NEMO-binding Domain Peptide Research Articles

Local treatment with the selective IκB kinase β inhibitor NEMO-binding domain peptide ameliorates synovial inflammation

Nuclear factor (NF)-κB is a key regulator of synovial inflammation. We investigated the effect of local NF-κB inhibition in rat adjuvant arthritis (AA), using the specific IκB kinase (IKK)-β blocking NF-κB essential modulator-binding domain (NBD) peptide. The effects of the NBD peptide on human fibroblast-like synoviocytes (FLS) and macrophages, as well as rheumatoid arthritis (RA) whole-tissue biopsies, were also evaluated. First, we investigated the effects of the NBD peptide on RA FLS in vitro. Subsequently, NBD peptides were administered intra-articularly into the right ankle joint of rats at the onset of disease. The severity of arthritis was monitored over time, rats were sacrificed on day 20, and tissue specimens were collected for routine histology and x-rays of the ankle joints. Human macrophages or RA synovial tissues were cultured ex vivo in the presence or absence of NBD peptides, and cytokine production was measured in the supernatant by enzyme-linked immunosorbent assay. The NBD peptide blocked interleukin (IL)-1-β-induced IκBα phosphorylation and IL-6 production in RA FLS. Intra-articular injection of the NBD peptide led to significantly reduced severity of arthritis (p < 0.0001) and reduced radiological damage (p = 0.04). This was associated with decreased synovial cellularity and reduced expression of tumor necrosis factor (TNF)-α and IL-1-β in the synovium. Incubation of human macrophages with NBD peptides resulted in 50% inhibition of IL-1-β-induced TNF-α production in the supernatant (p < 0.01). In addition, the NBD peptide decreased TNF-α-induced IL-6 production by human RA synovial tissue biopsies by approximately 42% (p < 0.01). Specific NF-κB blockade using a small peptide inhibitor of IKK-β has anti-inflammatory effects in AA and human RA synovial tissue as well as in two important cell types in the pathogenesis of RA: macrophages and FLS. These results indicate that IKK-β-targeted NF-κB blockade using the NBD peptide could offer a new approach for the local treatment of arthritis.

NFκB Activates in vivo the Synthesis of Inducible Cox-2 in the Brain

Interleukin-1beta (IL-1beta) induces cyclooxygenase-2 (Cox-2) expression in many of its cellular targets resulting in production and release of prostaglandins. Although IL-1beta-induced Cox-2 expression most likely requires activation of nuclear transcription factor kappa B (NFkappaB) pathway, this has never been formally demonstrated in vivo. We tested this using a specific inhibitor of NFkappaB activation, the NEMO binding domain (NBD) peptide, that has been shown previously to be effective in various in vivo models of acute inflammation. Incubation of rat glioma cells with the NBD peptide blocked IL-1beta-induced NFkappaB nuclear translocation. Furthermore, after injection of a biotinylated version of the NBD peptide into the lateral ventricle of the brain, we found that it readily diffused to its potential cellular targets in vivo. To test the effects of the peptide on NFkappaB activation and Cox-2 expression in the brain, we injected it intracerebroventricularly (36 microg/rat) into rats before intraperitoneal injection of IL-1beta (60 microg/kg). Treatment with NBD peptide completely abolished IL-1beta-induced NFkappaB activation and Cox-2 synthesis in microvasculature. In contrast, the peptide had no effect on constitutive neuronal Cox-2. These findings strongly support the hypothesis that IL-1beta-induced NFkappaB activation plays a major role in transmission of immune signals from the periphery to the brain.

Inactivation of the Cerebral NFκB Pathway Inhibits Interleukin-1β-Induced Sickness Behavior and c-Fos Expression in Various Brain Nuclei

The behavioral effects of peripherally administered interleukin-1beta (IL-1beta) are mediated by the production of cytokines and other proinflammatory mediators at the level of the blood-brain interface and by activation of neural pathway. To assess whether this action is mediated by NFkappaB activation, rats were injected into the lateral ventricle of the brain with a specific inhibitor of NFkappaB activation, the NEMO Binding Domain (NBD) peptide that has been shown previously to abolish completely IL-1beta-induced NFkappaB activation and Cox-2 synthesis in the brain microvasculature. NFkappaB pathway inactivation significantly blocked the behavioral effects of intraperitoneally administered IL-1beta in the form of social withdrawal and decreased food intake, and dramatically reduced IL-1beta-induced c-Fos expression in various brain regions as paraventricular nucleus, supraoptic nucleus, and lateral part of the central amygdala. These findings strongly support the hypothesis that IL-1beta-induced NFkappaB activation at the blood-brain interface is a crucial step in the transmission of immune signals from the periphery to the brain that underlies further events responsible of sickness behavior.

