IKK Complex Research Articles

NF-κB signalling is attenuated by the E7 protein from cutaneous human papillomaviruses

The high-risk Alpha-types of human papillomavirus (HPV) are the causative agent of cervical cancer, which is the second major cause of death among women worldwide. Recent investigations have shown that E7 from the Alpha-papillomavirus HPV-16 interacts with IKKα and IKKβ of the IKK complex in the NF-κB pathway leading to an attenuation of the activity. There is a possible link between development of non-melanoma skin cancer and cutaneous Beta-papillomavirus but if these HPV types attenuate the NF-κB pathway is unclear. Seven different E7 proteins, representing four out of the five different species of the Beta genus (HPV-20, -37, -38, -92, -93 and -96) and one from the Gamma genus (HPV-4) were investigated for potential modulation of the NF-κB pathway in U2OS cells. Our results demonstrate that E7 from all the cutaneous HPV types were capable of inhibiting the NF-κB activity as well as E7 from HPV-16. In addition, E7 proteins from the cutaneous HPV types demonstrated interaction with IKKα but not with IKKβ. The deregulation of the NF-κB pathway by cutaneous HPVs might contribute to the pathogenesis of non-melanoma skin cancers and its precursors.

Requirement of NEMO/IKKγ for effective expansion of KRAS-induced precancerous lesions in the pancreas

Pancreatic carcinoma, a leading cause of cancer death, is thought to develop out of pancreatic intraepithelial neoplasia (PanIN). PanIN lesions have not yet attained the fully malignant phenotype, but show increased proliferation and dysplasia, and frequently bear an oncogenic KRAS mutation. Pancreatic cancer development is associated with increased activity of the transcription factor NF-κB. NEMO (IKKγ) is a subunit of the IKK complex essential for the activation of canonical NF-κB signaling and has been ascribed both oncogenic and tumor-suppressive roles in gastrointestinal tumors. Here, we wanted to address the function of NEMO in pancreatic tumorigenesis. We therefore conditionally ablated NEMO in a mouse model for pancreatic carcinoma based on the expression of oncogenic KRAS in pancreatic precursor cells. Mice were analyzed for PanIN lesions and for the activation of associated signaling pathways. NEMO ablation in the pancreas, while in itself not causing any overt pathology, led to a drastic (>93%) decrease in the prevalence of both low-grade and high-grade PanIN in 10-month-old mice expressing oncogenic KRAS. Also, the inflammatory and fibrotic response associated with KRAS action in the pancreas was virtually abolished, including expression of inflammatory cytokines and activation of the interleukin-6/STAT3 axis. Moreover, the activation of MAPK signaling, Notch and KLF4 signaling normally observed in KRAS-induced PanIN was strongly reduced or absent when NEMO was ablated. Our study suggests that NEMO, an IKK subunit necessary for canonical NF-κB activation, is dispensable for normal pancreatic development and function, but essential for the propagation of KRAS-induced PanIN lesions.

Analysis of Nuclear Factor-κB (NF-κB) Essential Modulator (NEMO) Binding to Linear and Lysine-linked Ubiquitin Chains and Its Role in the Activation of NF-κB

Nuclear factor-κB (NF-κB) essential modulator (NEMO), a component of the inhibitor of κB kinase (IKK) complex, controls NF-κB signaling by binding to ubiquitin chains. Structural studies of NEMO provided a rationale for the specific binding between the UBAN (ubiquitin binding in ABIN and NEMO) domain of NEMO and linear (Met-1-linked) di-ubiquitin chains. Full-length NEMO can also interact with Lys-11-, Lys-48-, and Lys-63-linked ubiquitin chains of varying length in cells. Here, we show that purified full-length NEMO binds preferentially to linear ubiquitin chains in competition with lysine-linked ubiquitin chains of defined length, including long Lys-63-linked deca-ubiquitins. Linear di-ubiquitins were sufficient to activate both the IKK complex in vitro and to trigger maximal NF-κB activation in cells. In TNFα-stimulated cells, NEMO chimeras engineered to bind exclusively to Lys-63-linked ubiquitin chains mediated partial NF-κB activation compared with cells expressing NEMO that binds to linear ubiquitin chains. We propose that NEMO functions as a high affinity receptor for linear ubiquitin chains and a low affinity receptor for long lysine-linked ubiquitin chains. This phenomenon could explain quantitatively distinct NF-κB activation patterns in response to numerous cell stimuli.

