IκBα Superrepressor Research Articles

RelB/p52 NF-κB Complexes Rescue an Early Delay in Mammary Gland Development in Transgenic Mice with Targeted Superrepressor IκB-α Expression and Promote Carcinogenesis of the Mammary Gland

Classical NF-kappaB (p65/p50) transcription factors display dynamic induction in the mammary gland during pregnancy. To further elucidate the role of NF-kappaB factors in breast development, we generated a transgenic mouse expressing the IkappaB-alpha S32/36A superrepressor (SR) protein under control of the mouse mammary tumor virus (MMTV) long terminal repeat promoter. A transient delay in mammary ductal branching was observed in MMTV-SR-IkappaB-alpha mice early during pregnancy at day 5.5 (d5.5) and d7.5; however, development recovered by mid- to late pregnancy (d14.5). Recovery correlated with induction of nuclear cyclin D1 and RelB/p52 NF-kappaB complexes. RelB/p52 complexes induced cyclin D1 and c-myc promoter activities and failed in electrophoretic mobility shift assay to interact with IkappaB-alpha-glutathione S-transferase, indicating that their weak interaction with IkappaB-alpha can account for the observed recovery of mammary gland development. Activation of IKKalpha and NF-kappaB-inducing kinase was detected by d5.5, implicating the alternative NF-kappaB signaling pathway in RelB/p52 induction. Constitutively active IKKalpha induced p52, RelB, and cyclin D1 in untransformed mammary epithelial cells. Moreover, mouse mammary tumors induced by 7,12-dimethylbenz(a)anthracene treatment displayed increased RelB/p52 activity. Inhibition of RelB in breast cancer cells repressed cyclin D1 and c-Myc levels and growth in soft agar. These results implicate RelB/p52 complexes in mammary gland development and carcinogenesis.

Blockade of Histone Deacetylase Inhibitor-Induced RelA/p65 Acetylation and NF-κB Activation Potentiates Apoptosis in Leukemia Cells through a Process Mediated by Oxidative Damage, XIAP Downregulation, and c-Jun N-Terminal Kinase 1 Activation

NF-kappaB activation is reciprocally regulated by RelA/p65 acetylation and deacetylation, which are mediated by histone acetyltransferases (HATs) and deacetylases (HDACs). Here we demonstrate that in leukemia cells, NF-kappaB activation by the HDAC inhibitors (HDACIs) MS-275 and suberoylanilide hydroxamic acid was associated with hyperacetylation and nuclear translocation of RelA/p65. The latter events, as well as the association of RelA/p65 with IkappaBalpha, were strikingly diminished by either coadministration of the IkappaBalpha phosphorylation inhibitor Bay 11-7082 (Bay) or transfection with an IkappaBalpha superrepressor. Inhibition of NF-kappaB by pharmacological inhibitors or genetic strategies markedly potentiated apoptosis induced by HDACIs, and this was accompanied by enhanced reactive oxygen species (ROS) generation, downregulation of Mn-superoxide dismutase and XIAP, and c-Jun N-terminal kinase 1 (JNK1) activation. Conversely, N-acetyl L-cysteine blocked apoptosis induced by Bay/HDACIs by abrogating ROS generation. Inhibition of JNK1 activation attenuated Bay/HDACI lethality without affecting NF-kappaB inactivation and ROS generation. Finally, XIAP overexpression dramatically protected cells against the Bay/HDACI regimen but failed to prevent ROS production and JNK1 activation. Together, these data suggest that HDACIs promote the accumulation of acetylated RelA/p65 in the nucleus, leading to NF-kappaB activation. Moreover, interference with these events by either pharmacological or genetic means leads to a dramatic increase in HDACI-mediated lethality through enhanced oxidative damage, downregulation of NF-kappaB-dependent antiapoptotic proteins, and stress-related JNK1 activation.

