P65 Subunit Of NF-kappaB Research Articles

Linking Cardiac Mechanosensing at the Sarcomere M-Band, Nuclear Factor κB Signaling, and Cardiac Remodeling

How the heart senses increased biomechanic stress and converts this “input signal” to generalized downstream cardiac hypertrophy signaling paradigms remains an enigmatic but intriguing question in modern cardiovascular biology. Only recently have signaling molecules that can couple biomechanical stress to common pathways of cardiac hypertrophy been uncovered. Yeast-2-hybrid screens have identified several binding partners of sarcomeric proteins. These concerted actions revealed that especially the sarcomere Z-disc harbors key components that affect the activity of several notable kinases and phosphatase, including mitogen-activated protein kinases, protein kinases A and C, and calcineurin.1 Exemplary to this is the family of calsarcin proteins, which link the Z-disk protein α-actinin to calcineurin, a well-characterized Ca2+-responsive signaling molecule controlling activity of the transcription factor NFAT and both required and sufficient for cardiac hypertrophy after pleiotropic stimuli (Figure).2 However, interruption of the calcium overload typical for heart failure may not stop disease progression, and, therefore, Ca2+-independent signaling cascades might serve as useful targets to interfere with left ventricular (LV) remodeling on pressure overload or ischemia.3 Hypothetical signaling cascade linking Ang II signaling, cardiac mechanosensing at the sarcomere, and specific transcription factors in LV remodeling. Likewise, the giant sarcomeric protein titin is the starting point of a signaling complex where the zinc-finger protein nbr1 targets the ubiquitin-associated p62/SQSTM1 to sarcomeres. In turn, p62 …

Interplay of IKK/NF-κB signaling in macrophages and myofibers promotes muscle degeneration in Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a lethal X-linked disorder associated with dystrophin deficiency that results in chronic inflammation and severe skeletal muscle degeneration. In DMD mouse models and patients, we find that IkappaB kinase/NF-kappaB (IKK/NF-kappaB) signaling is persistently elevated in immune cells and regenerative muscle fibers. Ablation of 1 allele of the p65 subunit of NF-kappaB was sufficient to improve pathology in mdx mice, a model of DMD. In addition, conditional deletion of IKKbeta in mdx mice elucidated that NF-kappaB functions in activated macrophages to promote inflammation and muscle necrosis and in skeletal muscle fibers to limit regeneration through the inhibition of muscle progenitor cells. Furthermore, specific pharmacological inhibition of IKK resulted in improved pathology and muscle function in mdx mice. Collectively, these results underscore the critical role of NF-kappaB in the progression of muscular dystrophy and suggest the IKK/NF-kappaB signaling pathway as a potential therapeutic target for DMD.

Effect of a Cyclooxygenase‐2 Inhibitor on Interleukin‐1β–Stimulated Activation of the Transcription Factor Nuclear Factor‐Kappa B in Human Gingival Fibroblasts

In previous work, the cyclooxygenase-2 inhibitor NS-398 inhibited interleukin (IL)-1beta-stimulated prostaglandin E(2) (PGE(2)) production almost completely while partially inhibiting IL-6 production in aggressive periodontitis (AgP) human gingival fibroblasts. PGE(2) and the transcription factor nuclear factor-kappa B (NF-kappaB) regulate IL-1beta-stimulated IL-6 production. Cytoplasmic NF-kappaB is bound to inhibitors (IkappaB proteins). IL-1beta initiates a cascade resulting in phosphorylation and degradation of IkappaB, allowing nuclear translocation of NF-kappaB and target gene activation. The purpose of this study was to determine whether NS-398 inhibited phosphorylation of IkappaB and NF-kappaB activation. AgP fibroblasts (1 to 2 x 10(6)) were exposed to IL-1beta (1 x 10(11)M) with or without NS-398 (10 nM) in serum-free medium. The NF-kappaB subunit p65 and phospho-IkappaBalpha were measured in whole cell, cytoplasmic, or nuclear extracts, using colorimetric assays. Enzyme-linked immunosorbent assays were used to measure PGE(2) and IL-6 production by 2.5 x 10(4) cells after exposure to IL-1beta with or without NS-398 in serum-free medium. Consistent with previous results, NS-398 reduced IL-1beta-stimulated PGE(2) by approximately 98% (P <0.001) and IL-6 by approximately 65% (P <0.001). IL-1beta increased nuclear and cytoplasmic p65 ( approximately 8-fold [P <0.001] and approximately 2.5-fold [P <0.03], respectively) over control levels. NS-398 reduced IL-1beta-stimulated nuclear and cytoplasmic p65 to control levels. IL-1beta increased phospho-IkappaBalpha in whole cell extracts by a maximum of approximately 9.5 times (P = 0.0001), and this was inhibited significantly by NS-398 (P <or=0.008). NS-398 inhibited NF-kappaB activation and nuclear p65 levels in human gingival fibroblasts. This seemed to be due to inhibition of the phosphorylation cascade resulting in formation of phospho-IkappaBalpha and free p65. NF-kappaB inhibition may be useful in treating inflammatory diseases such as AgP.