Selective inhibition of NF‐κB in dendritic cells by the NEMO‐binding domain peptide blocks maturation and prevents T cell proliferation and polarization

Dendritic cells (DC) are the only antigen-presenting cells for naive T cells and, therefore, they are crucial players in the initiation of immune responses. Because DC maturation and cytokine production are NF-kappaB dependent, we hypothesized that blocking NF-kappaB activity in DC by selectively targeting the inhibitor of kappaB (IkappaB) kinase (IKK) complex using the novel NF-kappaB inhibitor NEMO-binding domain (NBD) peptide could inhibit DC maturation and other functional characteristics, resulting in modulation of the immune response. We used human monocyte-derived DC to test the biological effects of the NBD peptide in vitro. NF-kappaB inhibition by the NBD peptide resulted in blockade of IKK-mediated IkappaBalpha phosphorylation and subsequent nuclear translocation and DNA binding of NF-kappaB p65 in DC. In addition, IL-6, IL-12, and TNF-alpha production was dose-dependently blocked and NBD peptide treatment also led to a strong reduction of LPS-induced maturation. Functional analysis of these DC showed marked inhibition of T cell proliferation in the allogeneic mixed lymphocyte reaction, accompanied by less Th1 and Th2 polarization. The current study reveals for the first time the unique properties of this novel, highly specific NF-kappaB inhibitor in DC. Also, these data indicate that the NBD peptide could be used as an elegant tool in DC based immunotherapy for unwanted cellular immune responses.

Amelioration of acute inflammation by systemic administration of a cell‐permeable peptide inhibitor of NF‐κB activation

We used an experimental model of inflammation in mice, carrageenan-induced paw edema, to study the antiinflammatory effects of the NEMO-binding domain (NBD) peptide, which blocks activation of the inducible transcription factor NF-kappaB. Paw edema was induced by subplantar injection of 1% lambda-carrageenan into the mouse left hind paw. Test agents were given intraperitoneally immediately after carrageenan injection. The increase in footpad thickness was considered to be edema. In some experiments, the mice were killed and the paws were removed for histologic and molecular biology analysis. NF-kappaB DNA binding activity was evaluated in nuclear extracts by electrophoretic mobility shift assays. The expression levels of NF-kappaB-regulated cyclooxygenase 2 (COX-2) protein and tumor necrosis factor alpha (TNFalpha) messenger RNA (mRNA) were evaluated by immunoblot analysis and polymerase chain reaction amplification of reverse-transcribed mRNA, respectively. We found that systemically administered NBD peptide significantly inhibited edema formation and cellular infiltration in inflamed mouse paws. This antiinflammatory activity was most likely due to inhibition of expression of proinflammatory mediators, such as TNFalpha and COX-2, in inflamed tissues. These studies further establish NF-kappaB as a target for antiinflammatory therapy and provide support for the use of the NBD peptide as a possible therapeutic agent for inflammatory diseases.

IL-1-induced receptor activator of NF-κB ligand in human periodontal ligament cells involves ERK-dependent PGE 2 production

Periodontitis, an inflammatory disorder of the supporting tissue of teeth, is one of the most common infectious diseases in humans. Periodontal pathogens promote inflammatory cytokines such as interleukin-1 (IL-1) and prostaglandin E 2 (PGE 2), resulting in alveolar bone destruction. In the present study, we examined the cellular and molecular mechanisms of IL-1-induced osteoclastogenesis using a coculture system of human periodontal ligament (PDL) cells and mouse spleen cells. IL-1α induced tartrate-resistant acid phosphatase positive (TRAP +) cell formation in a dose-dependent manner. IL-1α up-regulated receptor activator of NF-κB ligand (RANKL) and down-regulated osteoprotegerin (OPG) mRNA expression in PDL cells. The addition of cell-permeable PKI, an inhibitor of the cAMP/PKA signaling pathway, to the cocultures 8 h after the IL-1α stimulation inhibited IL-1α-induced TRAP + cell formation. IL-1α-induced TRAP + cell formation was completely blocked by either NS398, a selective inhibitor of cyclooxygenase (COX)-2, or PD98059, a specific inhibitor of extracellular signal-regulated kinase (ERK). Pretreatment with NS398 and PD98059 also inhibited both the up-regulation of RANKL and the down-regulation of OPG expression by IL-1α in PDL cells. IL-1α activated ERK phosphorylation and PD98059 greatly inhibited both COX-2 mRNA expression and PGE 2 production induced by IL-1α in PDL cells. In contrast, NEMO binding domain (NBD) peptide, a specific inhibitor of NF-κB signaling, did not affect COX2, RANKL, or OPG mRNA expression induced by IL-1α. These results suggest that IL-1α stimulates osteoclast formation by increasing the expression level of RANKL versus OPG via ERK-dependent PGE 2 production in PDL cells.

