κB Sites Research Articles

Modulation of NF-κB–Dependent Gene Expression by H2O2: A Major Role for a Simple Chemical Process in a Complex Biological Response

We recently observed that H2O2 regulates inflammation via upexpression of a few NF-kappaB-dependent genes, while leaving expression of most NF-kappaB-dependent genes unaltered. Here we test the hypothesis that this differential gene expression depends on the apparent affinity of kappaB sites in the gene-promoter regions toward NF-kappaB. Accordingly, cells were transfected with three reporter plasmids containing kappaB sequences with different affinities for NF-kappaB. It was observed that the lower the affinity, the higher the range of TNF-alpha concentrations where H2O2 upregulated gene expression. Mathematical models reproduced the key experimental observations indicating that H2O2 upregulation ceased when NF-kappaB fully occupied the kappaB sites. In vivo, it is predicted that genes with high-affinity sites remain insensitive to H2O2, whereas genes with lower-affinity sites are upregulated by H2O2. In conclusion, a simple chemical mechanism is at the root of a complex biologic process such as differential gene expression caused by H2O2.

Synergistic Induction of Endothelin-1 by Tumor Necrosis Factor α and Interferon γ Is due to Enhanced NF-κB Binding and Histone Acetylation at Specific κB Sites

Endothelin-1 (ET-1) is a potent vasoconstrictor and co-mitogen for vascular smooth muscle and is implicated in pulmonary vascular remodeling and the development of pulmonary arterial hypertension. Vascular smooth muscle is an important source of ET-1. Here we demonstrate synergistic induction of preproET-1 message RNA and release of mature peptide by a combination of tumor necrosis factor alpha (TNFalpha) and interferon gamma (IFNgamma) in primary human pulmonary artery smooth muscle cells. This induction was prevented by pretreatment with the histone acetyltransferase inhibitor anacardic acid. TNFalpha induced a rapid and prolonged pattern of nuclear factor (NF)-kappaB p65 subunit activation and binding to the native preproET-1 promoter. In contrast, IFNgamma induced a delayed activation of interferon regulatory factor-1 without any effect on NF-kappaB p65 nuclear localization or consensus DNA binding. However, we found cooperative p65 binding and histone H4 acetylation at distinct kappaB sites in the preproET-1 promoter after stimulation with both TNFalpha and IFNgamma. This was associated with enhanced recruitment of RNA polymerase II to the ATG start site and read-through of the ET-1 coding region. Understanding such mechanisms is crucial in determining the key control points in ET-1 release. This has particular relevance to developing novel treatments targeted at the inflammatory component of pulmonary vascular remodeling.

Structural Basis of HIV-1 Activation by NF-κB—A Higher-Order Complex of p50:RelA Bound to the HIV-1 LTR

The activation and latency of human immunodeficiency virus type 1 (HIV-1) are tightly controlled by the transcriptional activity of its long terminal repeat (LTR) region. The LTR is regulated by viral proteins as well as host factors, including the nuclear factor kappaB (NF-κB) that becomes activated in virus-infected cells. The two tandem NF-κB sites of the LTR are among the most highly conserved sequence elements of the HIV-1 genome. Puzzlingly, these sites are arranged in a manner that seems to preclude simultaneous binding of both sites by NF-κB, although previous biochemical work suggests otherwise. Here, we have determined the crystal structure of p50:RelA bound to the tandem κB element of the HIV-1 LTR as a dimeric dimer, providing direct structural evidence that NF-κB can occupy both sites simultaneously. The two p50:RelA dimers bind the adjacent κB sites and interact through a protein contact that is accommodated by DNA bending. The two dimers clamp DNA from opposite faces of the double helix and form a topological trap of the bound DNA. Consistent with these structural features, our biochemical analyses indicate that p50:RelA binds the HIV-1 LTR tandem κB sites with an apparent anti-cooperativity but enhanced kinetic stability. The slow on and off rates we observe may be relevant to viral latency because viral activation requires sustained NF-κB activation. Furthermore, our work demonstrates that the specific arrangement of the two κB sites on the HIV-1 LTR can modulate the assembly kinetics of the higher-order NF-κB complex on the viral promoter. This phenomenon is unlikely restricted to the HIV-1 LTR but probably represents a general mechanism for the function of composite DNA elements in transcription.