Regulation of ATR-dependent pathways by the FHA domain containing protein SNIP1

The forkhead associated (FHA) domain-containing protein Smad nuclear interacting protein 1 (SNIP1) has multiple cellular functions, including the ability to interact with DNA-binding transcription factors and transcriptional coactivators. Moreover, we have demonstrated previously that SNIP1 regulates cyclin D1 expression and promoter activity. Here, we identify a new function for SNIP1 as a regulator of ATR checkpoint kinase-dependent pathways in human U-2 OS osteosarcoma cells: SNIP1 is required for p53 induction in response to ultraviolet light treatment and selectively regulates the phosphorylation of known ATR target proteins, including p53, Chk1 and the histone variant H2AX. These activities are independent of its ability to regulate cyclin D1 expression. Significantly, SNIP1 is also required for ATR-dependent functions of the human p14(ARF) tumour suppressor, including its ability to modulate the activity of the RelA(p65) NF-kappaB subunit. This, together with its other described functions, suggests that SNIP1 could have an important role during tumorigenesis and cancer therapy.

Gliadin Peptides Activate Blood Monocytes from Patients with Celiac Disease

To elucidate the role of innate immune responses in celiac disease, we investigated the effect of gliadin on blood monocytes from patients with celiac disease. Gliadin induced substantial TNF-alpha and IL-8 production by monocytes from patients with active celiac disease, lower levels by monocytes from patients with inactive celiac disease, and even lower levels by monocytes from healthy donors. In healthy donor monocytes gliadin induced IL-8 from monocytes expressing HLA-DQ2 and increased monocyte expression of the costimulatory molecules CD80 and CD86, the dendritic cell marker CD83, and the activation marker CD40. Gliadin also increased DNA binding activity of NF-kappaB p50 and p65 subunits in monocytes from celiac patients, and NF-kappaB inhibitors reduced both DNA binding activity and cytokine production. Thus, gliadin activation of HLA-DQ2(+) monocytes leading to chemokine and proinflammatory cytokine production may contribute to the host innate immune response in celiac disease.

An evaluation of anti-TNF-alpha-therapy in patients with ankylosing spondylitis: imbalanced activation of NF kappa B subunits in lymphocytes and modulation of serum cortisol concentration

The aim of this study was to analyse patients with ankylosing spondylitis (AS) during the course of infliximab therapy. The molecular effects were evaluated using lymphocytes and sera that were isolated before therapy began, then again after 2 and 12 weeks from 17 AS patients and compared to those of 24 healthy control individuals. All 17 AS patients responded to treatment with infliximab as assessed using BASDAI. Elevated serum levels of IL-6, CRP and cortisol were reduced to normal levels by the 12 weeks time point. The level of DNA-binding p65 was decreased during the course of infliximab therapy whereas the level of DNA-binding p50 remained elevated until the 12 weeks time point. Taken together, Infliximab is an effective treatment for AS and results in decreased levels of the inflammation markers IL-6 and CRP, and of endogenous cortisol concentration. Unequal alterations in the levels of activated NF-kappaB subunits p50 and p65 might provide insights into the mechanisms of NF-kappaB action and anti-TNF-alpha therapy in AS.