Heterochromatin protein 1 gamma and IκB kinase alpha interdependence during tumour necrosis factor gene transcription elongation in activated macrophages

IκB kinase α (IKKα) is part of the cytoplasmic IKK complex regulating nuclear factor-{kappa}B (NF-κB) release and translocation into the nucleus in response to pro-inflammatory signals. IKKα can also be recruited directly to the promoter of NF-κB-dependent genes by NF-κB where it phosphorylates histone H3 at serine 10, triggering recruitment of the bromodomain-containing protein 4 and the positive transcription elongation factor b. Herein, we report that IKKα travels with the elongating form of ribonucleic acid polymerase II together with heterochromatin protein 1 gamma (HP1γ) at NF-κB-dependent genes in activated macrophages. IKKα binds to and phosphorylates HP1γ, which in turn controls IKKα binding to chromatin and phosphorylation of the histone variant H3.3 at serine 31 within transcribing regions. Downstream of transcription end sites, IKKα accumulates with its inhibitor the CUE-domain containing protein 2, suggesting a link between IKKα inactivation and transcription termination.

Kaposi’s Sarcoma Associated Herpesvirus Encoded Viral FLICE Inhibitory Protein K13 Activates NF-κB Pathway Independent of TRAF6, TAK1 and LUBAC

BackgroundKaposi’s sarcoma associated herpesvirus encoded viral FLICE inhibitory protein (vFLIP) K13 activates the NF-κB pathway by binding to the NEMO/IKKγ subunit of the IκB kinase (IKK) complex. However, it has remained enigmatic how K13-NEMO interaction results in the activation of the IKK complex. Recent studies have implicated TRAF6, TAK1 and linear ubiquitin chains assembled by a linear ubiquitin chain assembly complex (LUBAC) consisting of HOIL-1, HOIP and SHARPIN in IKK activation by proinflammatory cytokines.Methodology/Principal FindingsHere we demonstrate that K13-induced NF-κB DNA binding and transcriptional activities are not impaired in cells derived from mice with targeted disruption of TRAF6, TAK1 and HOIL-1 genes and in cells derived from mice with chronic proliferative dermatitis (cpdm), which have mutation in the Sharpin gene (Sharpincpdm/cpdm). Furthermore, reconstitution of NEMO-deficient murine embryonic fibroblast cells with NEMO mutants that are incapable of binding to linear ubiquitin chains supported K13-induced NF-κB activity. K13-induced NF-κB activity was not blocked by CYLD, a deubiquitylating enzyme that can cleave linear and Lys63-linked ubiquitin chains. On the other hand, NEMO was required for interaction of K13 with IKK1/IKKα and IKK2/IKKβ, which resulted in their activation by “T Loop” phosphorylation.Conclusions/SignificanceOur results demonstrate that K13 activates the NF-κB pathway by binding to NEMO which results in the recruitment of IKK1/IKKα and IKK2/IKKβ and their subsequent activation by phosphorylation. Thus, K13 activates NF-κB via a mechanism distinct from that utilized by inflammatory cytokines. These results have important implications for the development of therapeutic agents targeting K13-induced NF-κB for the treatment of KSHV-associated malignancies.

