testify the accuracy of the model. Biological processes of putative targets of miRNAs were analyzed by bioinformatics methods such as GO (Go Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis. A total of 105 differentially expressed miRNAs were screened out in PRCC samples compared with healthy controls. Two critical miRNAs, hsa-mir-3199-2, and hsa-mir-1293, were screened out by LASSO (Least Absolute Shrinkage and Selection Operator), including 197 and 189 target genes, respectively. Furthermore, its' accuracy was testified by ROC analysis with the AUC (Area under the curve) value of 0.7774968 and 0.6743466. These miRNAs were significantly enriched in pathways as platelet activating factor biosynthetic process, epithelial cell maturation, and IkappaB kinase complex. In conclusion, hsa-mir-3199-2 and hsa-mir-1293 that can act as prognostic biomarkers of PRCC were screened out, which can provide new insights for the clinical treatment of the disease. J. Cell. Biochem. 118: 3488-3494, 2017. © 2017 Wiley Periodicals, Inc.

Immunodeficiency and disseminated mycobacterial infection associated with homozygous nonsense mutation of IKKβ

A, Genealogical tree for the kindred. Arrow highlights index case. Asterisks denote family members who died within the first 2 months of life with febrile illnesses. B, Histology showing granuloma in skin by hematoxylin and eosin staining.

A20-mediated negative regulation of canonical NF-κB signaling pathway

The nuclear factor kappa B (NF-κB) plays vital role in the immune system by regulating innate and adaptive immunity, development and survival of lymphocytes, and lymphoid organogenesis. All known NF-κB activators converge on the IkappaB kinase (IKK) complex to activate the canonical and non-canonical NF-κB pathways. The IKK complex contains two catalytic subunits (IKKα and IKKβ) and a regulatory subunit NEMO/IKKγ that regulates the canonical NF-κB pathway, whereas IKKα regulates the non-canonical pathway. The process of IKKα activation and its role in the regulation of canonical NF-κB activation remain elusive. The canonical pathway is rapidly activated and produces a potent inflammatory response to bacterial and viral infections as well as different types of stress; however, uncontrolled NF-κB activation can lead to autoimmune diseases and cancers. Therefore, to keep the inflammatory response in check, elaborate negative regulatory mechanisms operate to terminate NF-κB activation at multiple levels by de novo synthesis of NF-κB inhibitory proteins, and orchestration of protein ubiquitination and deubiquitination. The NF-κB target genes, IκBα and A20, play critical roles in termination of the active canonical NF-κB pathway. In this review, we discuss our recent findings describing a novel function for IKKα in nucleating the ubiquitin-editing enzyme A20 complex, a major negative regulator of canonical NF-κB signaling. Consistently with an inhibitory function of IKKα, it is targeted by the human T-cell leukemia virus 1 (HTLV-1) oncoprotein, Tax, to prevent assembly of the A20 complex to maintain persistent NF-κB activation that promotes transformation and survival of virus-transformed cells.

A Purine Scaffold HSP90 Inhibitor BIIB021 Has Selective Activity against KSHV-Associated Primary Effusion Lymphoma and Blocks vFLIP K13-Induced NF-κB

Kaposi sarcoma-associated herpes virus (KSHV)-associated primary effusion lymphomas (PEL) have extremely poor prognosis when treated with conventional chemotherapy. KSHV-encoded viral FLICE-inhibitory protein (vFLIP) K13 binds to the IkappaB kinase (IKK) complex to constitutively activate the NF-κB pathway, which has been shown to be essential for the survival and proliferation of PEL cells. The molecular chaperone HSP90 is a component of the IKK complex and is required for its activity. We have analyzed the effect of HSP90 inhibitors on the survival and proliferation of PEL cells and on the activity of the NF-κB pathway. We show that BIIB021, a purine scaffold-based orally administrable HSP90 inhibitor, shows preferential cytotoxicity toward PEL cells as compared with non-PEL cells. The cytotoxic effect of BIIB021 against PEL was associated with induction of cell-cycle arrest and apoptosis. BIIB021 blocked the expression of a number of cellular proteins involved in the regulation of cell cycle and apoptosis. BIIB021 also blocked constitutive NF-κB activity present in PEL cells, in part, by blocking the interaction of vFLIP K13 with the IKK complex subunits. In a xenograft model of PEL, BIIB021 significantly reduced tumor growth. BIIB021 blocks constitutive NF-κB activity in PEL and shows preferential antitumor activity against PEL in vitro and in vivo. BIIB021 may be a promising agent for treatment of PEL.