NF-κB inhibition sensitizes to starvation-induced cell death in high-risk myelodysplastic syndrome and acute myeloid leukemia

CD34(+) bone marrow blasts from high-risk myelodysplastic syndrome (MDS) patients as well as MDS patient-derived cell lines (P39 and MOLM13) constitutively activate the nuclear factor-kappaB (NF-kappaB) pathway and undergo apoptosis when NF-kappaB is inhibited. Here, we show that the combination of conventional chemotherapeutic agents (daunorubicin, mitoxantrone, 5-azacytidine or camptothecin) with the NF-kappaB inhibitor BAY11-7082 did not yield a synergistic cytotoxicity. In contrast, BAY11-7082 (which targets the NF-kappaB-activating I-kappaB kinase (IKK) complex) or knockdown of essential components of the NF-kappaB system (such as the IKK1 and IKK2 subunits of the IKK complex and the p65 subunit of NF-kappaB), by small interfering RNAs sensitized MDS cell lines to starvation-induced apoptosis. The combination of BAY11-7082 and nutrient depletion synergistically killed the acute myeloid leukemia (AML) cell line U937 as well as primary CD34(+) bone marrow blasts from AML and high-risk MDS patients. The synergistic killing by BAY11-7082, combined with nutrient depletion, led to cell death accompanied by all hallmarks of apoptosis, including an early loss of the mitochondrial transmembrane potential, the release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria, activation of caspase-3, phosphatidylserine exposure on the plasma membrane surface and nuclear chromatin condensation. Transmission electron microscopy revealed the presence of numerous autophagic vacuoles in the cytoplasm before cells underwent nuclear apoptosis. Nonetheless, cell death was neither inhibited by the pan-caspase inhibitor z-VAD-fmk nor by knockdown of AIF or of essential components of the autophagy pathway (ATG5, ATG6/Beclin-1, ATG10, ATG12). In contrast, external supply of glucose, insulin or insulin-like growth factor-I could retard the cell death induced by BAY11-7082 combined with starvation. These results suggest that in MDS cells, NF-kappaB inhibition can precipitate a bioenergetic crisis that leads to an autophagic stress response followed by apoptotic cell death.

Immunohistochemical localization of advanced glycation end-products (AGEs) and their receptor (RAGE) in polycystic and normal ovaries

The aim of the present study was to investigate the localization/immunohistochemical distribution of AGEs and RAGE, as well as their putative signalling mediator NF-kappaB in ovaries of women with polycystic ovary syndrome (PCOS) compared to normal. Archival ovarian-tissue samples from biopsies of six women with PCOS and from six healthy of similar age women, were examined immunohistochemically with monoclonal anti-AGEs, anti-RAGE and anti-NF-kappaB(p50/p65) specific antibodies. In healthy women, AGE immunoreactivity was observed in follicular cell layers (granulosa and theca) and luteinized cells, but not in endothelial cells. PCOS specimens displayed AGE immunoexpression in theca interna and granulosa cells as well as in endothelial cells, but staining of granulosa cells was stronger than in that of normal ovaries. RAGE was highly expressed in normal and PCOS tissues. Normal tissue exhibited no staining differences between granulosa cell layer and theca interna. However, in PCOS ovaries, granulosa cells displayed stronger RAGE expression compared to theca interna cells in comparison to controls. NF-kappaB(p50/p65) was expressed in the cytoplasm of theca interna and granulosa cells of both normal and PCOS ovaries; whereas the NF-kappaB p65 subunit was only observed in granulosa cells nuclei in PCOS tissue. In conclusion, these findings demonstrate for the first time that RAGE and AGE-modified proteins with activated NF-kappaB are expressed in human ovarian tissue. Furthermore, a differential qualitative distribution of AGE, RAGE and NF-kappaB p65 subunit was observed in women with PCOS compared to healthy controls, where a stronger localization of both AGE and RAGE was observed in the granulosa cell layer of PCOS ovaries.

Molecular Mechanisms of Cyclosporin A Inhibition of the Cytokine-Induced Matrix Metalloproteinase-9 in Glomerular Mesangial Cells