Inhibition of Type I Interferon-Mediated Antiviral Action in Human Glioma Cells by the IKK Inhibitors BMS-345541 and TPCA-1

The nuclear factor-kappa B (NFκB) signal transduction pathway plays an important role in immunity, inflammation, cell growth, and survival. Since dysregulation of this pathway results in high, constitutive NFκB activation in various cancers and immune disorders, the development of specific drugs to target this pathway has become a focus for treating these diseases. NFκB regulates various aspects of the cellular response to interferon (IFN). However, the role of the upstream regulator of the NFκB signaling pathway, the inhibitor of κB kinase (IKK) complex, on IFN function has not been examined. In the present study, we examined the effects of 2 IKK inhibitors, N-(1,8-Dimethylimidazo[1,2-a]quinoxalin-4-yl)-1,2-ethanediamine hydrochloride (BMS-345541) and 2-[(aminocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide (TPCA-1), on IFN action in several human glioma cell lines. IKK inhibitors inhibit glioma cell proliferation, as well as TNF-induced RelA (p65) nuclear translocation and NFκB-dependent IL8 gene expression. Importantly, BMS-345541 and TPCA-1 differentially inhibit IFN-induced gene expression, completely suppressing MX1 and GBP1 gene expression, while having only a minor effect on ISG15 expression. Furthermore, these IKK inhibitors displayed marked differences in blocking IFN-induced antiviral action against cytopathic effects and replication of vesicular stomatitis virus (VSV) and encephalomyocarditis virus (EMCV). Our results show that the IKK complex plays an important function in IFN-induced gene expression and antiviral activity. Since VSV and EMCV are oncolytic viruses used in cancer therapy, our results indicate the potential synergy in combining IKK inhibitors with oncolytic viruses.

Abstract 4829: A purine scaffold Hsp90 inhibitor has antitumor activity in KSHV-associated malignancies by suppressing vFLIP

Abstract Background: Kaposi's sarcoma herpesvirus (KSHV/ HHV-8), a member of the α-herpesvirus family of human DNA viruses, is the etiologic agent of several malignancies in immune-compromised individuals, such as Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). Activated NF-κB is a critical mechanism by which KSHV-infected PEL cells are protected from apoptosis. KSHV vFLIP has been identified as a viral oncogene that is responsible for NF-κB-dependent anti-apoptotic gene expression in PEL cells, and also prevents autophagy in KSHV-infected endothelial cells. In particular, the IKK signaling complex consisting of IKKα, IKKα, IKKα, vFLIP and Hsp90 was shown to be essential for survival in PEL cells. The chaperone protein Hsp90 binds client proteins involved in the regulation of cell survival and apoptosis signal transduction, including Akt, IKK complex, and KSHV vFLIP. A lack of Hsp90 causes protein misfolding, ubiquitination and degradation. The Hsp90 inhibitor geldanamycin was previously tested in PEL cells and shown to inhibit activity of the IKK complex in vitro. However, geldanamycin is of limited therapeutic potential due to its undesirable pharmacophysiology. We tested a new purine-scaffold Hsp90 inhibitor with high selectivity for tumor versus normal cell Hsp90, which is water-soluble with high oral bioavailability and excellent therapeutic window. Materials and methods: We evaluated the sensitivity of several KSHV infected and uninfected cell lines to treatment with this inhibitor, designated PU-H71. We performed viability and NF-κB reporter luciferase assays, and immunoblot and qRT-PCR analyses of cellular and viral proteins. Finally, we used a mouse PEL xenograft model and in vivo imaging to assess tumor responses to PU-H71. Results: We found all KSHV-positive PEL cell lines to be exquisitely sensitive when compared to uninfected lymphoma cells, with LC50s in the nanomolar range. PU-H71 was shown to induce PEL cell death by apoptosis and autophagy within 48 hours of treatment. PU-H71 also showed preferential toxicity in an in vitro model of KS. Pulldown demonstrated association of the compound with the active vFLIP-IKKα signalosome. Western blot analysis indicated that the IKK signaling complex was destabilized, and components vFLIP and IKKα were degraded upon PU-H71 treatment, resulting in inhibition of NF-κB signaling, as confirmed by reporter luciferase assay. qRT-PCR and reporter analysis indicated low levels of concomitant lytic reactivation. PU-H71 was further shown to inhibit progression of tumor spread and confer a significant survival advantage (p<0.02) in a mouse xenograft model of PEL, with persistence of IKKα destabilization ex vivo. Conclusions: Our findings demonstrate that Hsp90 inhibition with PU-H71 leads to reduced vFLIP levels in KSHV-infected cells, and to tumor responses. Thus, PU-H71 is a promising targeted approach for the treatment of KS and PEL. 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 4829. doi:1538-7445.AM2012-4829

