NEMO-binding Domain Research Articles

Role of NF-κB activation in LPS-induced endothelial barrier breakdown

Endothelial barrier breakdown contributes to organ failure in sepsis. The key mechanism by which the potent sepsis inductor lipopolysaccharide (LPS) disrupts the endothelial barrier is controversial. Here, we tested the hypothesis that NF-κB activation is critically involved in endothelial barrier breakdown. Application of LPS to monolayers of porcine pulmonary artery endothelial cells (PAEC) and human dermal microvascular endothelial cells (HDMEC) induced a rapid and sustained activation of NF-κB as revealed by translocation of its subunit p65 into the nuclei in nuclear extraction assays and by immunostaining. Measurements of transendothelial electrical resistance (TER) and intercellular gap formation demonstrated significant breakdown of endothelial barrier properties following LPS treatment for 3h. Interestingly, monolayers recovered spontaneously beginning after 10h. Increased cAMP prevented LPS-induced loss of endothelial barrier properties, but did not block NF-κB activation. Application of the cell-permeable NEMO-binding domain (NBD) synthetic peptide was effective to prevent NF-κB activation, but did neither block LPS-induced loss of TER nor intercellular gap formation. NBD peptide alone did not alter endothelial barrier properties, but enhanced the barrier-compromising effects when applied in combination with LPS. Similarly, siRNA-mediated knock-down of p65 in HDMECs did not prevent LPS-induced barrier breakdown. Known targets of NF-κB-derived protein expression of caveolin or vasodilator-stimulated phosphoprotein (VASP) remained unaltered by LPS treatment of endothelial cells. In summary, our data indicate that NF-κB activation by LPS is not critically involved in disruption of endothelial barrier properties. Rather, our data suggest that NF-κB activation acts as a part of a rescue mechanism.

Testing NF-κB-based Therapy in Hemiparkinsonian Monkeys

Parkinson's disease (PD) is the most common human neurodegenerative disorder affecting movement, balance, flexibility, and coordination. Despite intense investigation, no effective therapy is available to stop the onset PD or halt its progression. The primate model of PD is considered to be one of the best available models for human PD. Since neuroinflammation plays an important role in the pathogenesis of PD and NF-κB, a proinflammatory transcription factor, participates in the transcription of many proinflammatory molecules, this study evaluates the ability of a peptide corresponding to the NF-κB essential modifier (NEMO)-binding domain (NBD) of IκB kinase (IKK)α or IKKβ to protect dopaminergic neurons in hemiparkinsonian monkeys. First, we found that NF-κB was activated within the substantia nigra pars compacta of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxicated hemiparkinsonian monkeys. However, intramuscular injection of wild type NBD (wtNBD) peptide reduced nigral activation of NF-κB and expression of inducible nitric oxide synthase, protected both the nigrostriatal axis and neurotransmitters, and improved motor functions in hemiparkinsonian monkeys. These findings were specific as mutated NBD peptide did not exhibit such effects. These results may help in the translation of NF-κB-based therapy to PD clinics.

Enhanced targeted anticancer effects and inhibition of tumor metastasis by the TMTP1 compound peptide TMTP1-TAT-NBD

Micromolecular agents that block tumor development and metastasis hold great promise as cancer-targeted therapies. Tumor molecular targeted peptide 1 (TMTP1) was previously shown to target primary tumors and metastatic foci specifically. In this study, a group of composite peptides were incorporated to TMPT1. The NF-κB essential modulator-binding domain (NBD), and the trans-activator of transcription (TAT) peptide, were synthesized to enhance the targeted anti-tumor effects of TMTP1. TMTP1-NBD did not exhibit strong affinity to tumor cells as we had expected. Conjugating TAT with TMTP1-NBD ameliorated the poor hydrophilicity and negative charge of TMTP1-NBD. Therefore TMTP1-TAT-NBD displayed strong affinity and anti-tumor effects as we expected in vivo and in vitro. Interestingly cytoplasmic glycogen accumulation as well as apoptosis was observed in TMTP1-TAT-NBD treated PC-3M-1E8 cells. The downstream signaling pathways including AKT, GSK-3β, IκBα and NF-κB activity were verified to decrease by TMTP1-TAT-NBD. The pharmacokinetics and distribution of TMTP1-TAT-NBD in MDA-MB-231 tumor-bearing mice model provided some evidence for safety of the composite peptide, which showed the fluorescence of the peptide peaked in the tumor 6h after injection, with little fluorescence detected in normal organs except for very weak fluorescence in kidney. In conclusion, TMTP1-TAT-NBD may be a promising targeted anti-tumor agent for primary tumor and metastatic foci, which enhances the anticancer effects through inhibiting the AKT/GSK-3β/NF-κB pathway comparing with TMTP1.