The IκB Kinase (IKK) Inhibitor, NEMO-binding Domain Peptide, Blocks Osteoclastogenesis and Bone Erosion in Inflammatory Arthritis

Activation of NF-kappaB leads to expression of ample genes that regulate inflammatory and osteoclastogenic responses. The process is facilitated by induction of IkappaB kinase (IKK) complex that phosphorylates IkappaB and leads to its dissociation from the NF-kappaB complex, thus permitting activation of NF-kappaB. The IKK complex contains primarily IKKalpha, IKKbeta, and the regulatory kinase IKKgamma, also known as NEMO. NEMO regulates the IKK complex activity through its binding to carboxyl-terminal region of IKKalpha and IKKbeta, termed NEMO-binding domain (NBD). In this regard, a cell-permeable NBD peptide has been shown to block association of NEMO with the IKK complex and inhibit activation of NF-kappaB. Given the pivotal role of cytokine-induced NF-kappaB in osteoclastogenesis and inflammatory bone loss, we deduced that cell-permeable TAT-NBD peptide may hinder osteoclastogenesis and bone erosion in inflammatory arthritis. Using NBD peptides, we show that wild type, but not mutant, NBD blocks IKK activation and reduces cytokine-induced promoter and DNA binding activities of NF-kappaB and inhibits cytokine-induced osteoclast formation by osteoclast precursors. Consistent with the key role of NF-kappaB in osteoinflammatory responses in vivo, wild type TAT-NBD administered into mice prior to induction of inflammatory arthritis efficiently block in vivo osteoclastogenesis, inhibits focal bone erosion, and ameliorates inflammatory responses in the joints of arthritic mice. The mutant NBD peptide fails to exert these functions. These results provide strong evidence that IKKs are potent regulators of cytokine-induced osteoclastogenesis and inflammatory arthritis. More importantly, blockade of NEMO assembly with the IKK complex is a viable strategy to avert inflammatory osteolysis.

The effects of NF-κB inhibition by a NEMO binding domain peptide in vitro and in vivo

The effects of NF-κB inhibition by a NEMO binding domain peptide in vitro and in vivo

Tumor Necrosis Factor-α Induces Nuclear Factor-κB-dependent TRPC1 Expression in Endothelial Cells

We investigated the role of tumor necrosis factor-alpha (TNF-alpha) in activating the store-operated Ca2+ channels in endothelial cells via the expression of transient receptor potential channel (TRPC) isoforms. We observed that TNF-alpha exposure of human umbilical vein endothelial cells resulted in TRPC1 mRNA and protein expression, whereas it had no effect on TRPC3, TRPC4, or TRPC5 expression. The TRPC1 expression was associated with increased Ca2+ influx after intracellular Ca2+ store depletion with either thrombin or thapsigargin. We cloned the 5'-regulatory region of the human TRPC1 (hTRPC1) gene which contained a TATA box and CCAAT sequence close to the transcription initiation site. We also identified four nuclear factor-kappaB (NF-kappaB)-binding sites in the 5'-regulatory region. To address the contribution of NF-kappaB in the mechanism of TRPC1 expression, we determined the effects of TNF-alpha on expression of the reporter luciferase after transfection of hTRPC1 promoter-luciferase (hTRPC1-Pro-Luc) construct in the human dermal microvascular endothelial cell line. Reporter activity increased >4-fold at 4 h after TNF-alpha challenge. TNF-alpha-induced increase in reporter activity was markedly reduced by co-expression of either kinase-defective IKKbeta kinase mutant or non-phosphorylatable IkappaB mutant. Treatment with NEMO-binding domain peptide, which prevents NF-kappaB activation by selectively inhibiting IKKgamma interaction with IKK complex, also blocked the TNF-alpha-induced TRPC1 expression. Thus, TNF-alpha induces TRPC1 expression through an NF-kappaB-dependent pathway in endothelial cells, which can trigger augmented Ca2+ entry following Ca2+ store depletion. The augmented Ca2+ entry secondary to TRPC1 expression may be an important mechanism of endothelial injury induced by TNF-alpha.

Protection of Islets by in SituPeptide-mediated Transduction of the IκB Kinase Inhibitor Nemo-binding Domain Peptide

We have previously demonstrated that adenoviral gene transfer of the NF-kappaB inhibitor IkappaB to human islets results in protection from interleukin (IL)-1beta-mediated dysfunction and apoptosis. Here we report that human and mouse islets can be efficiently transduced by a cationic peptide transduction domain (PTD-5) without impairment of islet function. PTD mediated delivery of a peptide inhibitor of the IL-1beta-induced IkappaB kinase (IKK), derived from IKKbeta (NBD; Nemo-binding domain), and completely blocked the detrimental effects of IL-1beta on islet function and NF-kappaB activity, in a similar manner to Ad-IkappaB. We also demonstrate that mouse islets can be transduced in situ by infusion of the transduction peptide through the bile duct prior to isolation, resulting in 40% peptide transduction of the beta-cells. Delivery of the IKK inhibitor transduction fusion peptide (PTD-5-NBD) in situ to mouse islets resulted in improved islet function and viability after isolation. These results demonstrate the feasibility of using PTD-mediated delivery to transiently modify islets in situ to improve their viability and function during isolation, prior to transplantation.