Abstract 110: Role of CARD 10 mediated NF-kB activation in colorectal carcinoma cells

Abstract Colorectal carcinoma is the fourth most common cancer in men and the third in women worldwide, accounting for 8% of all cancer deaths. NF-κB gene plays a role in tumorgenesis through the transcriptional activation of genes associated with cell proliferation, angiogenesis, metastasis, tumor promotion, inflammation and suppression of apoptosis. NF-κB protein activation, defined as nuclear expression of NF-κB by immunohistochemistry was an independent prognostic marker for poor survival. CARD10 gene is known positive regulator of the NF-κB activation pathway and can activate the NF-κB activity. CARD10 is an enzyme that is encoded by the CARD10 gene and it associates with IKK complex resulting in NF-κB activation. Considering the potential therapeutic utility of targeting NF-κB and its key modulators, we previously studied CARD10 genetic alterations and immunohistochemical expression of CARD10 in a large cohort cases1. CARD10 protein expression associated significantly with NF-κB activation, and CARD10 amplification was significantly associated with CARD10 protein expression and presence of distance metastasis. To extend and confirm our clinical finding, we performed in vitro studies using a panel of CRC cell lines to investigate the biological role of CARD10 alterations in CRC carcinogenesis. Our data showed that TNF-α treatment caused efficient translocation of p56 in CARD 10 muatation harboring cell line -DLD-1 cells to nucleus as compared to HCT-15 cells. Furthermore, the luciferase activity was significantly higher in CARD 10 amplified HCT-116 cells as compared to DLD1 and HCT-15 cells which carry normal CRAD10 gene copy number after 48 hours of transfection with an NF-κB reporter. In addition, it was also observed that the formation of colonies was significantly higher in HCT-116 as compared to DLD-1 and HCT-15. Finally CARD 10 siRNA transfection knocked down the expression of CARD10 and abrogated the expression of NF-κB, and resulted in apoptosis through activation of caspase-3. These data suggest that CARD10 mutation and amplification mediated NFκB activation play an important role in cellular transformation of CRC cells. Thus, the study raises the possibility that inhibition of CARD10 might have significant therapeutic value in CRC. Reference: 1: Jehad Abubaker, Prashant Bavi, Zeenath Jehan, Maqbool Ahmed, Wael Al-Haqawi, Mehar Sultana, Sarita Prabhakaran, Nasser Al-Sanea, Alaa Abduljabbar, Luai H Ashari, Samar Alhomoud, Fouad Al-Dayel, Shahab Uddin, Khawla S. Al-Kuraya. Clinicopathological significance of CARD10 alterations in Middle Eastern colorectal cancer. 102nd AACR Annual Meeting, April2-6, 2011, Orlando, Florida. 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 110. doi:1538-7445.AM2012-110

Delphinidin blocks IL-1β-induced activation of NF-κB by modulating the IKKβ gene expression and the activation of NIK in human chondrocytes