Colon-Targeted Cell-Permeable NFκB Inhibitory Peptide Is Orally Active against Experimental Colitis

For the purpose of development of orally active peptide therapeutics targeting NFκB for treatment of inflammatory bowel disease (IBD), two major barriers in oral delivery of therapeutic peptides, metabolic lability and tissue impermeability, were circumvented by introduction of a colon-targeted delivery system and cell permeable peptides (CPP) to NFκB inhibitory peptides (NIP). Suppression of NFκB activation was compared following treatment with various CPP conjugated NIPs (CPP-NIP). The most potent CPP-NIP was loaded in a capsule coated with a colon specific polymer, which was administered orally to colitic rats. The anti-inflammatory activity of the colon-targeted CPP-NIP was evaluated by measuring inflammatory indices in the inflamed colonic tissue. For confirmation of the local action of the CPP-NIP, the same experiment was done after rectal administration. Tissue permeability of the CPP-NIP was examined microscopically and spectrophotometrically using FITC-labeled CPP-NIP (CPP-NIP-FITC). NEMO binding domain peptide (NBD, TALDWSWLQTE) fused with a cell permeable peptide CTP (YGRRARRRARR), CTP-NBD, was most potent in inhibiting NFκB activity in cells. Colon-targeted CTP-NBD, but not colon-targeted NBD and CTP-NBD in an enteric capsule, ameliorated the colonic injury, which was in parallel with decrease in MPO activity and the levels of inflammatory mediators. Intracolonic treatment with CTP-NBD alleviated rat colitis and improved all the inflammatory indicators. CTP-NBD-FITC was detected at much greater level in the inflamed tissue than was NBD-FITC. Taken together, introduction of cell permeability and colon targetability to NIP may be a feasible strategy for an orally active peptide therapy for treatment of IBD.

Selective inhibition of nuclear factor‐κB by nuclear factor‐κB essential modulator‐binding domain peptide suppresses the metastasis of highly metastatic oral squamous cell carcinoma

Nuclear factor-κB (NF-κB) activation contributes to the development of metastasis, thus leading to a poor prognosis in many cancers, including OSCC. However, little in vivo experimental data are available about the effects of NF-κB inhibition on OSCC metastasis. OSCC sublines were established from a GFP-expressing parental cell line, GSAS, and designated GSAS/N3 and N5 according to the in vivo passage number after cervical lymph node metastasis by a serial orthotopic transplantation model. In vitro migration and invasion were assessed in these cells, and the NF-κB activities and expression of NF-κB-regulated metastasis-related molecules were also examined. In in vivo experiments, the metastasis and survival of tumor-engrafted mice were monitored. Furthermore, the effects of a selective NF-κB inhibitor, NEMO-binding domain (NBD) peptide, on metastasis in GSAS/N5-engrafted mice were assessed, and engrafted tongue tumors were immunohistochemically examined. Highly metastatic GSAS/N3 and N5 cells showed an enhanced NF-κB activity, thus contributing to increased migration, invasion, and a poor prognosis compared with the parent cells. Furthermore, the expression levels of NF-κB-regulated metastasis-related molecules, such as fibronectin, β1 integrin, MMP-1, -2, -9, and -14, and VEGF-C, were upregulated in the highly metastatic cells. The NBD peptide suppressed metastasis and tongue tumor growth in GSAS/N5-inoculated mice, and was accompanied by the downregulation of the NF-κB-regulated metastasis-related molecules in engrafted tongue tumors. Our results suggest that the selective inhibition of NF-κB activation by NBD peptide may provide an effective approach for the treatment of highly metastatic OSCC.