The effects of the immunosuppressants cyclosporin A (CsA) and tacrolimus (FK506) on the IL-1beta-induced matrix metalloproteinase-9 (MMP-9) were investigated. Impairment of the protease-antiprotease balance contributes to renal fibrosis, which is observed collectively under long-term treatment with either immunosuppressant. It is demonstrated that CsA, in contrast to FK506, reduced the IL-1beta-induced MMP-9 content in conditioned media of mesangial cells, which coincides with a reduction in the cytokine-induced MMP-9 mRNA level. Similar to FK506, the VIVIT peptide, a specific inhibitor of the nuclear factor of activated T cells, did not affect the cytokine-induced MMP-9 level. Moreover, CsA caused a dose-dependent inhibition on the IL-1beta-induced luciferase activity of a 1.3-kb MMP-9 promoter fragment. Concomitant, electrophoretic mobility shift assay revealed that CsA selectively inhibits the cytokine-induced DNA binding of activator protein-1 and NF-kappaB. The effects on NF-kappaB binding were accompanied by a marked reduction in the nuclear content of the p65 subunit of NF-kappaB. Accordingly, CsA specifically impaired the IL-1beta-triggered degradation of inhibitory NF-kappaB. The suppressive effects by CsA on MMP-9 expression were accompanied by a reduction in the cytokine-induced phosphorylation of p42/p44 and c-Jun N-terminal Kinase (JNK). It is interesting that only the JNK inhibitor SP600125 impaired the cytokine-triggered MMP-9 level, suggesting that CsA, via inhibition of the JNK pathway, negatively interferes with the NF-kappaB-dependent transcriptional control of MMP-9. Interference with MMP-9 transcription may account for the accumulation of extracellular matrix underlying the high fibrotic potential of CsA during anti-inflammatory therapies with calcineurin inhibitors.

Evidence for BAG3 modulation of HIV‐1 gene transcription

A family of co-chaperone proteins that share the Bcl-2-associated athanogene (BAG) domain are involved in a number of cellular processes, including proliferation and apoptosis. Among these proteins, BAG3 has received increased attention due to its high levels in several disease models and ability to associate with Hsp70 and a number of other molecular partners. BAG3 expression is stimulated during cell response to stressful conditions, such as exposure to high temperature, heavy metals, and certain drugs. Here, we demonstrate that BAG3 expression is elevated upon HIV-1 infection of human lymphocytes and fetal microglial cells. Furthermore, BAG3 protein was detectable in the cytoplasm of reactive astrocytes in HIV-1-associated encephalopathy biopsies, suggesting that induction of BAG3 is part of the host cell response to viral infection. To assess the impact of BAG3 upregulation on HIV-1 gene expression, we performed transcription assays and demonstrated that BAG3 can suppress transcription of the HIV-1 long terminal repeat (LTR) in microglial cells. This activity was mapped to the kappaB motif of the HIV-1 LTR. Results from in vitro and in vivo binding assays revealed that BAG3 suppresses interaction of the p65 subunit of NF-kappaB with the kappaB DNA motif of the LTR. Results from binding and transcriptional assay identified the C-terminus of BAG3 as a potential domain involved in the observed inhibitory effect of BAG3 on p65 activity. These observations reveal a previously unrecognized cell response, that is, an increase in BAG3, elicited by HIV-1 infection, and may provide a new avenue for the suppression of HIV-1 gene expression.

Role of IKK and oscillatory NFκB kinetics in MMP‐9 gene expression and chemoresistance to 5‐fluorouracil in RKO colorectal cancer cells

Nuclear factor kappa B (NFkappaB) is a central participant in the metastasis and chemoresistance of colorectal cancer (CRC). However, it is not fully understood to what extent NFkappaB contributes to induction of the metastasis-associated matrix metalloprotease-9 (MMP-9) gene and sensitivity to the commonly used chemotherapeutic 5-fluorouracil (5-Fu) in CRC. Using the RKO human CRC cell line and two NFkappaB signaling deficient RKO mutants, we investigated NFkappaB's role in the induction of MMP-9 and 5-Fu sensitivity in RKO CRC cells. NFkappaB plays a predominant role in MMP-9 gene induction in RKO cells, as evidenced by the failure of tumor necrosis factor alpha (TNFalpha) to induce MMP-9 in either of the NFkappaB signaling mutants. RKO cells exhibit a robust, oscillatory NFkappaB activity in response to TNFalpha not seen in either of the NFkappaB mutant cell lines, which instead demonstrate diminished, nonoscillatory NFkappaB activation. Analysis of TNFalpha-induced phosphorylation and MMP-9 promoter recruitment of the p65 NFkappaB subunit revealed a significant reduction in p65 phosphorylation as well as reduced and altered recruitment of p65 to the MMP-9 gene promoter in the mutants compared to the parental RKO cell line. 5-Fu only activated NFkappaB in the parental RKO cells through induction of IkappaB-kinase (IKK) activity and increased sensitivity to 5-Fu is observed in both NFkappaB mutant lines. Our results suggest that TNFalpha-dependent induction of MMP-9 gene expression is tightly regulated by oscillatory/cumulative activation of NFkappaB and that 5-Fu stimulates NFkappaB and RKO CRC cell survival through induction of IKK activity.