Inhibition of MAPK and NF-κB Pathways Is Necessary for Rapid Apoptosis in Macrophages Infected with Yersinia

Macrophages respond to infection with pathogenic Yersinia species by activating MAPK- and NF-kappaB-signaling pathways. To counteract this response, Yersiniae secrete a protease (Yersinia outer protein J (YopJ)) that is delivered into macrophages, deactivates MAPK- and NF-kappaB-signaling pathways, and induces apoptosis. NF-kappaB promotes cell survival by up-regulating expression of several apoptosis inhibitor genes. Previous studies show that deactivation of the NF-kappaB pathway by YopJ is important for Yersinia-induced apoptosis. To determine whether deactivation of the NF-kappaB pathway is sufficient for Yersinia-induced apoptosis, two inhibitors of the NF-kappaB pathway, IkappaBalpha superrepressor or A20, were expressed in macrophages. Macrophages expressing these proteins were infected with Yersinia pseudotuberculosis strains that secrete functionally active or inactive forms of YopJ. Apoptosis levels were substantially higher (5- to 10-fold) when active YopJ was delivered into macrophages expressing IkappaBalpha superrepressor or A20, suggesting that deactivation of the NF-kappaB pathway is not sufficient for rapid Yersinia-induced apoptosis. When macrophages expressing A20 were treated with specific inhibitors of MAPKs, similar levels of apoptosis (within approximately 2-fold) were observed when active or inactive YopJ were delivered during infection. These results suggest that MAPK and NF-kappaB pathways function together to up-regulate apoptosis inhibitor gene expression in macrophages in response to Yersinia infection and that YopJ deactivates both pathways to promote rapid apoptosis. In addition, treating macrophages with a proteasome inhibitor results in higher levels of infection-induced apoptosis than can be achieved by blocking NF-kappaB function alone, suggesting that proapoptotic proteins are stabilized when proteasome function is blocked in macrophages.

The IκB Kinase Is a Key Factor in Triggering Influenza A Virus-induced Inflammatory Cytokine Production in Airway Epithelial Cells

Influenza A viruses continue to represent a severe threat worldwide, causing large epidemics and pandemics responsible for thousands of deaths every year. Excessive inflammation due to overabundant production of proinflammatory cytokines by airway epithelial cells is considered an important factor in disease pathogenesis. Here we report that influenza A virus induced IkappaB kinase (IKK) activity in human airway epithelial A549 cells, resulting in persistent activation of nuclear factor-kappaB (NF-kappaB), a critical regulator of the inflammatory response. Although lung epithelial cells are highly sensitive to stimulation of the IKK/NF-kappaB pathway by influenza virus infection, NF-kappaB was not activated in several non-pulmonary cells permissive to the virus, indicating a cell-specific response. Moreover, NF-kappaB was not essential for virus replication but triggered the expression of proinflammatory cytokines in infected lung cells and was directly responsible for production of high levels of interleukin-8, a chemokine associated with influenza-induced inflammation and airway pathology. We also report that 9-deoxy-delta9,delta12-13,14-dihydro-prostaglandin D2, a cyclopentenone prostanoid with therapeutic efficacy against influenza in preclinical studies, was a powerful inhibitor of influenza virus-induced IKK activity and interleukin-8 production by human pulmonary cells. The results identify IKK as an important factor in triggering influenza virus-induced inflammatory reactions in pulmonary epithelium, suggesting novel therapeutic approaches in the treatment of influenza.

Apoptosis Induced by the Toll-Like Receptor Adaptor TRIF Is Dependent on Its Receptor Interacting Protein Homotypic Interaction Motif