Abstract 3945: IKK-Epsilon, a novel factor identified by proteomics, contributes to prostate cancer cell-induced osteoclast activity in vitro.

Abstract Prostate cancer preferentially metastasizes to skeleton resulting in osteoblastic lesions with underlying osteoclast activity. Novel strategies to inhibit the tumor skeletal metastasis involve targeting not only tumor cells but also bone microenvironment which is composed of osteoclasts, osteoblasts and stromal cells that include fibroblasts, immune and inflammatory cells, adipocytes, glial cells, smooth muscle cells, and resident and recruited vascular cells. The microenvironment can exert both stimulatory and inhibitory influences on proliferation and malignant properties of tumor cells. In this study, we intended to identify factors that may play key roles in this bidirectional and dynamic interaction between tumor cells and bone microenvironment. First, we fractionated the prostate cancer PC3 cell conditioned media by the molecular weight of proteins in media using centricon column. Then we applied the fractions to culturing osteoclast precursor RAW 264.7 cells in vitro. We repeated and scaled up the above steps to collect enough conditioned media, and concentrated the fractions of interest. The fractions that showed activity of osteoclast induction were determined by mass spectrometry. We found that the conditioned media contained a large amount of IKK-Epsilon, a subunit of PMA-inducible IKK (I-KappaB Kinase) complex. We further observed that recombinant IKK-Epsilon could induce osteoclast activity in vitro, but unexpectedly it had no effect on the activation of NF-kappa-B by TNF or IL-1 in these cells. Together with the findings that several identified factors in the conditioned media such as MCP-1 and IL-8 that induced osteoclast activity, in vitro validation of the identified factor IKK-Epsilon was confirmed by using neutralizing antibody on the functional assays. We have not yet identified unknown factors in the current study, however it is clear that unknown factors in the conditioned media may also play key roles in the tumor-induced osteoclast activation since blocking MCP-1, IL-8, and IKK-Epsilon did not completely block the tumor-induced osteoclast activity. We conclude that IKK-Epsilon, a novel factor identified by proteomics, may contribute to prostate cancer cell-induced osteoclast activity in vitro. Supported by NSF key project 81130046; NSF projects 81171993 and 81272415; Guangxi projects 201201ZD004 and 2012GXNSFCB53004. Citation Format: Xin Yang, Zhen Xiao, Jianhua Wang, Yi Lu, Yang Xu, Xin Huang, Atsushi Mizokami, Evan T. Keller, Jian Zhang. IKK-Epsilon, a novel factor identified by proteomics, contributes to prostate cancer cell-induced osteoclast activity in vitro. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3945. doi:10.1158/1538-7445.AM2013-3945

Abstract 1510: Blockade of the mTOR/NF-κB signaling axis augments prostate cancer treatment responses through suppression of a microenvironment-derived DNA damage secretory program