Purpose: Osteoarthritis (OA) is a highly prevalent disease that affects the quality of life of its victims. Interleukin-1 β (IL-1β) plays a major role in the pathogenesis of OA and is present in the arthritic joints at high levels. IL-1β has been shown to cause increased production of catabolic enzymes such as MMPs, and inflammatory mediators such as prostaglandins and nitric oxide through the activation of several signaling pathways including NF-κB. Novel approaches using plant derived polyphenols to inhibit signal transduction pathways have shown promise and could overcome some of the toxicity issues related to current therapies. One key signaling mechanism implicated in OA that could be a target of orally bioavailable inhibitors is the NF-κB pathway. Delphinidin (2-(3,4,5-trihydroxyphenyl)chromenylium-3,5,7-triol) is an anthocyanidin that is widely distributed in pigmented fruits and flowers and has been shown to possess anti-inflammatory activity and block the activation of NF-κB in cancer cells. No such study with human chondrocytes has been reported. In the present study using primary human chondrocytes we determined (a) the chondroprotective effect of Delphinidin; and (b) mechanism of NF-κB inhibition in IL-1β-stimulated human chondrocytes. Methods: Chondrocytes were derived by enzymatic digestion of human cartilage (OA chondrocytes) from patients undergoing total knee arthroplasty. OA chondrocytes were pretreated with Delphinidin for different time points and were then stimulated with IL-1β (10ng/ml) in vitro and total RNA or cell lysate was prepared following our standard protocols. To determine the gene expression, single stranded cDNA was synthesized and the expression of target mRNAs (COX-2, MMP-13, iNOS) was quantified using TaqMan Assays. HeLa cells were transfected with promoter reporter vectors and negative control vectors using the Fugene-6 reagent and the effect on promoter activity was verified by Luciferase assay. Activation of NF-κB p65 was determined by specific ELISA-based DNA binding assay. Total protein levels were determined by detergent compatible Biorad Protein Assay. Nitric oxide production was determined by Griess reaction. Protein expression and the phosphorylated forms of different kinases were determined by Western immunoblotting. Data were analyzed using Origin 6.1 software package and p<0.05 was considered significant. Results: Pretreatment of chondrocytes with Delphinidin (50 μg/ml) for 2 hrs significantly inhibited the IL-1β induced expression of COX-2, iNOS and MMP-13 and the production of nitric oxide. Delphinidin also inhibited the phosphorylation of IKKβ and was also a potent suppressor of IKKβ gene and protein expression in OA chondrocytes. IKK promoter had a strong activity in HeLa cells and this activity and production of the Luciferase reporter enzyme was blocked by pre-treatment with Delphinidin. Functional pathway analyses showed that IL-1β-induced phosphorylation of NF-κB-inducing Kinase (NIK), which interacts with and activates IKKα and IKKβ which then phosphorylate IκB, was inhibited by pretreatment of OA chondrocytes with Delphinidin. In agreement with these findings, pre-treatment with Delphinidin inhibited the phosphorylation and degradation of IκB, as determined by Western immunoblotting, and the activation and the DNA binding activity of NF-κB p65 determined by a highly specific ELISA based assay. Conclusions: Taken together, the data presented here identifies a novel mechanism of NF-κB inhibition by Delphinidin by inhibiting NIK, a kinase upstream of IKK complex in the NF-κB activation pathway. Given the important role played by IL-1β and NF-κB in OA, these results may provide important clues to develop useful pharmacological inhibitors of NF-κB for the prevention and/or treatment of OA.

Thiocolchicoside suppresses osteoclastogenesis induced by RANKL and cancer cells through inhibition of inflammatory pathways: a new use for an old drug

Most patients with cancer die not because of the tumour in the primary site, but because it has spread to other sites. Common tumours, such as breast, multiple myeloma, and prostate tumours, frequently metastasize to the bone. To search for an inhibitor of cancer-induced bone loss, we investigated the effect of thiocolchicoside, a semi-synthetic colchicoside derived from the plant Gloriosa superba and clinically used as a muscle relaxant, on osteoclastogenesis induced by receptor activator of NF-κB ligand (RANKL) and tumour cells. We used RAW 264.7 (murine macrophage) cells, a well-established system for osteoclastogenesis, and evaluated the effect of thiocolchicoside on RANKL-induced NF-κB signalling and osteoclastogenesis as well as on osteoclastogenesis induced by tumour cells. Thiocolchicoside suppressed osteoclastogenesis induced by RANKL, and by breast cancer and multiple myeloma cells. Inhibition of the NF-κB pathway was responsible for this effect since the colchicoside inhibited RANKL-induced NF-κB activation, activation of IκB kinase (IKK) and suppressed inhibitor of NF-κBα (IκBα) phosphorylation and degradation, an inhibitor of NF-κB. Furthermore, an inhibitor of the IκBα kinase γ or NF-κB essential modulator, the regulatory component of the IKK complex, demonstrated that the NF-κB signalling pathway is mandatory for osteoclastogenesis induced by RANKL. Together, these data suggest that thiocolchicoside significantly suppressed osteoclastogenesis induced by RANKL and tumour cells via the NF-κB signalling pathway. Thus, thiocolchicoside, a drug that has been used for almost half a century to treat muscle pain, may also be considered as a new treatment for bone loss.