TLRs detect specific molecular features of microorganisms and subsequently engage distinct signaling networks through the differential use of Toll/IL-1R (TIR)-domain-containing adapter proteins. In this study, we investigated the control of apoptosis by the TIR domain-containing adapter proteins MyD88, TIR-domain containing adapter protein (TIRAP), TIR-domain-containing adapter-inducing IFN-beta (TRIF), TRIF-related adapter molecule (TRAM), and sterile alpha motifs and beta-catenin/armadillo repeats (SARM). Upon overexpression, TRIF was the sole TIR-adapter to potently engage mammalian cell death signaling pathways. TRIF-induced cell death required caspase activity initiated by the Fas/Apo-1-associated DD protein-caspase-8 axis and was unaffected by inhibitors of the intrinsic apoptotic machinery. The proapoptotic potential of TRIF mapped to the C-terminal region that was found to harbor a receptor interacting protein (RIP) homotypic interaction motif (RHIM). TRIF physically interacted with the RHIM-containing proteins RIP1 and RIP3, and deletion and mutational analyses revealed that the RHIM in TRIF was essential for TRIF-induced apoptosis and contributed to TRIF-induced NF-kappa B activation. The domain that was required for induction of apoptosis could activate NF-kappa B but not IFN regulatory factor-3, yet the activation of NF-kappa B could be blocked by superrepressor I kappa B alpha without blocking apoptosis. Thus, the ability of TRIF to induce apoptosis was not dependent on its ability to activate either IFN regulatory factor-3 or NF-kappa B but was dependent on the presence of an intact RHIM. TRIF serves as an adaptor for both TLR3 and TLR4, receptors that are activated by dsRNA and LPS, respectively. These molecular motifs are encountered during viral and bacterial infection, and the apoptosis that occurs when TRIF is engaged represents an important host defense to limit the spread of infection.

Expression of Rac1b stimulates NF-κB-mediated cell survival and G1/S progression

The small GTPase Rac1 can stimulate various signaling pathways following a tightly controlled GDP–GTP exchange. A splicing variant designated Rac1b was found to exist predominantly in the active GTP-bound state but the functional consequences of its expression remain unknown. Here we used mouse fibroblasts as a model to assess the signaling properties of Rac1b. We show that, in contrast to Rac1, expression of wild-type Rac1b is sufficient to stimulate cyclin D1 accumulation and G1/S progression in these cells. Moreover, expression of wild-type Rac1b, but not of wild-type Rac1, dramatically increased cell survival in the presence of only minimal growth stimuli. Both cellular responses were blocked by the NF-κB super-repressor IκBα(A32A36). Active Rac1b induced the phosphorylation and membrane translocation of IκBα, a prerequisite for the activation of NF-κB. These data demonstrate that Rac1b is a highly active Rac1 variant that stimulates cell cycle progression and cell survival in pathways involving NF-κB.

NF-κB signalling regulates the growth of neural processes in the developing PNS and CNS

The proper growth and elaboration of neural processes is essential for the establishment of a functional nervous system during development and is an integral feature of neural plasticity throughout life. Nuclear factor-kappa B (NF-kappaB) is classically known for its ubiquitous roles in inflammation, immune and stress-related responses and regulation of cell survival in all tissues, including the nervous system. NF-kappaB participation in other cellular processes remains poorly understood. Here we report a mechanism for controlling the growth of neural processes in developing peripheral and central neurons involving the transcription factor NF-kappaB. Inhibiting NF-kappaB activation with super-repressor IkappaB-alpha, BAY 11 7082 (IkappaB-alpha phosphorylation inhibitor) or N-acetyl-Leu-Leu-norleucinal (proteosomal degradation inhibitor), or inhibiting NF-kappaB transcriptional activity with kappaB decoy DNA substantially reduced the size and complexity of the neurite arbors of sensory neurons cultured with brain-derived neurotrophic factor while having no effect on their survival. NF-kappaB exerted this effect during a restricted period of development following the phase of naturally occurring neuronal death when the processes and connections of the remaining neurons are extensively modified and refined. Inhibiting NF-kappaB activation or NF-kappaB transcriptional activity in layer 2 pyramidal neurons in postnatal somatosensory cortical slices reduced dendritic arbor size and complexity. This function of NF-kappaB has important implications for neural development and may provide an explanation for reported involvement of NF-kappaB in learning and memory.