Distinct patterns of intracerebral hemorrhage‐induced alterations in NF‐κB subunit, iNOS, and COX‐2 expression

Transcription factor nuclear factor-kappaB (NF-kappaB), plays a key role in regulating inflammation in brain pathologies. The goal of this study was to characterize temporal changes in NF-kappaB activation, NF-kappaB subunit expression, and expression of selected NF-kappaB-regulated gene products [inducible form of nitric oxide synthase (iNOS) and cyclooxygenase-2], at the transcriptional and translational level in the brain after intracerebral hemorrhage (ICH). Employing the intrastriatal injection of autologous blood in rats to model ICH, we demonstrated, using NF-kappaB-DNA binding assay, a robust and prolonged NF-kappaB activation, starting as early as 15 min after the onset of ICH. Consequently, we demonstrated that the mRNA and protein for p50, p52, p65, c-Rel, and RelB NF-kappaB subunits, as well as IkappaBalpha were all up-regulated, with a time course ranging from minutes to days following ICH, depending on the subunit. Using reverse transcription-polymerase chain reaction to analyze mRNA and immunoblotting to analyze protein in ICH-affected tissue, we found robust induction of iNOS at both mRNA and protein levels that followed a time-course similar to changes in p65, p52, and RelB mRNA. Oddly, in contrast to iNOS, cyclooxygenase-2 mRNA and protein following an early transient increase demonstrated significant reduction in response to ICH. In summary, NF-kappaB activation occurs within minutes and persists for at least a week in response to ICH. This reaction utilizes different NF-kappaB regulatory subunits and is associated with the expression of selected target genes.

Regulation of neuronal nitric oxide synthase exon 1f gene expression by nuclear factor‐κB acetylation in human neuroblastoma cells

The neuronal nitric oxide synthase (nNOS) is predominantly expressed in nervous tissues and subject to complex transcriptional controls. To determine the effect of acetylation on nNOS expression, human neuroblastoma SK-N-SH cells were treated with trichostatin A (TSA), a histone deacetylase inhibitor. As a consequence, total and exon 1f-specific nNOS mRNA, nNOS protein and nNOS-derived nitric oxide production were increased. Immunoprecipitation and western blot showed both nuclear factor-kappaB (NF-kappaB) subunits p65 and p50 were acetylated in the presence of TSA. The enhancement of the p65 and p50 acetylation was in accordance with their increased binding affinities to the NF-kappaB responsive element, which was identified at position -893 to -884 of the nNOS exon 1f promoter. Luciferase assays revealed that TSA up-regulated the transcriptional activity of the nNOS 1f promoter through NF-kappaB-mediated transactivation. Taken together, we demonstrate that acetylation plays a crucial role in nNOS expression and suggest that acetylation of NF-kappaB p65 and p50 subunits by TSA treatment may augment their DNA-binding affinities, thereby activating the nNOS exon 1f promoter. It may be one of the mechanisms by which acetylation modulates nNOS expression and nitric oxide output in SK-N-SH cells and may be the molecular basis for certain neurological disorders.

Transcriptional profiles of chicken embryo cell cultures following infection with infectious bursal disease virus

Infectious bursal disease virus (IBDV) is the causative agent of infectious bursal disease in chickens and causes a significant economic loss for the poultry industry. Little is understood about the mechanism involved in the host responses to IBDV infection. For better understanding the IBDV-host interaction, we measured steady-state levels of transcripts from 28 cellular genes of chicken embryo (CE) cell cultures infected with IBDV vaccine stain Bursine-2 during a 7-day infection course by use of the quantitative real-time RT-PCR SYBR green method. Of the genes tested, 21 genes (IRF-1, IFN 1-2 promoter, IFNAR-1, IRF-10, IFN-gamma, 2',5'-OAS, IAP-1, caspase 8, TRAIL-like, STAT-3, IL-6, IL-8, MIP-3 alpha, MHC-I, MHC-II, TVB, GLVR-1, OTF, IL-13R alpha, ST3GAL-VI and PGK) showed an increased expression. The remaining seven genes (IFNAR-2, IFN-alpha, NF-kappaB subunit p65, BLRcp38, DDX1, G6PDH and UB) showed a constant expression or only slight alteration. Apparently, the host genes involved in pro-inflammatory response and apoptosis, interferon-regulated proteins, and the cellular immune response were affected by IBDV infection, indicating involvement in the complex signaling pathways of host responses to the infection. This study thus contributes to the understanding of the pathogenesis of IBD and provides an insight into the virus-host interaction.