P65/RelA Modulates BECN1 Transcription and Autophagy

Recently, autophagy has emerged as a critical process in the control of T-cell homeostasis. Given the pivotal role of NF-kappaB in the signaling events of T cells, we have analyzed and unveiled a conserved NF-kappaB binding site in the promoter of the murine and human BECN1 autophagic gene (Atg6). Accordingly, we demonstrate that the NF-kappaB family member p65/RelA upregulates BECN1 mRNA and protein levels in different cellular systems. Moreover, p65-mediated upregulation of BECN1 is coupled to increased autophagy. The newly identified kappaB site in the BECN1 promoter specifically interacts with p65 both in vitro and in living Jurkat cells upon phorbol myristate acetate (PMA)-ionomycin stimulation, where p65 induction is coupled to BECN1 upregulation and autophagy induction. Finally, anti-CD3- and PMA-ionomycin-mediated activation of T-cell receptor signaling in peripheral T cells from lymph nodes of healthy mice results in an upregulation of BECN1 expression that can be blocked by the NF-kappaB inhibitor BAY 11-7082. Altogether, these data suggest that autophagy could represent a novel route modulated by p65 to regulate cell survival and control T-cell homeostasis.

E2F1 Induces Tumor Cell Survival via Nuclear Factor-κB–Dependent Induction of EGR1 Transcription in Prostate Cancer Cells

Transcription factor E2F1 has been implicated in both apoptosis-promoting and apoptosis-suppressing effects. However, factors that mediate its antiapoptotic effects are still not identified. Using prostate tumor-derived cell lines, we showed here that E2F1 activated the expression of transcription factor EGR1 for promoting cell survival. E2F1 up-regulated the production of EGR1-induced growth factors, epidermal growth factor, platelet-derived growth factor, and insulin-like growth factor II, which in turn activated the phosphoinositide-3-kinase/Akt pathway to resist drug-induced apoptosis. Moreover, E2F1 directly induced the transcription of the Egr1 gene using the kappaB site located in its proximal promoter. E2F1 physically interacted with the RelA subunit of nuclear factor-kappaB and modulated its transactivity to fully activate EGR1 transcription. Together, these studies uncovered a novel mechanism for E2F1-induced suppression of apoptosis in prostate cancer. [Cancer Res 2009;69(6):2324-31].

RECK Negatively Regulates Matrix Metalloproteinase-9 Transcription

RECK, a glycosylphosphatidylinositol-anchored glycoprotein, inhibits the enzymatic activities of some matrix metalloproteinases (MMP), thereby suppressing tumor cell metastasis; however, the detailed mechanism is still obscure. In this study, we compared the gene expression profiles between mock- and RECK-transfected HT1080 cells and showed that RECK decreases MMP-9 mRNA levels but not other MMP mRNA levels. Moreover, treatment with RECK-specific siRNA increased MMP-9 mRNA in RECK-expressing cells. The promoter assay showed that MMP-9 promoter activity was suppressed by RECK and that RECK-mediated suppression of MMP-9 promoter activity requires 12-O-tetradecanoylphorbol-13-acetate-responsive element (TRE) and kappaB sites. Moreover, the binding ability of Fra-1 and c-Jun to TRE within the MMP-9 promoter region was suppressed by RECK. Thus, these results show that RECK is a negative regulator of MMP-9 transcription.

Induction of histidine decarboxylase in macrophages inhibited by the novel NF-κB inhibitor (−)-DHMEQ

Histamine often causes inflammation, and this amine is produced by histidine decarboxylase (HDC). We found that (−)-DHMEQ, an NF-κB inhibitor, inhibited lipopolysaccharide (LPS)-induced histamine production and HDC induction in mouse macrophage cell line RAW264.7. However, as there is no κB site in the HDC promoter, we studied the mechanism of inhibition. Knockdown of the transcription factor C/EBPβ reduced the HDC expression in LPS-treated cells. (−)-DHMEQ inhibited the C/EBPβ transcriptional activity in a reporter assay and in an electrophoresis mobility shift assay. But it did not inhibit the in vitro binding of C/EBPβ to DNA. It also did not lower the nuclear amount of C/EBPβ. On the other hand, the addition of recombinant p65, a component of NF-κB, enhanced the activity of C/EBPβ acting as a cofactor in vitro. Then, we found that (−)-DHMEQ lowered the nuclear amount of p65. Thus, inhibition of the C/EBPβ activity by (−)-DHMEQ would be due to a reduction in the amount of nuclear p65, which has a co-activator activity for C/EBPβ that is essential for the HDC induction. (−)-DHMEQ may be useful as an anti-inflammatory agent by lowering the histamine production in the body.