NBD Peptides Protect Against Ischemia Reperfusion After Orthotopic Liver Transplantation in Rats

NBD (NEMO binding domain) peptides could selectively inhibit the inflammation induced NF-κB activity, while sparing the protective functions of basal NF-κB activity. The aim of this study was to determine whether NBD peptides inhibited the transcriptional activity of nuclear factor-κB (NF-κB) during liver transplant ischemia-reperfusion injury (IRI), without affecting its basal function. Sprague Dawley (SD) rats were performed orthotropic liver transplantation according to the Kamada technique. Donors were given NBD peptides (8 mg/kg, intraperitoneal) 2 h before surgery (n = 24) and the controls were treated with the same volume of physiologic saline (n = 24). An additional 16 animals in normal condition (did not undergo any surgery) were also divided into two groups and given the same treatment as above to assess the effect of NBD peptides on basal function. We analyzed levels of alanine aminotransferase (ALT), tumor necrosis factor (TNF-α), IKK (IκB kinase) complex phosphorylation, IκBα degradation, NF-κB transcriptional activity, apoptosis, and performed a morphologic study of liver tissues at 3, 6, and 24 h after portal vein reperfusion and in normal condition (n = 8). Pretreatment with NBD peptides significantly improved liver function, attenuating liver parenchymal cell damage, apoptosis by down-regulating TNF-α level, inhibiting IKK complex phosphorylation, IκBα degradation, and NF-κB transcriptional activity, but had no effect in normal condition. NBD peptides attenuated hepatic IRI by preventing NF-κB activation, without affecting basal NF-κB activity.

Disruption of IκB Kinase (IKK)-mediated RelA Serine 536 Phosphorylation Sensitizes Human Multiple Myeloma Cells to Histone Deacetylase (HDAC) Inhibitors

Post-translational modifications of RelA play an important role in regulation of NF-κB activation. We previously demonstrated that in malignant hematopoietic cells, histone deacetylase inhibitors (HDACIs) induced RelA hyperacetylation and NF-κB activation, attenuating lethality. We now present evidence that IκB kinase (IKK) β-mediated RelA Ser-536 phosphorylation plays a significant functional role in promoting RelA acetylation, inducing NF-κB activation, and limiting HDACI lethality in human multiple myeloma (MM) cells. Immunoblot profiling revealed that although basal RelA phosphorylation varied in MM cells, Ser-536 phosphorylation correlated with IKK activity. Exposure to the pan-HDACIs vorinostat or LBH-589 induced phosphorylation of IKKα/β (Ser-180/Ser-181) and RelA (Ser-536) in MM cells, including cells expressing an IκBα "super-repressor," accompanied by increased RelA nuclear translocation, acetylation, DNA binding, and transactivation activity. These events were substantially blocked by either pan-IKK or IKKβ-selective inhibitors, resulting in marked apoptosis. Consistent with these events, inhibitory peptides targeting either the NF-κB essential modulator (NEMO) binding domain for IKK complex formation or RelA phosphorylation sites also significantly increased HDACI lethality. Moreover, IKKβ knockdown by shRNA prevented Ser-536 phosphorylation and significantly enhanced HDACI susceptibility. Finally, introduction of a nonphosphorylatable RelA mutant S536A, which failed to undergo acetylation in response to HDACIs, impaired NF-κB activation and increased cell death. These findings indicate that HDACIs induce Ser-536 phosphorylation of the NF-κB subunit RelA through an IKKβ-dependent mechanism, an action that is functionally involved in activation of the cytoprotective NF-κB signaling cascade primarily through facilitation of RelA acetylation rather than nuclear translocation.

NEMO-Binding Domain Peptide Inhibits Constitutive NF-κB Activity and Reduces Tumor Burden in a Canine Model of Relapsed, Refractory Diffuse Large B-Cell Lymphoma

Activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL) is an aggressive, poorly chemoresponsive lymphoid malignancy characterized by constitutive canonical NF-κB activity that promotes lymphomagenesis and chemotherapy resistance via overexpression of antiapoptotic NF-κB target genes. Inhibition of the canonical NF-κB pathway may therefore have therapeutic relevance in ABC-DLBCL. Here, we set out to determine whether dogs with spontaneous DLBCL have comparative aberrant constitutive NF-κB activity and to determine the therapeutic relevance of NF-κB inhibition in dogs with relapsed, resistant DLBCL. Canonical NF-κB activity was evaluated by electrophoretic mobility shift assays and immunoblot analyses, and NF-κB target gene expression was measured by quantitative real time PCR. Primary malignant canine B lymphocytes were treated with the selective IKK complex inhibitor NF-κB essential modulator-binding domain (NBD) peptide and evaluated for NF-κB activity and apoptosis. NBD peptide was administered intranodally to dogs with relapsed B-cell lymphoma and NF-κB target gene expression and tumor burden were evaluated pre- and post-treatment. Constitutive canonical NF-κB activity and increased NF-κB target gene expression were detected in primary DLBCL tissue. NBD peptide inhibited this activity and induced apoptosis of primary malignant B cells in vitro. Intratumoral injections of NBD peptide to dogs with relapsed DLBCL inhibited NF-κB target gene expression and reduced tumor burden. This work shows that dogs with spontaneous DLBCL represent a clinically relevant, spontaneous, large animal model for human ABC-DLBCL and shows the therapeutic relevance of NF-κB inhibition in the treatment of ABC-DLBCL. These results have important translational relevance for ABC-DLBCL treatment in human patients.