The Tumor Necrosis Factor-like Weak Inducer of Apoptosis (TWEAK)-Fibroblast Growth Factor-inducible 14 (Fn14) Signaling System Regulates Glioma Cell Survival via NFκB Pathway Activation and BCL-XL/BCL-W Expression

The Fn14 gene encodes a type Ia transmembrane protein that belongs to the tumor necrosis factor receptor superfamily. We recently showed that fibroblast growth factor-inducible 14 (Fn14) is overexpressed in migrating glioma cells in vitro and in glioblastoma multiforme clinical specimens in vivo. To determine the biological role of Fn14 in brain cancer progression, we examined the activity of Fn14 as a potential mediator of cell survival. Tumor necrosis factor-like weak inducer of apoptosis (TWEAK)-stimulated glioma cells had increased cellular resistance to cytotoxic therapy-induced apoptosis. Either TWEAK treatment or Fn14 overexpression in glioma cells resulted in the activation of NFkappaB and subsequently the translocation of NFkappaB from the cytoplasm to the nucleus. In addition, Fn14 activation induced BCL-XL and BCL-W mRNA and protein levels, and this effect was dependent upon NFkappaB transcriptional activity. Substitution of a putative NFkappaB binding site identified in the BCL-X promoter significantly decreased Fn14-induced transactivation. Furthermore Fn14-induced transactivation of the BCL-X promoter was also diminished by the super-repressor IkappaBalpha mutant, which specifically inhibits NFkappaB activity, and by mutations in the NFkappaB binding motif of the BCL-X promoter. Additionally small interfering RNA-mediated depletion of either BCL-XL or BCL-W antagonized the TWEAK protective effect on glioma cells. Our results suggest that NFkappaB-mediated up-regulation of BCL-XL and BCL-W expression in glioma cells increases cellular resistance to cytotoxic therapy-induced apoptosis. We propose that the Fn14 protein functions, in part, through the NFkappaB signaling pathway to up-regulate BCL-XL and BCL-W expression to foster malignant glioblastoma cell survival. Targeted therapy against Fn14 as an adjuvant to surgery may improve management of invasive glioma cells and advance the outcome of this devastating cancer.

Neuronal Activation of NF-κB Contributes to Cell Death in Cerebral Ischemia

The transcription factor NF-kappaB is a key regulator of inflammation and cell survival. NF-kappaB is activated by cerebral ischemia in neurons and glia, but its function is controversial. To inhibit NF-kappaB selectively in neurons and glial cells, we have generated transgenic mice that express the IkappaBalpha superrepressor (IkappaBalpha mutated at serine-32 and serine-36, IkappaBalpha-SR) under transcriptional control of the neuron-specific enolase (NSE) and the glial fibrillary acidic protein (GFAP) promoter, respectively. In primary cortical neurons of NSE-IkappaBalpha-SR mice, NF-kappaB activity was partially inhibited. To assess NF-kappaB activity in vivo after permanent middle cerebral artery occlusion (MCAO), we measured the expression of NF-kappaB target genes by real-time polymerase chain reaction (PCR). The induction of c-myc and transforming growth factor-beta2 by cerebral ischemia was inhibited by neuronal expression of IkappaBalpha-SR, whereas induction of GFAP by MCAO was reduced by astrocytic expression of IkappaBalpha-SR. Neuronal, but not astrocytic, expression of the NF-kappaB inhibitor reduced both infarct size and cell death 48 hours after permanent MCAO. In summary, the data show that NF-kappaB is activated in neurons and astrocytes during cerebral ischemia and that NF-kappaB activation in neurons contributes to the ischemic damage.

The death receptor antagonist FAIM promotes neurite outgrowth by a mechanism that depends on ERK and NF-κB signaling

Fas apoptosis inhibitory molecule (FAIM) is a protein identified as an antagonist of Fas-induced cell death. We show that FAIM overexpression fails to rescue neurons from trophic factor deprivation, but exerts a marked neurite growth–promoting action in different neuronal systems. Whereas FAIM overexpression greatly enhanced neurite outgrowth from PC12 cells and sympathetic neurons grown with nerve growth factor (NGF), reduction of endogenous FAIM levels by RNAi decreased neurite outgrowth in these cells. FAIM overexpression promoted NF-κB activation, and blocking this activation by using a super-repressor IκBα or by carrying out experiments using cortical neurons from mice that lack the p65 NF-κB subunit prevented FAIM-induced neurite outgrowth. The effect of FAIM on neurite outgrowth was also blocked by inhibition of the Ras–ERK pathway. Finally, we show that FAIM interacts with both Trk and p75 neurotrophin receptor NGF receptors in a ligand-dependent manner. These results reveal a new function of FAIM in promoting neurite outgrowth by a mechanism involving activation of the Ras–ERK pathway and NF-κB.