Anti-inflammatory effects of a novel peptide designed to bind with NF-?B p50 subunit

To explore the anti-inflammatory effects of a novel peptide designed to bind with the NF-kappaB p50 subunit. The affinity of the peptide binding with p50 was measured with a biosensor. Levels of tumor necrosis factor-alpha(TNF-alpha) and interleukin 6 (IL-6) from a human acute monocytic leukemia cell line (THP-1) treated with lipopolysaccharide (LPS) were measured using the ELISA method. In vivo anti-inflammatory effects of the peptide were tested with phorbol myristate acetate (PMA)-induced ear edema and zymosan A-induced peritonitis in mice. The peptide has the ability to interact with the NF-kappaB p50 subunit and can effectively inhibit TNF-alpha and IL-6 production in the THP-1 cell line, PMA-induced ear edema and zymosan A-induced peritonitis in mice. The peptide may have therapeutic potential for the treatment of local acute inflammation.

Silibinin polarizes Th1/Th2 immune responses through the inhibition of immunostimulatory function of dendritic cells

Silibinin is the primary active compound in silymarin. It has been demonstrated to exert anti-carcinogenic effects and hepato-protective effects. However, the effects of silibinin on the maturation and immunostimulatory activities exhibited by dendritic cells (DCs) remain, for the most part, unknown. In this study, we have attempted to determine whether silibinin can influence surface molecule expression, dextran uptake, cytokine production, capacity to induce T-cell differentiation, and the signaling pathways underlying these phenomena in murine bone marrow-derived DCs. Silibinin was shown to significantly suppress the expression of CD80, CD86, MHC class I, and MHC class II in the DCs, and was also associated with impairments of LPS-induced IL-12 expression in the DCs. Silibinin-treated DCs proved highly efficient with regard to Ag capture via mannose receptor-mediated endocytosis. Silibinin also inhibited the LPS-induced activation of MAPKs and the nuclear translocation of the NF-kappaB p65 subunit. Additionally, silibinin-treated DCs evidenced an impaired induction of Th1 response, and a normal cell-mediated immune response. These findings provide new insight into the immunopharmacological functions of silibinin, especially with regard to their impact on the DCs. These findings expand our current understanding of the immunopharmacological functions of silibinin, and may prove useful in the development of therapeutic adjuvants for acute and chronic DC-associated diseases.

Inhibition of NEMO, the regulatory subunit of the IKK complex, induces apoptosis in high-risk myelodysplastic syndrome and acute myeloid leukemia

In high-risk myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), blasts constitutively activate the antiapoptotic transcription factor nuclear factor-kappaB (NF-kappaB). Here, we show that this NF-kappaB activation relies on the constitutive activation of the IkappaB kinase (IKK) complex, which is formed by the IKKalpha, IKKbeta and IKKgamma/NF-kappaB essential modulator (NEMO) subunits. A cell-permeable peptide that mimics the leucine zipper subdomain of IKKgamma, thus preventing its oligomerization, inhibited the constitutive NF-kappaB activation and induced apoptotic cell death in a panel of human MDS and AML cell lines (P39, MOLM13, THP1 and MV4-11). Small interfering RNA-mediated knockdown of the p65 NF-kappaB subunit or the three IKK subunits including IKKgamma/NEMO also induced apoptotic cell death in P39 cells. Cell death induced by the IKKgamma/NEMO-antagonistic peptide involved the caspase-independent loss of the mitochondrial transmembrane potential as well as signs of outer mitochondrial membrane permeabilization with the consequent release of cytochrome c, apoptosis-inducing factor and endonuclease G. Primary bone marrow CD34(+) cells from high-risk MDS and AML patients also succumbed to the IKKgamma/NEMO-antagonistic peptide, but not to a mutated control peptide. Altogether, these data indicate that malignant cells in high-risk MDS and AML cells critically depend on IKKgamma/NEMO to survive. Moreover, our data delineate a novel procedure for their therapeutic removal, through inhibition of IKKgamma/NEMO oligomerization.