NF‐κB p52:RelB heterodimer recognizes two classes of κB sites with two distinct modes

The X‐ray structure of the nuclear factor‐κB (NF‐κB) p52:RelB:κB DNA complex reveals a new recognition feature not previously seen in other NF‐κB:κB DNA complexes. Arg 125 of RelB is in contact with an additional DNA base pair. Surprisingly, the p52:RelB R125A mutant heterodimer shows defects both in DNA binding and in transcriptional activity only to a subclass of κB sites. We found that the Arg 125‐sensitive κB sites contain more contiguous and centrally located A:T base pairs than do the insensitive sites. A protein‐induced kink observed in this complex, which used an AT‐rich κB site, might allow the DNA contact by Arg 125; such a kink might not be possible in complexes with non‐AT‐rich κB sites. Furthermore, we show that the p52:RelB heterodimer binds to a broader spectrum of κB sites when compared with the p50:RelA heterodimer. We suggest that the p52:RelB heterodimer is more adaptable to complement sequence and structural variations in κB sites when compared with other NF‐κB dimers.

A Positive Feedback Loop Between C/EBPα and NF-κB p50 Inhibits Both the Intrinsic and Extrinsic Apoptosis Pathways

Resistance to apoptosis is a hallmark of leukemia, representing a major therapeutic obstacle. We previously showed that CCAAT/enhancer binding protein α (C/EBPα) and its AML mutants inhibit apoptosis in hematopoietic cells dependent upon induction of bcl-2 in cooperation with NF-κB p50. We now identify the genes encoding mcl-1, an anti-apoptotic bcl-2 member and FLIP, an inhibitor of the extrinsic apoptosis pathway, as additional direct genetic targets of the C/EBPα:NF-κB complex. In particular, C/EBPα binds to the BCL2, FLIP or MCL1 promoters, as detected by ChIP assay in both Ba/F3 cells and normal murine marrow or spleen cells. In contrast, C/EBPα does not bind to the promoters of these genes in marrow or spleen cells obtained from nfkb1(−/−) mice that lack NF-κB p50. In addition, induction of C/EBPα from the zinc-responsive MT promoter in Ba/F3 cells leads to induction of BCL2, FLIP, and MCL1 mRNAs. Furthermore, C/EBPα(ΔK312), an AML derived leucine zipper (LZ) mutant that does not bind DNA but can interact with NF-κB p50, retains the ability to localize to the BCL2, FLIP, and MCL1 promoters and to induce their corresponding mRNAs. The promoters for each of these genes also bind endogenous NF-κB p50 as assessed by ChIP assay. We previously demonstrated that the ability of C/EBPα to block the intrinsic apoptotic pathway and protect cells from radiation- or cytokine withdrawal-induced apoptosis is dependent upon induction of bcl-2. We now show that in C/EBPα transgenic splenocytes bcl-2 protein is induced only in the presence of NF-κB p50. As expected from the induction of FLIP by C/EBPα, we now also find that transgenic C/EBPα protects wild-type but not nfkb1(−/−) splenocytes from apoptosis induced by FasL activation of the extrinsic apoptotic pathway. In addition, we now demonstrate direct interaction of C/EBPα with NF-κB p50 using highly purified proteins isolated from E. coli. Together these data indicate that BCL2, MCL1, or FLIP are induced cooperatively by C/EBPα or C/EBPα(ΔK312) proteins that tether to the corresponding promoters via bound NF-κB p50. Additionally, we found that C/EBPα or C/EBPα(ΔK312) bind to the nfkb1 promoter and induce its mRNA expression. Conversely, we also demonstrate that immature, lineage-negative marrow cells isolated from nfkb1(−/−) mice have an approximately 3-fold reduced level of C/EBPα and that NF-κB p50 binds the endogenous CEBPA promoter in normal murine marrow cells in the ChIP assay, indicating cross-regulation and a potential positive feedback loop between C/EBPα and NF-κB p50. Indeed, induction of exogenous C/EBPα in Ba/F3 cells increases nuclear localization of NF-κB as assessed by gel shift assay. In summary, C/EBPα and its AML mutants interact directly with NF-κB p50 via their basic regions; through binding to a DNA κB site the complex activates several anti-apoptotic genes including BCL2, MCL1 and FLIP, thereby inhibiting both the extrinsic and intrinsic apoptotic pathways. In addition, through positive feedback NF-κB p50 induces C/EBPα expression, and C/EBPα induces NF-κB p50. These effects are retained by the C/EBPα AML mutants and do not depend on DNA binding, contributing to NF-κB deregulation in malignant blasts. As C/EBPα or mutant C/EBPα variants and activated NF-κB are commonly expressed together in AML stem cells, targeting the C/EBPα:NF-κB p50 interaction with small molecules may provide a novel therapeutic approach to this and other malignancies.