Abstract Background/Rationale: Resistance to cytotoxic chemo- and radiotherapy is a common event in advanced prostate cancers. While resistance is established as a consequence of cancer cell intrinsic genetic and epigenetic changes, emerging evidence indicates that therapy failure is also caused by cell extrinsic factors such as cytokines, growth factors, proteases and other soluble ligands generated from damaged stromal cells in the tumor microenvironment (TMEN). We recently reported a unique feature of stromal cells, namely a DNA damage secretory phenotype (DDSP) resulting from genotoxic therapeutics. Thus, anti-cancer therapies that cause DNA damage responses (DDR), unexpectedly, trigger pro-survival signaling in neoplastic or pre-neoplastic cells and may promote subsequent therapy resistance. Results: In this study, we show that molecular inhibition of the mammalian target of rapamycin (mTOR) through genetic or chemical approaches suppresses a cellular secretory program resulting from exposure to genotoxic agents. We identified a physical association between mTOR and the I-kappa-B kinase (IKK) complex which is activated by DNA damage stimuli, and found IKKα to be the major subunit that directly interacts with mTOR while assembling the IKK complex. Rapamycin abrogates IKKα phosphorylation, IαBβ degradation and the central action-p65/p50 nuclear translocation. Thus, IKK catalytic subunits are not equally implicated in propagating damage signals to NF-κB, with IKKα playing the pivotal role. We determined that the mTOR/Raptor complex is activated by genotoxic stress and promotes NF-κB signaling. Suppressing mTOR dramatically abolished transcriptional levels of NF-κB target genes. We also determined that paracrine-acting DDSP factors are regulated through both transcriptional and post-transcriptional mechanisms, as upregulation of S6K and downregulation of 4E-BP1 play essential and synergistical roles in synthesis of DDSP factors. Finally, we determined that mTORC1 is the predominant mediator of the development of the DDSP phenotype, and it receives signals from upstream kinases. Interestingly, PI3K is not required for signal transduction of damage responses in prostate fibroblasts, which occurs through Akt phosphorylation caused by activated p38 MAPK signaling. Conclusions: These studies establish that mTOR is a novel target for therapeutic intervention in the mitigation of resistance mechanisms derived from the tumor microenvironment following genotoxic cancer treatments. These studies suggest that mTOR inhibitors may have efficacy in augmenting tumor responses to ionizing radiation and/or chemotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1510. doi:1538-7445.AM2012-1510

The Cytoplasmic Domain of the HIV-1 Glycoprotein gp41 Induces NF-κB Activation through TGF-β-Activated Kinase 1

The human and simian immunodeficiency viruses (HIV and SIV) primarily infect lymphocytes, which must be activated for efficient viral replication. We show that the cytoplasmic domain of the transmembrane glycoprotein gp41 (gp41CD) of both HIV-1 and SIV induces activation of NF-κB, a cellular factor important for proviral genome transcription and lymphocyte activation. This NF-κB activating property localized to a region 12-25 (SIV) or 59-70 (HIV-1) residues from the gp41 membrane-spanning domain. An siRNA-based screen of 42 key NF-κB regulators revealed that gp41CD-mediated activation occurs through the canonical NF-κB pathway via TGF-β-activated kinase 1 (TAK1). TAK1 activity was required for gp41CD-mediated NF-κB activation, and HIV-1-derived gp41CD physically interacted with TAK1 through the same region required for NF-κB activation. Importantly, an NF-κB activation-deficient HIV-1 mutant exhibited increased dependence on cellular activation for replication. These findings demonstrate an evolutionarily conserved role for gp41CD in activating NF-κB to promote infection.

HTLV Tax: A Fascinating Multifunctional Co-Regulator of Viral and Cellular Pathways

Human T-cell lymphotropic virus type 1 (HTLV-1) has been identified as the causative agent of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The virus infects between 15 and 20 million people worldwide of which approximately 2–5% develop ATL. The past 35 years of research have yielded significant insight into the pathogenesis of HTLV-1, including the molecular characterization of Tax, the viral transactivator, and oncoprotein. In spite of these efforts, the mechanisms of oncogenesis of this pleiotropic protein remain to be fully elucidated. In this review, we illustrate the multiple oncogenic roles of Tax by summarizing a recent body of literature that refines our understanding of cellular transformation. A focused range of topics are discussed in this review including Tax-mediated regulation of the viral promoter and other cellular pathways, particularly the connection of the NF-κB pathway to both post-translational modifications (PTMs) of Tax and subcellular localization. Specifically, recent research on polyubiquitination of Tax as it relates to the activation of the IkappaB kinase (IKK) complex is highlighted. Regulation of the cell cycle and DNA damage responses due to Tax are also discussed, including Tax interaction with minichromosome maintenance proteins and the role of Tax in chromatin remodeling. The recent identification of HTLV-3 has amplified the importance of the characterization of emerging viral pathogens. The challenge of the molecular determination of pathogenicity and malignant disease of this virus lies in the comparison of the viral transactivators of HTLV-1, -2, and -3 in terms of transformation and immortalization. Consequently, differences between the three proteins are currently being studied to determine what factors are required for the differences in tumorogenesis.