IKKβ/NF-κB Activation Causes Severe Muscle Wasting in Mice

Muscle wasting accompanies aging and pathological conditions ranging from cancer, cachexia, and diabetes to denervation and immobilization. We show that activation of NF-κB, through muscle-specific transgenic expression of activated IκB kinase β (MIKK), causes profound muscle wasting that resembles clinical cachexia. In contrast, no overt phenotype was seen upon muscle-specific inhibition of NF-κB through expression of IκBα superrepressor (MISR). Muscle loss was due to accelerated protein breakdown through ubiquitin-dependent proteolysis. Expression of the E3 ligase MuRF1, a mediator of muscle atrophy, was increased in MIKK mice. Pharmacological or genetic inhibition of the IKKβ/NF-κB/MuRF1 pathway reversed muscle atrophy. Denervation- and tumor-induced muscle loss were substantially reduced and survival rates improved by NF-κB inhibition in MISR mice, consistent with a critical role for NF-κB in the pathology of muscle wasting and establishing it as an important clinical target for the treatment of muscle atrophy.

15-deoxy-Delta12,14-prostaglandin J2 inhibits IFN-inducible protein 10/CXC chemokine ligand 10 expression in human microglia: mechanisms and implications.

Regulation of cytokine and chemokine expression in microglia may have implications for CNS inflammatory disorders. In this study we examined the role of the cyclopentenone PG 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) in microglial inflammatory activation in primary cultures of human fetal microglia. 15d-PGJ(2) potently inhibited the expression of microglial cytokines (IL-1, TNF-alpha, and IL-6). We found that 15d-PGJ(2) had differential effects on the expression of two alpha-chemokines; whereas the Glu-Lys-Arg (ELR)(-) chemokine IFN-inducible protein-10/CXCL10 was inhibited, the ELR(+) chemokine IL-8/CXCL8 was not inhibited. These findings were shown in primary human microglia and the human monocytic cells line THP-1 cells, using diverse cell stimuli such as bacterial endotoxin, proinflammatory cytokines (IL-1 and TNF-alpha), IFN-beta, and HIV-1. Furthermore, IL-8/CXCL8 expression was induced by 15d-PGJ(2) alone or in combination with TNF-alpha or HIV-1. Combined results from EMSA, Western blot analysis, and immunocytochemistry showed that 15d-PGJ(2) inhibited NF-kappaB, Stat1, and p38 MAPK activation in microglia. Adenoviral transduction of super-repressor IkappaBalpha, dominant negative MKK6, and dominant negative Ras demonstrated that NF-kappaB and p38 MAPK were involved in LPS-induced IFN-inducible protein 10/CXCL10 production. Interestingly, although LPS-induced IL-8/CXCL8 was dependent on NF-kappaB, the baseline or 15d-PGJ(2)-mediated IL-8/CXCL8 production was NF-kappaB independent. Our results demonstrate that 15d-PGJ(2) has opposing effects on the expression of two alpha-chemokines. These data may have implications for CNS inflammatory diseases.

Inhibition of NF-Kappa B augments sensitivity to 5-Fluorouracil/Folinic acid in colon cancer 1

Background 5-fluorouracil (5-FU), the most common antimetabolite used for the treatment of colorectal cancer, exerts its cytotoxic effects through the induction of apoptosis. Folinic acid potentiates the effect of 5-FU. Drug activity is currently limited as a result of inducible chemoresistance. Limited research suggests that the transcription factor nuclear factor kappa-B (NF-κB), which has antiapoptotic properties, may play a major role in inducible chemoresistance. Materials and methods SW48 colon cancer cells were used for all experiments. Cell growth was determined by cell proliferation assay. Apoptosis was assessed by measuring caspase 3 activity. Activation of NF-κB was ascertained by electrophoretic mobility shift assay, luciferase reporter assay, and Western blot analysis. Results Treatment with 5-FU (0.001–10 m m), not only inhibited growth and induced apoptosis but significantly activated NF-κB in SW48 cells. Folinic acid alone (0.01–100 mg/L) did not inhibit growth but improved the cytotoxic effect of 5-FU in a dose-dependent manner. Likewise, folinic acid alone did not activate NF-κB or induce apoptosis but enhanced 5-FU-mediated NF-κB activation and cell apoptosis. Transfection with adenovirus IκBα super-repressor strongly inhibited constitutive activation of NF-κB and significantly enhanced 5-FU and 5-FU/Folinic acid-mediated growth inhibition ( P < 0.05). Conclusions Treatment with 5-FU activates NF-κB. Folinic acid enhances 5-FU-mediated activation of NF-κB. Inhibition of NF-κB enhances the cytotoxic effect of 5-FU with or without Folinic acid in colon cancer cells.