Systemic delivery of NEMO binding domain/IKKγ inhibitory peptide to young mdx mice improves dystrophic skeletal muscle histopathology

The activation of nuclear factor κB (NF-κB) contributes to muscle degeneration that results from dystrophin deficiency in human Duchenne muscular dystrophy (DMD) and in the mdx mouse. In dystrophic muscle, NF-κB participates in inflammation and failure of muscle regeneration. Peptides containing the NF-κB Essential Modulator (NEMO) binding domain (NBD) disrupt the IκB kinase complex, thus blocking NF-κB activation. The NBD peptide, which is linked to a protein transduction domain to achieve in vivo peptide delivery to muscle tissue, was systemically delivered to mdx mice for 4 or 7weeks to study NF-κB activation, histological changes in hind limb and diaphragm muscle and ex vivo function of diaphragm muscle. Decreased NF-κB activation, decreased necrosis and increased regeneration were observed in hind limb and diaphragm muscle in mdx mice treated systemically with NBD peptide, as compared to control mdx mice. NBD peptide treatment resulted in improved generation of specific force and greater resistance to lengthening activations in diaphragm muscle ex vivo. Together these data support the potential of NBD peptides for the treatment of DMD by modulating dystrophic pathways in muscle that are downstream of dystrophin deficiency.

Improvement of cardiac contractile function by peptide-based inhibition of NF-κB in the utrophin/dystrophin-deficient murine model of muscular dystrophy

BackgroundDuchenne muscular dystrophy (DMD) is an inherited and progressive disease causing striated muscle deterioration. Patients in their twenties generally die from either respiratory or cardiac failure. In order to improve the lifespan and quality of life of DMD patients, it is important to prevent or reverse the progressive loss of contractile function of the heart. Recent studies by our labs have shown that the peptide NBD (Nemo Binding Domain), targeted at blunting Nuclear Factor κB (NF-κB) signaling, reduces inflammation, enhances myofiber regeneration, and improves contractile deficits in the diaphragm in dystrophin-deficient mdx mice.MethodsTo assess whether cardiac function in addition to diaphragm function can be improved, we investigated physiological and histological parameters of cardiac muscle in mice deficient for both dystrophin and its homolog utrophin (double knockout = dko) mice treated with NBD peptide. These dko mice show classic pathophysiological hallmarks of heart failure, including myocyte degeneration, an impaired force-frequency response and a severely blunted β-adrenergic response. Cardiac contractile function at baseline and frequencies and pre-loads throughout the in vivo range as well as β-adrenergic reserve was measured in isolated cardiac muscle preparations. In addition, we studied histopathological and inflammatory markers in these mice.ResultsAt baseline conditions, active force development in cardiac muscles from NBD treated dko mice was more than double that of vehicle-treated dko mice. NBD treatment also significantly improved frequency-dependent behavior of the muscles. The increase in force in NBD-treated dko muscles to β-adrenergic stimulation was robustly restored compared to vehicle-treated mice. However, histological features, including collagen content and inflammatory markers were not significantly different between NBD-treated and vehicle-treated dko mice.ConclusionsWe conclude that NBD can significantly improve cardiac contractile dysfunction in the dko mouse model of DMD and may thus provide a novel therapeutic treatment for heart failure.