Ubiquitin ligase CHIP induces TRAF2 proteasomal degradation and NF-κB inactivation to regulate breast cancer cell invasion

Transcriptional factor nuclear factor-kappaB (NF-κB) plays a crucial role in human breast cancer cell invasion and metastasis. The carboxyl terminus of Hsc70-interacting protein (CHIP) is a U-box-type ubiquitin ligase that induces ubiquitination and proteasomal degradation of its substrate proteins. In this study, we investigated the role of CHIP in the NF-κB pathway in the invasion of MDA-MB-231 cells, a highly aggressive breast cancer cell line. We showed that overexpression of CHIP significantly inhibits the invasion of the MDA-MB-231 cells. The overexpression of CHIP suppressed expression of urokinase plasminogen activator (uPA) and matrix metalloproteinase-9 (MMP-9) in MDA-MB-231 cells. Moreover, CHIP strongly inhibited the nuclear localization and the transcriptional activity of NF-κB. The activation of the IkappaB kinase complex (IKK) was also blocked by CHIP overexpression. Importantly, CHIP overexpression resulted in a significant decrease in the level of TNF receptor-associated factor 2 (TRAF2), an upstream key player in the NF-κB pathway. However, the level of TRAF2 was restored after treatment with a proteasome inhibitor, MG-132. Moreover, CHIP overexpression promoted the ubiquitination of TRAF2. We also found cell invasion significantly decreased in cells transfected with TRAF2 small interfering RNA (siRNA). In contrast, when CHIP expression was suppressed by siRNA in poorly invasive MCF-7 cells, cell invasion significantly increased in conjunction with enhanced NF-κB activation and TRAF2 levels. Taken together, these results suggest that CHIP regulates NF-κB-mediated cell invasion via the down-regulation of TRAF2.

Specific correction of a splice defect in brain by nutritional supplementation

Recent studies emphasize the importance of mRNA splicing in human genetic disease, as 20-30% of all disease-causing mutations are predicted to result in mRNA splicing defects. The plasticity of the mRNA splicing reaction has made these mutations attractive candidates for the development of therapeutics. Familial dysautonomia (FD) is a severe neurodegenerative disorder, and all patients have an intronic IVS20+6T>C splice site mutation in the IKBKAP gene, which results in tissue-specific skipping of exon 20 and a corresponding reduction in ikappaB kinase complex associated protein (IKAP) levels. We created transgenic mouse lines using a human IKBKAP bacterial artificial chromosome (BAC) into which we inserted the IKBKAP splice mutation (FD BAC) and have shown that the transgenic mice exhibit the same tissue-specific aberrant splicing patterns as seen in FD patients. We have previously demonstrated that the plant cytokinin kinetin can significantly improve the production of wild-type IKBKAP transcripts in FD lymphoblast cell lines by improving exon inclusion. In this study, we tested the ability of kinetin to alter IKBKAP splicing in the transgenic mice carrying the FD BAC and show that it corrects IKBKAP splicing in all major tissues assayed, including the brain. The amount of wild-type IKBKAP mRNA and IKAP protein was significantly higher in the kinetin-treated mice. These exciting results prove that treatment of FD, as well as other mechanistically related splicing disorders, with kinetin holds great promise as a potential therapeutic aimed at increasing normal protein levels, which may, in turn, slow disease progression.