Characterization of the IκB-kinase NEMO Binding Domain

Proinflammatory activation of NF-kappaB requires an upstream kinase complex (IkappaB-kinase; IKK) composed of two catalytic subunits (IKKalpha and IKKbeta) and a noncatalytic regulatory component named NEMO (NF-kappaB essential modulator). NEMO interacts with a COOH-terminal sequence within both IKKs termed the NEMO-binding domain (NBD), and a cell-permeable NBD peptide blocks NEMO/IKKbeta interactions and inhibits tumor necrosis factor-alpha-induced NF-kappaB. We report here that a peptide encompassing the NBD not only blocked association of both IKKs with NEMO but also disrupted preformed NEMO/IKK complexes in vitro. Furthermore, peptide blocking and alanine-scanning mutation studies revealed differences between the NBDs of IKKalpha and IKKbeta, and mutational analysis of the IKKbeta NBD identified the physical properties required at each position to maintain association with NEMO. Finally, we demonstrate that loss of NEMO-binding by IKKbeta through deletion of the NBD renders it catalytically active and that potential phosphorylation within the IKKbeta NBD may serve as a signal to down-regulate IKK activity. Our findings therefore provide critical insight into the physical properties of the NBD that will be valuable for the design of drugs aimed at disrupting the IKK complex and also reveal potential regulatory mechanisms controlling the function of the IKK complex.

Selective targeting of the nuclear factor-κB pathway enhances tumor necrosis factor–related apoptosis-inducing ligand-mediated pancreatic cancer cell death

Background. Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL), a member of the tumor necrosis factor family, selectively induces apoptosis in various cancer cells; however, certain cancers can evade TRAIL-mediated apoptosis. FLICE-like inhibitory protein (FLIP), an inhibitor of caspase-8, (also known as FLICE) is regulated by the transcription factor nuclear factor-kappaB (NF-κB) and can contribute to TRAIL resistance. The purpose of our study was to determine whether inhibition of NF-κB can enhance TRAIL-mediated pancreatic cancer cell death and decrease FLIP levels. Methods. The human pancreatic cancer cell lines MIA PaCa-2 and L3.6 were treated with TRAIL, NEMO-binding domain (NBD) peptide (a novel selective NF-κB inhibitor), or a combination of both. Cell viability and apoptosis were measured. Gel mobility shift assays were performed to assess NF-κB binding activity. Western blots were performed to assess FLIP levels after treatment with NBD or infection with an adenovirus encoding mutated IkappaBalpha. Results. The aggressive L3.6 cell line was resistant to TRAIL treatment, whereas MIA PaCa-2 cells were sensitive to TRAIL. The combination of TRAIL and NBD significantly decreased cell viability and increased apoptosis in L3.6 cells. Cellular levels of FLIP were decreased by inhibition of NF-κB (either by NBD treatment or mutant IkappaBalpha infection). Conclusions. Our findings demonstrate resistance of the aggressive L3.6 pancreatic cell line to TRAIL treatment alone; inhibition of NF-κB by NBD increased TRAIL-mediated cell death and decreased FLIP protein levels. Novel agents that selectively target the NF-κB pathway may be useful adjuvant therapies for chemoresistant pancreatic cancers. (Surgery 2002;132:127-34.)

Selective Inhibition of NF-κB Activation by a Peptide That Blocks the Interaction of NEMO with the IκB Kinase Complex

Activation of the transcription factor nuclear factor (NF)–κB by proinflammatory stimuli leads to increased expression of genes involved in inflammation. Activation of NF-κB requires the activity of an inhibitor of κB (IκB)-kinase (IKK) complex containing two kinases (IKKα and IKKβ) and the regulatory protein NEMO (NF-κB essential modifier). An amino-terminal α-helical region of NEMO associated with a carboxyl-terminal segment of IKKα and IKKβ that we term the NEMO-binding domain (NBD). A cell-permeable NBD peptide blocked association of NEMO with the IKK complex and inhibited cytokine-induced NF-κB activation and NF-κB–dependent gene expression. The peptide also ameliorated inflammatory responses in two experimental mouse models of acute inflammation. The NBD provides a target for the development of drugs that would block proinflammatory activation of the IKK complex without inhibiting basal NF-κB activity.