Protection from diabetes development by single-chain antibody-mediated delivery of a NF-κB inhibitor specifically to β-cells in vivo

Recently, we reported the generation of single-chain antibodies (SCAs) highly specific for rodent and human β-cells. Our current report describes the generation of a fusion protein of one of these SCAs (SCA B1) with a NF-κB essential modifier (NEMO)-binding domain (NBD) peptide, thereby creating a selective inhibitor of NF-κB activation in β-cells. The SCA B1-NBD fusion protein was cloned in the pIRES-EGFP, expressed in bacteria, and purified by metal affinity chromatography; the newly generated complex was then administered intravenously to rodents and evaluated for its ability to protect β-cells against cytokines in vitro and diabetogenic agents in vivo. First, it was shown clearly that our SCA B1-NBD fusion protein binds highly selective to CD rat β-cells in vivo. Second, we observed that SCA B1-mediated in vivo delivery of the NBD peptide completely blocked IL-1β + IFNγ- and TNFα + IFNγ-mediated induction of NF-κB as well as islet dysfunction in culture. Finally, repeated intravenous injection of SCA B1-NBD prior to multiple low-dose administration of streptozotocin in CD mice not only induced a striking resistance to diabetes development but also preserved β-cell mass. In conclusion, our data show for the first time that a SCA B1-NBD fusion peptide reliably protects β-cells against cytokines in vitro and allows protection from diabetes development in CD mice in vivo.

Targeting Inflammation in Cardiovascular Diseases. Still a Neglected field?

Prevention and treatment of atherosclerosis is still a clinical challenge in the cardiovascular medicine. The classical belief that atherosclerotic lesion development solely depends on lipid deposition has been replaced by the current concept that activation of immune and inflammatory responses plays a central role in plaque initiation and progression. In this review we summarize studies on human and genetically modified animals describing a finite number of cellular and molecular mechanisms that underlie immunoinflammation in atherosclerotic plaques. We focus on the pro- and antiinflammatory mediators activated during atherogenesis and the intracellular signaling pathways regulating these events. Besides the advances on established pharmacological agents, we propose potential strategies for reduction/stabilization of atherosclerotic plaques based on the clinical data in inflammatory-associated pathologies and on the encouraging studies in experimental models of atherosclerosis. We emphasize the potential of such novel inhibitors comprising receptor antagonists, neutralizing antibodies, kinase inhibitors, peptide-based technologies, and chemicals as emerging antiinflammatory strategies for the treatment of atherosclerotic disease complications.

Maintenance of IKKβ Activity Is Necessary to Protect Lung Grafts From Acute Injury

Signaling pathways that target I-κB kinase β (IKKβ) activation stimulate the expression of nuclear factor (NF)-κB-dependent genes and are thus believed to primarily promote inflammation and injury in solid organ grafts. We examined the role of IKKβ in a mouse model of lung transplantation-mediated ischemia-reperfusion injury using NF-κB essential modulator (NEMO)-binding domain (NBD) peptide to pharmacologically inhibit IKK activation. As myeloid cells are primarily responsible for the production of acute inflammatory mediators after lung transplantation, we also investigated the effects of myeloid cell-specific IKKβ gene deletion on acute lung graft injury by transplanting mutant mice. When NBD was administered at a dose that partially inhibits IKKβ activation, we observed attenuated lung graft injury and blunted expression of intragraft proinflammatory mediators. Surprisingly, when the dose of NBD was increased to a level that ablates intragraft IKKβ activation, graft inflammation, and injury were significantly worse compared with recipients treated with control peptide. Similar to lung recipients with pharmacologically ablated IKKβ activity, donor-recipient transplant combinations with a myeloid cell-specific IKKβ gene deletion had marked intragraft inflammation and poor lung function. Our data show maintenance of IKKβ activity is critical for promoting graft homeostasis with important implications for targeting NF-κB-dependent signaling pathways for treating acute lung injury.