Screening of substrate peptide sequences for tissue-type transglutaminase (TGase 2) using T7 phage cDNA library

Transglutaminase (TGase) is a family of enzymes that catalyzes cross-linking reaction between glutamine- and lysine residue of substrate proteins in several mammalian biological events. Substrate proteins for TGase and their physiological relevance have been still in research, continuously expanding. In this study, we have established a novel screening system that enables identification of cDNA sequence encoding favorable primary structure as a substrate for tissue-type transglutaminase (TGase 2), a multifunctional and ubiquitously expressing isozyme. By the screening, we identified several T7 phage clones that displayed substrate peptides for TGase 2 as a translated product from human brain cDNA library. Among the selected clones, the C-terminal region of IKAP, IkappaB kinase complex associated protein, appeared as a highly reactive substrate sequence for TGase 2. This system will open possibility of rapid identification of substrate sequences for transglutaminases at a genetic level.

Cutting edge: association with I kappa B kinase beta regulates the subcellular localization of Homer3.

The signaling and adaptor protein Homer3 plays a role in controlling immune homeostasis and self-reactivity. Homer3 is recruited to the immune synapse (IS) following TCR ligation, although the mechanisms regulating this subcellular localization are unknown. We show that Homer3 specifically associates with a novel ubiquitin-like domain in the IkappaB kinase (IKK) beta subunit of the IKK complex. Homer3 associates with IKKbeta in T cells and colocalizes with the IKK complex at the IS. However, Homer3 is not required for IKK activation, as NF-kappaB signaling is intact in Homer3-deficient T cells. Instead, the IKK complex recruits Homer3 to the IS following TCR engagement, and we present evidence that this association regulates actin dynamics in T cells. These findings identify a novel interaction between two major signaling proteins and reveal an unexpected NF-kappaB-independent function for the IKK complex in regulating the subcellular localization of Homer3.

Nuclear IKKβ Is an Adaptor Protein for IκBα Ubiquitination and Degradation in UV-Induced NF-κB Activation

Proinflammatory cytokines activate NF-kappaB using the IkappaB kinase (IKK) complex that phosphorylates inhibitory proteins (IkappaBs) at N-terminal sites resulting in their ubiquitination and degradation in the cytoplasm. Although ultraviolet (UV) irradiation does not lead to IKK activity, it activates NF-kappaB by an unknown mechanism through IkappaBalpha degradation without N-terminal phosphorylation. Here, we describe an adaptor function of nuclear IKKbeta in UV-induced IkappaBalpha degradation. UV irradiation induces the nuclear translocation of IkappaBalpha and association with IKKbeta, which constitutively interacts with beta-TrCP through heterogeneous ribonucleoprotein-U (hnRNP-U) leading to IkappaBalpha ubiquitination and degradation. Furthermore, casein kinase 2 (CK2) and p38 associate with IKKbeta and promote IkappaBalpha degradation by phosphorylation at C-terminal sites. Thus, nuclear IKKbeta acts as an adaptor protein for IkappaBalpha degradation in UV-induced NF-kappaB activation. NF-kappaB activated by the nuclear IKKbeta adaptor protein suppresses anti-apoptotic gene expression and promotes UV-induced cell death.

CC2D1A, a DM14 and C2 Domain Protein, Activates NF-κB through the Canonical Pathway

CC2D1A is an evolutionarily conserved protein that contains four DM14 domains at the N terminus and a C2 domain at the C terminus. Loss-of-function mutations in CC2D1A have been linked to mental retardation in human, but the biochemical function of this protein is largely unknown. Here, we show that CC2D1A is a potent activator of NF-κB. The activation of NF-κB by CC2D1A requires its C2 domain. CC2D1A activates NF-κB in a manner that depends on the ubiquitin-conjugating enzyme Ubc13, TNF receptor-associated factor TRAF2, the protein kinase TAK1, and the IκB kinase (IKK) complex. In addition, the deubiquitination enzyme Cylindromatosis (CYLD) negatively regulates the activity of CC2D1A. These results suggest that CC2D1A activates NF-κB through the canonical IKK pathway.