Ubiquitination in signaling to and activation of IKK

A role for polyubiquitination in the activation of inhibitor of NF-κB (IκB) kinase (IKK) through a proteasome-independent mechanism was first reported in 1996, but the physiological significance of this finding was not clear until 2000 when TRAF6 was found to be a ubiquitin E3 ligase that catalyzes lysine-63 (K63) polyubiquitination. Since then, several proteins known to regulate IKK have been linked to the ubiquitin pathway. These include the deubiquitination enzymes CYLD and A20 that inhibit IKK, and the ubiquitin binding proteins NEMO and TAB2 which are the regulatory subunits of IKK and TAK1 kinase complexes, respectively. Now accumulating evidence strongly supports a central role of K63 polyubiquitination in IKK activation by multiple immune and inflammatory pathways. Interestingly, recent research suggests that some alternative ubiquitin chains such as linear or K11 ubiquitin chains may also play a role in certain pathways such as the TNF pathway. Here I present a historical narrative of the discovery of the role of ubiquitin in IKK activation, review recent advances in understanding the role and mechanism of ubiquitin-mediated IKK activation, and raise some questions to be resolved in future research.

IKK biology

Summary: The inhibitor of nuclear factor‐κB (IκB) kinase (IKK) complex is the master regulator of the NF‐κB signaling pathway. The activation of the IKK complex is a tightly regulated, highly stimulus‐specific, and target‐specific event that is essential for the plethora of functions attributed to NF‐κB. More recently, NF‐κB‐independent roles of IKK members have brought increased complexity to its biological function. This review highlights some of the major advances in the studies of the process of IKK activation and the biological roles of IKK family members, with a focus on NF‐κB‐independent functions. Understanding these complex processes is essential for targeting IKK for therapeutics.

The role of mRNA degradation in immunity and inflammation

The innate immune system is an evolutionally conserved host defense mechanism against pathogens. Innate immune responses are initiated by pattern recognition receptors (PRRs), which recognize specific structures of microorganisms. Among them, Toll-like receptors (TLRs) are capable of sensing organisms ranging from bacteria to fungi, protozoa and viruses, and play a major role in innate immunity. Individual TLRs recognize different microbial components, and give rise to different patterns in gene expression. We are now focusing on the role of genes induced in response to TLR stimulation, particularly the genes that are rapidly induced in a MyD88-dependent manner within 30 min after LPS stimulation. Among them, we have recently identified a novel gene named Zc3h12a which has a CCCH-type zinc finger domain. The knockout mice developed spontaneous autoimmune diseases accompanied by splenomegaly and lymphadenopathy. Subsequent studies showed that Zc3h12a is a nuclease involved in destabilization of IL-6 and IL-12mRNA. We renamed it Regulatory RNase-1 (Regnase-1) based on the function. We recently found that the IKK complex controls Il6 mRNA stability by phosphorylating Regnase-1 in response to IL-1R/TLR stimulation. Phosphorylated Regnase-1 underwent ubiquitination and degradation. Regnase-1 re-expressed in IL-1R/TLR-activated cells exhibited delayed kinetics, and Regnase-1 mRNA was found to be negatively regulated by Regnase-1 itself via a stem-loop region present in the Regnase-1 3' untranslated region. These data demonstrate that the IKK complex phosphorylates not only IkBalpha, activating transcription, but also Regnase-1, releasing the brake on Il6 mRNA expression.