Glucocorticoids and tumor necrosis factor alpha cooperatively regulate toll-like receptor 2 gene expression.

Tumor necrosis factor alpha (TNF-alpha) and glucocorticoids are widely recognized as mutually antagonistic regulators of adaptive immunity and inflammation. Surprisingly, we show here that they cooperatively regulate components of innate immunity. The Toll-like receptor 2 (TLR2) gene encodes a transmembrane receptor critical for triggering innate immunity. Although TLR2 mRNA and protein are induced by inflammatory molecules such as TNF-alpha, we show that TLR2 is also induced by the anti-inflammatory glucocorticoids in cells where they also regulate MKP-1 mRNA and protein levels. TNF-alpha and glucocorticoids cooperate to regulate the TLR2 promoter, through the involvement of a 3' NF-kappaB site, a STAT-binding element, and a 3' glucocorticoid response element (GRE). Molecular studies show that the IkappaBalpha superrepressor or a STAT dominant negative element prevented TNF-alpha and dexamethasone stimulation of TLR2 promoter. Similarly, an AF-1 deletion mutant of glucocorticoid receptor or ablation of a putative GRE notably reduced the cooperative regulation of TLR2. Using chromatin immunoprecipitation assays, we demonstrate that all three transcription factors interact with both endogenous and transfected TLR2 promoters after stimulation by TNF-alpha and dexamethasone. Together, these studies define novel signaling mechanism for these three transcription factors, with a profound impact on discrimination of innate and adaptive immune responses.

Carboplatin induces apoptotic cell death through downregulation of constitutively active nuclear factor-κB in human HPV-18 E6-positive HEp-2 cells

Because the role of nuclear factor κB (NF-κB) is in cellular growth control and neoplasia, we explored the status of NF-κB and investigated its role in survival of human HPV-18 E6-positive HEp-2 cells. We observed accumulation of p65 in the nucleus. Moreover, without any external stimulus constitutive NF-κB DNA binding and transactivation activity were detected in HEp-2 cells. Treatment with NF-κB inhibitor curcumin (diferuloylmethane) and transient transfection of the mutant form of IκBα, IκBα super repressor (IκBα-SR), suppressed constitutive NF-κB activity as well as proliferation, suggesting that constitutive NF-κB activity is required for the survival of HEp-2 cells. Carboplatin treatment downregulated constitutive NF-κB activity and prevented nuclear retention of p65. Further, carboplatin also suppressed the constitutive IκBα phosphorylation leading to stabilization of IκBα protein in the cells. Carboplatin inhibited NF-κB binding to its response element present in Bcl-2 promoter resulting in downregulation of antiapoptotic Bcl-2 protein. Thus, our results for the first time indicate that constitutive NF-κB has a significant role in the survival of HPV-18 E6-positive HEp-2 cells. Moreover, inactivation of NF-κB is one of the mechanisms underlying the induction of carboplatin-mediated apoptosis in HPV-18 E6-positive cancer cells.

Identification of a negative regulatory cis-element in the enhancer core region of the prostate-specific antigen promoter: implications for intersection of androgen receptor and nuclear factor-kappaB signalling in prostate cancer cells.