IKKβ Leads to an Inflammatory Skin Disease Resembling Interface Dermatitis

IKKbeta is a subunit of the IkappaB kinase (IKK) complex required for NF-kappaB activation in response to pro-inflammatory signals. NF-kappaB regulates the expression of many genes involved in inflammation, immunity, and apoptosis, and also controls cell proliferation and differentiation in different tissues; however, its function in skin physiopathology remains controversial. In this study we report the alterations caused by increased IKKbeta activity in skin basal cells of transgenic mice. These animals suffered chronic inflammation with abundant macrophages and other CD45(+) infiltrating cells in the skin, which resulted in epidermal basal cell injury and degeneration of hair follicles. They showed histological features characteristic of interface dermatitis (ID). This phenotype is accompanied by an increased production of inflammatory cytokines by transgenic keratinocytes. Accordingly, transcriptome studies show upregulation of genes associated with inflammatory responses. The inflammatory phenotype observed as a consequence of IKKbeta overexpression is independent of T and B lymphocytes, as it also arises in mice lacking these cell types. In summary, our data indicate the importance of IKKbeta in the development of ID and in the homeostasis of stratified epithelia. Our results also support the idea that IKKbeta might be a valid therapeutic target for the treatment of skin inflammatory diseases.

Novel Insights into the Cellular Mechanisms of the Anti-inflammatory Effects of NF-κB Essential Modulator Binding Domain Peptides

The classical nuclear factor κB (NF-κB) signaling pathway is under the control of the IκB kinase (IKK) complex, which consists of IKK-1, IKK-2, and NF-κB essential modulator (NEMO). This complex is responsible for the regulation of cell proliferation, survival, and differentiation. Dysregulation of this pathway is associated with several human diseases, and as such, its inhibition offers an exciting opportunity for therapeutic intervention. NEMO binding domain (NBD) peptides inhibit the binding of recombinant NEMO to IKK-2 in vitro. However, direct evidence of disruption of this binding by NBD peptides in biological systems has not been provided. Using a cell system, we expanded on previous observations to show that NBD peptides inhibit inflammation-induced but not basal cytokine production. We report that these peptides cause the release of IKK-2 from an IKK complex and disrupt NEMO-IKK-2 interactions in cells. We demonstrate that by interfering with NEMO-IKK-2 interactions, NBD peptides inhibit IKK-2 phosphorylation, without affecting signaling intermediates upstream of the IKK complex of the NF-κB pathway. Furthermore, in a cell-free system of IKK complex activation by TRAF6 (TNF receptor-associated factor 6), we show that these peptides inhibit the ability of this complex to phosphorylate downstream substrates, such as p65 and inhibitor of κBα (IκBα). Thus, consistent with the notion that NEMO regulates IKK-2 catalytic activity by serving as a scaffold, appropriately positioning IKK-2 for activation by upstream kinase(s), our findings provide novel insights into the molecular mechanisms by which NBD peptides exert their anti-inflammatory effects in cells.

Natural products as targeted modulators of the nuclear factor-kappaB pathway.

The use of plant extracts to alleviate inflammatory diseases is centuries old and continues to this day. This review assesses the current understanding of the use of such plants and natural products isolated from them in terms of their action against the ubiquitous transcription factor, nuclear factor kappa B (NF-kappaB). As an activator of many pro-inflammatory cytokines and inflammatory processes the modulation of the NF-kappaB transduction pathway is a principal target to alleviate the symptoms of such diseases as arthritis, inflammatory bowel disease and asthma. Two pathways of NF-kappaB activation will first be summarised, leading to the IKK (IkappaB kinase) complex, that subsequently initiates phosphorylation of the NF-kappaB inhibitory protein (IKB). Natural products and some extracts are reviewed and assessed for their activity and potency as NF-kappaB inhibitors. A large number of compounds are currently known as NF-kappaB modulators and include the isoprenoids, most notably kaurene diterpenoids and members of the sesquiterpene lactones class, several phenolics including curcumin and flavonoids such as silybin. Additional data on cellular toxicity are also highlighted as an exclusion principle for pursuing such compounds in clinical development. In addition, where enough data exists some conclusions on structure-activity relationship are provided.