NF-κB Inhibition Protects against Tumor-Induced Cardiac Atrophy in Vivo

Cancer cachexia is a severe wasting syndrome characterized by the progressive loss of lean body mass and systemic inflammation. It occurs in approximately 80% of patients with advanced malignancy and is the cause of 20% to 30% of all cancer-related deaths. The mechanism by which striated muscle loss occurs is the tumor release of pro-inflammatory cytokines, such as IL-1, IL-6, and TNF-α. These cytokines interact with their cognate receptors on muscle cells to enhance NF-κB signaling, which then mediates muscle loss and significant cardiac dysfunction. Genetic inhibition of NF-κB signaling has demonstrated its predominant role in skeletal muscle loss. Therefore, we tested two novel drugs designed to specifically inhibit NF-κB by targeting the IκB kinase (IKK) complex: Compound A and NEMO binding domain (NBD) peptide. Using an established mouse model of cancer cachexia (C26 adenocarcinoma), we determined how these drugs affected the development of tumor-induced cardiac atrophy and function. Echocardiographic and histological analysis revealed that both Compound A and NBD inhibit cardiac NF-κB activity and prevent the development of tumor-induced systolic dysfunction and atrophy. This protection was independent of any effects of the tumor itself (Compound A) or tumor-secreted cytokines (NBD). This study identifies for the first time, to our knowledge, that drugs targeting the IKK complex are cardioprotective against cancer cachexia-induced cardiac atrophy and systolic dysfunction, suggesting therapies that may help reduce cardiac-associated morbidities found in patients with advanced malignancies.

Peptide-Based Inhibition of NF-κB Rescues Diaphragm Muscle Contractile Dysfunction in a Murine Model of Duchenne Muscular Dystrophy

Deterioration of diaphragm function is one of the prominent factors that contributes to the susceptibility of serious respiratory infections and development of respiratory failure in patients with Duchenne Muscular Dystrophy (DMD). The NF-κB signaling pathway has been implicated as a contributing factor of dystrophic pathology, making it a potential therapeutic target. Previously, we demonstrated that pharmacological inhibition of NF-κB via a small NEMO Binding Domain (NBD) peptide was beneficial for reducing pathological features of mdx mice. Now, we stringently test the effectiveness and clinical potential of NBD by treating mdx mice with various formulations of NBD and use diaphragm function as our primary outcome criteria. We found that administering DMSO-soluble NBD rescued 78% of the contractile deficit between mdx and wild-type (WT) diaphragm. Interestingly, synthesis of a GLP NBD peptide as an acetate salt permitted its solubility in water, but as a negative consequence, also greatly attenuated functional efficacy. However, replacing the acetic acid counterion of the NBD peptide with trifluoroacetic acid retained the peptide's water solubility and significantly restored mdx diaphragm contractile function and improved histopathological indices of disease in both diaphragm and limb muscle. Together, these results support the feasibility of using a mass-produced, water-soluble NBD peptide for clinical use.

Diazoxide potentiates mesenchymal stem cell survival via NF-κB-dependent miR-146a expression by targeting Fas

We have previously reported that preconditioning of bone marrow-derived mesenchymal stem cells (MSCs) with diazoxide (DZ) significantly improved cell survival via NF-κB signaling. Since micro-RNAs (miRNAs) are critical regulators of a wide variety of biological events, including apoptosis, proliferation, and differentiation, it is likely that DZ-induced survival is mediated by miRNAs. Here we show that miR-146a expressed during preconditioning with DZ is a key regulator of stem cell survival. Treatment of MSCs with DZ (200 μM) markedly increased miR-146a expression and promoted cell survival, as evaluated by lactate dehydrogenase release and transferase-mediated dUTP nick-end labeling staining. Interestingly, blocking NF-κB by IKK-γ NEMO binding domain inhibitor peptide did not induce miR-146a expression, indicating NF-κB regulates miR-146a expression. Moreover, blockade of miR-146a expression by antisense miR-146a inhibitor abolished DZ-induced cytoprotective effects, suggesting a critical role of miR-146a in MSC survival. Computational analysis found a consensus putative target site of miR-146a relevant to apoptosis in the 3' untranslated region of Fas mRNA. The role of Fas as a target gene was substantiated by abrogation of miR-146a, which markedly increased Fas protein expression. This was verified by luciferase reporter assay, which showed that forced expression of miR-146a downregulated Fas expression via targeting its 3'-UTR of this gene. Taken together, these data demonstrated that cytoprotection afforded by preconditioning of MSCs with DZ was regulated by miR-146a induction, which may be a novel therapeutic target in cardiac ischemic diseases.