The NF-kappaB (nuclear factor-kappaB) transcription factors mediate activation of a large number of gene promoters containing diverse kappaB-site sequences. Here, PSA (prostate-specific antigen) was used as an AR (androgen receptor)-responsive gene to examine the underlying mechanism by which the NF-kappaB p65 transcription factor down-regulates the transcriptional activity of AR in cells. We observed that activation of NF-kappaB by TNFalpha (tumour necrosis factor alpha) inhibited both basal and androgen-stimulated PSA expression, and that this down-regulation occurred at the promoter level, as confirmed by the super-repressor IkappaBalpha (S32A/S36A), a dominant negative inhibitor of NF-kappaB. Using a linker-scanning mutagenesis approach, we identified a cis -element, designated XBE (X-factor-binding element), in the AREc (androgen response element enhancer core) of the PSA promoter, which negatively regulated several AR-responsive promoters, including that of PSA. When three copies of XBE in tandem were juxtaposed to GRE4 (glucocorticoid response element 4), a 4-6-fold reduction of inducible GRE4 activity was detected in three different cell lines, LNCaP, ARCaP-AR and PC3-AR. Bioinformatics and molecular biochemical studies indicated that XBE is a kappaB-like element that binds specifically to the NF-kappaB p65 subunit; consistent with these observations, only NF-kappaB p65, but not the NF-kappaB p50 subunit, was capable of inhibiting AR-mediated PSA promoter transactivation in LNCaP cells. In addition, our data also showed that AR binds to XBE, as well as to the kappaB consensus site, and that the transfection of AR inhibits the kappaB-responsive promoter in transient co-transfection assays. Collectively, these data indicate that cross-modulation between AR and NF-kappaB p65 transcription factors may occur by a novel mechanism involving binding to a common cis -DNA element.

NF-κB and c-Jun-dependent regulation of macrophage inflammatory protein-2 gene expression in response to lipopolysaccharide in RAW 264.7 cells

Macrophage inflammatory protein-2 (MIP-2) is a mouse C–X–C chemokine that plays an important role in the recruitment of neutrophils. Transcription of the MIP-2 gene is rapidly induced by lipopolysaccharide (LPS) stimulation in cells of macrophage lineage. We show here that the MIP-2 promoter is transcriptionally activated in a macrophage cell line RAW 264.7 by LPS through a sequence located between −450 and −54 and this region contains two copies of activator protein-1 (AP-1) and one copy of nuclear factor-κB (NF-κB) binding site. A MIP-2 promoter-reporter was activated by ectopical expression of NF-κB p65 or c-Jun transcription factors. Inhibition of NF-κB nuclear localization by co-expression of a mutant IκBα protein (IκBα super repressor, IκBαSR) blocked LPS-induced transcription from a MIP-2 promoter-reporter construct, showing that NF-κB activation is required for MIP-2 gene expression in the LPS-signaling pathway. By deletion analysis of the MIP-2 promoter region, we show that NF-κB and c-Jun binding sites are essential for LPS-induced MIP-2 gene expression. Using transient transfection, NF-κB and c-Jun transcription factors were found to synergistically activate the MIP-2 promoter. In summary, our data suggest that both NF-κB and c-Jun contribute to LPS-induced mouse MIP-2 gene expression in RAW 264.7 cells.

Drug-sensitivity pattern analysis for study of functional relationship between gene products

We have developed a method that we call ‘drug-sensitivity pattern analysis’, or DSPA, for analysis of protein function. Cells are transfected with cDNA of the test molecule, followed by analysis of the sensitivity of the transfected cells to multiple growth-inhibitory drugs. If two cDNA products have similar functions, their transfected cells should show similar drug-sensitivity patterns. The cDNAs of some signaling molecules were transfected into NIH3T3 or Ha- ras-transformed NIH3T3 (ras-NIH) cells and stable transfectants, which expressed high amounts of the gene product, were isolated. Chemosensitivity of the transfected clone was compared with the parental cells by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide method using more than 40 drugs. The chemosensitivity changes caused by the transfected gene were calculated and expressed numerically as ‘drug chemosensitivity index’ (DCI). When the DCI values were analyzed by regression analysis, a significant positive relationship between IκBα superrepressor and dominant-negative IKKβ and an inverse relationship between p53 and Mdm2 were consistent with previous reports. Thus, the DSPA method is useful for identifying functional similarities between gene products.