Association of p65 and C/EBPβ with HIV‐1 LTR modulates transcription of the viral promoter

In human immunodeficiency virus type 1 (HIV-1) latently infected cells, NF-kappaB (NF-kappaB) plays a critical role in the transcriptional induction of the HIV-1 promoter. The trans-activating ability of NF-kappaB can be modified by another nuclear factor C/EBPbeta that can physically bind to NF-kappaB and regulate its activity. Because the HIV-1 promoter also contains a C/EBPbeta site adjacent to the NF-kappaB site, the present study examined cooperative functional in vivo interaction of the p65 subunit of NF-kappaB and C/EBPbeta, and the impact of Tat in this event. We demonstrated that ectopic expression of p65 along with Tat increases p65 binding to HIV-1 LTR, and that this increase correlates with enhanced HIV-1 promoter activity. Further, co-expression of C/EBPbeta and Tat leads to a decrease in p65 binding, which allows C/EBPbeta to bind more efficiently to the LTR. Inhibition of p65 expression by siRNA significantly decreases C/EBPbeta-binding and LTR expression. Using ChIP assay, we confirmed the existence of an interchange between p65 and C/EBPbeta and their abilities to bind to the LTR in vivo. These observations demonstrate that a delicate balance of interaction between p65, C/EBPbeta, and Tat can dictate the level of HIV-1 LTR transcription.

Alleviation of neuropathic pain by intrathecal injection of antisense oligonucleotides to p65 subunit of NF-κB

Treatment of neuropathic pain remains a challenge. The current study investigated the therapeutic effect of intrathecal administration of NF-kappaB antisense oligodeoxynucleotides (ODNs) on mechanical allodynia and thermal hyperalgesia in a chronic constriction injury (CCI) model of rats. Lumbar intrathecal catheters were implanted in male Sprague-Dawley rats and a CCI model was established. Thermal and mechanical nociceptive thresholds were assessed with paw withdrawal latency (PWL) to radiant heat and von Frey filaments. The phosphorothioate-modified antisense ODNs to p65 subunit of NF-kappaB were administered intrathecally on each of five consecutive days post-CCI. Nuclear NF-kappaB p65 expression was assessed by western blot. CCI induced mechanical allodynia and thermal hyperalgesia and significantly increased NF-kappaB p65 protein expression. Intrathecal injection of antisense ODN markedly suppressed the expression of NF-kappaB p65 protein and significantly attenuated CCI-induced mechanical allodynia and thermal hyperalgesia. The activation of NF-kappaB pathway may contribute to neuropathic pain in CCI rats. Suppression of NF-kappaB could be a potential new strategy for the treatment of neuropathic pain.

Bacteroides fragilisenterotoxin induces cyclooxygenase‐2 and fluid secretion in intestinal epithelial cells through NF‐κB activation

Bacteroides fragilis produces an approximately 20-kDa heat-labile toxin (B. fragilis enterotoxin, BFT) which is known to be associated with diarrhea. To determine whether cyclooxygenase (COX)-2, via NF-kappaB activation, can contribute to BFT-induced diarrhea, the relationship between COX-2 expression and fluid secretion in BFT-stimulated human intestinal epithelial cells was examined. BFT stimulation increased the expression of COX-2, but not COX-1, in human intestinal epithelial cells. Suppression of the NF-kappaB signal significantly decreased COX-2 expression in response to BFT stimulation. Prostaglandin E2 (PGE2) levels were increased in parallel with COX-2 expression, and, conversely, PGE2 production was significantly inhibited when COX-2 or NF-kappaB activities were suppressed using COX-2 small interfering RNA (siRNA), p65 NF-kappaB subunit siRNA, or a retrovirus encoding the IkappaBalpha superrepressor. In addition, a selective COX-2 inhibitor, NS-398, significantly inhibited the increased cAMP level induced by BFT stimulation. Furthermore, a selective COX-2 inhibitor prevented BFT-induced PGE2 production and ileal fluid secretion in a mouse ileal loop model. These results suggest that the secretory response to BFT stimulation may be mediated by the production of PGE2, through NF-kappaB activation and the up-regulation of COX-2 in intestinal epithelial cells.