A homogeneous time-resolved fluorescence-based high-throughput screening system for discovery of inhibitors of IKKβ–NEMO interaction

The nuclear transcription factor NF-κB is crucial to the expression of numerous cytokines, enzymes, and cell adhesion molecules, all of which can drive inflammatory and autoimmune disorders such as rheumatoid arthritis. The IKK complex plays the most important role in the signal cascade leading to NF-κB activation. Recently, inhibition of the interaction between NEMO (NF-κB essential modulator) and the catalytic subunits of IKK, especially IKKβ, has received particular attention as a possible new therapeutic approach to treatment of inflammatory disorders, and several reports have shown the efficacy of cell permeable NEMO binding domain (NBD)-containing peptides in blocking the IKK/NF-κB pathway. In this article, we describe in detail the development and validation of two novel binding assays, a homogeneous time-resolved fluorescence (HTRF)-based assay and an enzyme-linked immunosorbent assay (ELISA)-based assay, suitable for the discovery of small molecules that inhibit IKKβ–NEMO interaction. Using the HTRF-based assay, we screened approximately 15,000 compounds from our chemical library and eliminated false positive hits by the ELISA-based assay and IKK complex kinase assay. As a result, seven positive hit compounds that inhibit IKK complex activity through inhibition of IKKβ–NEMO interaction were identified. These hit compounds may have a good potential in the treatment of inflammatory and autoimmune disorders such as rheumatoid arthritis.

Detrimental Effect of the Proteasome Inhibitor, Bortezomib in Bacterial Superantigen- and Lipopolysaccharide-induced Systemic Inflammation

Bacterial superantigen (BSAg)-induced toxic shock syndrome (TSS) and bacterial lipopolysaccharide (LPS)-induced shock are characterized by severe systemic inflammation. As nuclear factor kappaB (NF kappaB) plays an important role in inflammation and bortezomib, a proteasome inhibitor widely used in cancer chemotherapy, is a potent inhibitor of NF kappaB activation, we evaluated the therapeutic and prophylactic use of bortezomib in these conditions using murine models. Bortezomib prophylaxis significantly reduced serum levels of many cytokines and chemokines induced by BSAg. However, at 3 hours, serum level of TNF-a, an important cytokine implicated in TSS, was significantly reduced but not abolished. At 6 hours, there was no difference in the serum TNF-a levels between bortezomib treated and untreated mice challenged with staphylococcal enterotoxin B (SEB). Paradoxically, all mice treated with bortezomib either before or after BSAg challenge succumbed to TSS. Neither bortezomib nor BSAg was lethal if given alone. Serum biochemical parameters and histopathological findings suggested acute liver failure as the possible cause of mortality. Liver tissue from SEB-challenged mice treated with bortezomib showed a significant reduction in NF kappaB activation. Because NF kappaB-dependent antiapoptotic pathways protect hepatocytes from TNF-alpha-induced cell death, inhibition of NF kappaB brought forth by bortezomib in the face of elevated TNF-alpha levels caused by BSAg or LPS is detrimental.

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.

Preconditioning Promotes Survival and Angiomyogenic Potential of Mesenchymal Stem Cells in the Infarcted Heart via NF-κB Signaling

We proposed that pharmacological manipulation of mesenchymal stem cells (MSCs) with diazoxide enhanced their survival and regenerative potential via NFkappaB regulation. MSCs preconditioned ((PC)MSCs) with diazoxide and later subjected to oxidant stress with 100 micromol/L H(2)O(2) either immediately or after 24 h exhibited higher survival (p < 0.01 vs nonpreconditioned MSCs; (Non-PC)MSCs) with concomitantly increased phosphorylation of PI3K, Akt, GSK3beta (cytoplasmic), and NF-kappaB (p65) (nuclear). Akt kinase activity was determined as a function of GSK3beta activity. Pretreatment of (PC)MSCs with Wortmannin (Wt), NEMO-binding domain (NBD), or NF-kappaB (p50) siRNA abolished NF-kappaB (p65) activity. Preconditioning increased NF-kappaB-dependent elevation of secretable growth factors associated with their paracrine effects. Inhibition of PI3K activity with Wt reduced (PC)MSCs viability at both early and 24 h time-points. However, inhibition of NF-kappaB reduced viability of (PC)MSCs only at 24 h time-point. For in vivo studies, DMEM without cells (group-1) or containing 1 x 10(6) male (Non-PC)MSCs (group-2), (PC)MSCs (group-3), (PC)MSCs pretreated with Wortmannin (group-4) or NF-kappaB decoy (group-5) were transplanted in a female rat model of acute myocardial infarction. Group-3 showed highest cell survival and growth factor expression, increased angiomyogenesis, and functional improvement. We conclude that activation of NF-kappaB by preconditioning promoted (PC)MSCs survival and angiomyogenic potential in the infarcted heart.