HCC chemo-resistance to camptothecin is through NF-κB-independent pathway

The transcription factor NF-KB has anti-apoptotic properties and may confer chemo-resistance to cancer cells, NF-KB is constitutively activated in bladder cancer, breast cancer, and melanoma, in which cancer cell lines, inhibition of NF-KB induces cell death in bepatocellular carcinoma (HCC), constitutive activation of NF-KB was tbund more frequently in tumor tissue compared with non-tumor tissue. Our AIM is to clarify the role of NF-KB in camptothecin-induced apoptosis of HCC. METHODS: Studies were performed in 3 hepatoma cell lines, Hep3B, HepG2 and Huh7. Camptothecin-induced apoptosis was evaluated wath DNA [addering assay and DNA fragmentation ELISA. Western blot analysis for NF-KB (p65) and IKB were performed with nuclear extracts and whole lysates, respectively, to examine the activation of NF-KB by, camptothecin. To block NF-KB activation, adenov~rus expressing IKB super-repressor (Ad5IgBsf) was intected with ceils 12 hours befm'e the treatment of camptothecin We examined whether camptothecin (0.5-2 t~M)-iuduced apoptosis was enhanced under transt~ction of Ad5IKBsr (50-200 MOI) DNA fragmentation ELISA. RESULTS DNA laddering was clearly shown alter 1-2 p.M of camptnthecin treatment in all 3 HCC cells. DNA fragmentation ELISA demonstrated that the rate of apoptotic cell death in Hep3B was 20% lower than that of HepG2, indicating that Hep3B is resistant to camptothecininduced apoptosis. The pretreatment of cyclobeximide (to block the protein symthesis) with Hep3B did not enhance camptothecin-induced apoptosis. NF-KB was constitutively activated in Hep3B and camptothecni enhanced the translocation of NF-KB to the nucleus from 30 to 120min after camptothecin. After 12 hours of AdSIKBsr infection, we confirmed the expression of IKB super-repressor in Hep3B However, inactivation of NF-KB by Ad5lKBsr did not sensitize Hep3B to camptothecin-induced apoptosis in Hep3B in Conchision, HCC chemo-resistance to camptothecin is through NF-,~rB-independent pathway.

Inhibition of IkappaB kinase by a new class of retinoid-related anticancer agents that induce apoptosis.

The transcription factor NF-kappaB is overexpressed or constitutively activated in many cancer cells, where it induces expression of antiapoptotic genes correlating with resistance to anticancer therapies. Small molecules that inhibit the NF-kappaB signaling pathway could therefore be used to induce apoptosis in NF-kappaB-overexpressing tumors and potentially serve as anticancer agents. We found that retinoid antagonist MX781 inhibited the activation of NF-kappaB-dependent transcriptional activity in different tumor cell lines. MX781 was able to completely inhibit tumor necrosis factor alpha-mediated activation of IkappaB kinase (IKK), the upstream regulator of NF-kappaB. Inhibition of IKK activity resulted from direct binding of MX781 to the kinase, as demonstrated by in vitro inhibition studies. Two other molecules, MX3350-1 and CD2325, which are retinoic acid receptor gamma-selective agonists, were capable of inhibiting IKK in vitro, although they exerted variable inhibition of IKK and NF-kappaB activities in intact cells in a cell type-specific manner. However, N-(4-hydroxyphenyl)-retinamide, another apoptosis-inducing retinoid, and retinoic acid as well as other nonapoptotic retinoids did not inhibit IKK. Inhibition of IKK by the retinoid-related compounds and other small molecules correlated with reduced cell proliferation and increased apoptosis. Reduced cell viability was also observed after overexpression of an IKKbeta kinase-dead mutant or the IkappaBalpha superrepressor. The induction of apoptosis by the retinoid-related molecules that inhibited IKK was dependent on caspase activity but independent of the retinoid receptors. Thus, the presence of an excess of retinoic acid or a retinoid antagonist did not prevent the inhibition of IKK activation by MX781 and CD2325, indicating a retinoid receptor-independent mechanism of action.