Calpastatin upregulation in Mycoplasma hyorhinis-infected cells is promoted by the mycoplasma lipoproteins via the NF-κB pathway

Mycoplasma hyorhinis frequently contaminates cultured cells, with effects on synthetic and metabolic pathways. We demonstrated for the first time that contamination of cells by a strain of M. hyorhinis (NDMh) results in increased levels of calpastatin (the endogenous inhibitor of the ubiquitous Ca(2+) -dependent protease calpain). We now show that the calpastatin upregulation by NDMh in neuroblastoma SH-SY5Y cells resides in the NDMh lipoprotein fraction (LPP), via the NF-κB transcription pathway. NF-κB activation requires dissociation of the cytoplasmic NF-κB/IκB complex followed by NF-κB translocation to the nucleus. NDMh-LPP induced translocation of the NF-κB RelA subunit to the nucleus and upregulated calpastatin. RelA translocation and calpastatin elevation were prevented when dissociation of the NF-κB/IκB complex was inhibited either by transfection with the non-phosphorylatable IκB mutant ΔNIκBα, or by using PS1145, an inhibitor of the IκB kinase (IKK complex). Increased calpastatin levels attenuate calpain-related amyloid-β-peptide and Ca(2+) -toxicity (these are central to the pathogenesis of Alzheimer's Disease). LPP-induced elevation of calpastatin provides an example of effects on non-inflammatory intracellular proteins, the outcome being significant alterations in host cell functions. Since calpastatin level is important in the control of calpain activity, mycoplasmal LPP may be of interest in treating some pathological processes involving excessive calpain activation.

Differential usage of NF-κB activating signals by IL-1β and TNF-α in pancreatic beta cells

The cytokines interleukin (IL)-1β and tumor necrosis factor (TNF)-α induce β-cell death in type 1 diabetes via NF-κB activation. IL-1β induces a more marked NF-κB activation than TNF-α, with higher expression of genes involved in β-cell dysfunction and death. We show here a differential usage of the IKK complex by IL-1β and TNF-α in β-cells. While TNF-α uses IKK complexes containing both IKKα and IKKβ, IL-1β induces complexes with IKKα only; this effect is achieved by induction of IKKβ degradation via the proteasome. Both IKKγ and activation of the TRAF6-TAK1-JNK pathway are involved in IL-1β-induced IKKβ degradation.

Viral Mediated Redirection of NEMO/IKKγ to Autophagosomes Curtails the Inflammatory Cascade

The early host response to viral infections involves transient activation of pattern recognition receptors leading to an induction of inflammatory cytokines such as interleukin-1β (IL-1β) and tumor necrosis factor α (TNFα). Subsequent activation of cytokine receptors in an autocrine and paracrine manner results in an inflammatory cascade. The precise mechanisms by which viruses avert an inflammatory cascade are incompletely understood. Nuclear factor (NF)-κB is a central regulator of the inflammatory signaling cascade that is controlled by inhibitor of NF-κB (IκB) proteins and the IκB kinase (IKK) complex. In this study we show that murine cytomegalovirus inhibits the inflammatory cascade by blocking Toll-like receptor (TLR) and IL-1 receptor-dependent NF-κB activation. Inhibition occurs through an interaction of the viral M45 protein with the NF-κB essential modulator (NEMO), the regulatory subunit of the IKK complex. M45 induces proteasome-independent degradation of NEMO by targeting NEMO to autophagosomes for subsequent degradation in lysosomes. We propose that the selective and irreversible degradation of a central regulatory protein by autophagy represents a new viral strategy to dampen the inflammatory response.

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.

Dangkwisoo-san, an herbal medicinal formula, ameliorates acute lung inflammation via activation of Nrf2 and suppression of NF-κB

Ethnopharmacological relevanceDangkwisoo-san (DS), an herbal medicinal formula, has long been used in Korea for the treatment of inflammatory complications caused by physical trauma. Although the therapeutic effect of DS is likely associated with anti-inflammatory activity, the precise underlying mechanisms are largely unknown. Here we sought to elucidate the possible mechanisms of anti-inflammatory activity of DS. Materials and methodsThe water extract of DS was orally fed to C57BL/6 mice for 14 days prior to LPS intranasal instillation for lung inflammation. The effects of DS on lung inflammation were determined by differential cell counting, lung histology, and semi-quantitative RT-PCR of lung sections. The effects of DS on the activities of Nrf2 and NF-κB were assessed by western blotting, semi-quantitative RT-PCR, and luciferase reporter assays in RAW 264.7, an NF-κB reporter cell line, and HEK 293 transfected with an NF-κB reporter construct. ResultsMice that were treated with a water extract of DS showed significant attenuation of lung inflammation induced by intranasal lipopolysaccharide (LPS) compared to control mice treated with vehicle. In vitro experiments show that DS activated Nrf2, an anti-oxidant transcription factor that protects from various inflammatory diseases, and induced Nrf2-regulated genes including GCLC, NQO-1 and HO-1. In addition, DS suppressed NF-κB activity and reduced the production of pro-inflammatory cytokines. Transfection experiment indicates that inhibition of NF-κB likely occurred upstream of IKK complex. Furthermore, DS enhanced the expression of HO-1 and suppressed that of IL-1β and TNF-α in inflamed mouse lungs. ConclusionsThese results suggest that the therapeutic effects of DS are related with suppression of inflammation, which is, at least in part, mediated by activation of anti-inflammatory factor Nrf2 and inhibition of pro-inflammatory factor NF-κB.

3-Formylchromone Interacts with Cysteine 38 in p65 Protein and with Cysteine 179 in IκBα Kinase, Leading to Down-regulation of Nuclear Factor-κB (NF-κB)-regulated Gene Products and Sensitization of Tumor Cells

3-Formylchromone (3-FC) has been associated with anticancer potential through a mechanism yet to be elucidated. Because of the critical role of NF-κB in tumorigenesis, we investigated the effect of this agent on the NF-κB activation pathway. Whether activated by inflammatory agents (such as TNF-α and endotoxin) or tumor promoters (such as phorbol ester and okadaic acid), 3-FC suppressed NF-κB activation. It also inhibited constitutive NF-κB expressed by most tumor cells. This activity correlated with sequential inhibition of IκBα kinase (IKK) activation, IκBα phosphorylation, IκBα degradation, p65 phosphorylation, p65 nuclear translocation, and reporter gene expression. We found that 3-FC inhibited the direct binding of p65 to DNA, and this binding was reversed by a reducing agent, thus suggesting a role for the cysteine residue. Furthermore, mutation of Cys38 to Ser in p65 abolished this effect of the chromone. This result was confirmed by a docking study. 3-FC also inhibited IKK activation directly, and the reducing agent reversed this inhibition. Furthermore, mutation of Cys179 to Ala in IKK abolished the effect of the chromone. Suppression of NF-κB activation led to inhibition of anti-apoptotic (Bcl-2, Bcl-xL, survivin, and cIAP-1), proliferative (cyclin D1 and COX-2), invasive (MMP-9 and ICAM-1), and angiogenic (VEGF) gene products and sensitization of tumor cells to cytokines. Thus, this study shows that modification of cysteine residues in IKK and p65 by 3-FC leads to inhibition of the NF-κB activation pathway, suppression of anti-apoptotic gene products, and potentiation of apoptosis in tumor cells.

Context-Dependent Regulation of Autophagy by IKK-NF-κB Signaling: Impact on the Aging Process

The NF-κB signaling system and the autophagic degradation pathway are crucial cellular survival mechanisms, both being well conserved during evolution. Emerging studies have indicated that the IKK/NF-κB signaling axis regulates autophagy in a context-dependent manner. IKK complex and NF-κB can enhance the expression of Beclin 1 and other autophagy-related proteins and stimulate autophagy whereas as a feedback response, autophagy can degrade IKK components. Moreover, NF-κB signaling activates the expression of autophagy inhibitors (e.g., A20 and Bcl-2/xL) and represses the activators of autophagy (BNIP3, JNK1, and ROS). Several studies have indicated that NF-κB signaling is enhanced both during aging and cellular senescence, inducing a proinflammatory phenotype. The aging process is also associated with a decline in autophagic degradation. It seems that the activity of Beclin 1 initiation complex could be impaired with aging, since the expression of Beclin 1 decreases as does the activity of type III PI3K. On the other hand, the expression of inhibitory Bcl-2/xL proteins increases with aging. We will review the recent literature on the control mechanisms of autophagy through IKK/NF-κB signaling and emphasize that NF-κB signaling could be a potent repressor of autophagy with ageing.