C/EBPα or C/EBPα oncoproteins regulate the intrinsic and extrinsic apoptotic pathways by direct interaction with NF-κB p50 bound to the bcl-2 and FLIP gene promoters

CCAAT/enhancer binding protein α (C/EBPα) is mutated in 10% of acute myeloid leukemias, resulting in either a truncated protein or an altered leucine zipper (C/EBPαLZ) that prevents DNA-binding. C/EBPα induces bcl-2 in cooperation with NF-κB p50 to inhibit apoptosis. We now demostrate that C/EBPα or a C/EBPαLZ oncoprotein bind the bcl-2 P2 promoter in chromatin immunoprecipitation assays and induce the promoter dependent on the integrity of a κB site. C/EBPα expressed as a transgene in B cells binds and activates the bcl-2 promoter, but not in nfkb1−/− mice lacking NF-κB p50. Bcl-2 is central to the intrinsic apoptotic pathway, while FLICE inhibitory protein (FLIP) modulates caspase-8, the initiator caspase of the extrinsic pathway. C/EBPα and C/EBPαLZ also bind the FLIP promoter and induce its expression dependent upon NF-κB p50. Moreover, induction of FLIP by C/EBPα protects splenocytes from Fas ligand-induced apoptosis, but only if p50 is present. We also demonstrate direct interaction between bacterially produced C/EBPα and NF-κB p50, mediated by the C/EBPα basic region. These findings indicate that C/EBPα or its oncoproteins activate the bcl-2 and FLIP genes by tethering to their promoters via bound NF-κB p50. Targeting their interaction may favor apoptosis of transformed cells.

The LIM-homeodomain transcription factor LMX1B regulates expression of NF-kappa B target genes

LMX1B is a LIM-homeodomain transcription factor essential for development. Putative LMX1B target genes have been identified through mouse gene targeting studies, but their identity as direct LMX1B targets remains hypothetical. We describe here the first molecular characterization of LMX1B target gene regulation. Microarray analysis using a tetracycline-inducible LMX1B expression system in HeLa cells revealed that a subset of NF-κB target genes, including IL-6 and IL-8, are upregulated in LMX1B-expressing cells. Inhibition of NF-κB activity by short interfering RNA-mediated knock-down of p65 impairs, while activation of NF-κB activity by TNF-α synergizes induction of NF-κB target genes by LMX1B. Chromatin immunoprecipitation demonstrated that LMX1B binds to the proximal promoter of IL-6 and IL-8 in vivo, in the vicinity of the characterized κB site, and that LMX1B recruitment correlates with increased NF-κB DNA association. IL-6 promoter–reporter assays showed that the κB site and an adjacent putative LMX1B binding motif are both involved in LMX1B-mediated transcription. Expression of NF-κB target genes is affected in the kidney of Lmx1b −/− knock-out mice, thus supporting the biological relevance of our findings. Together, these data demonstrate for the first time that LMX1B directly regulates transcription of a subset of NF-κB target genes in cooperation with nuclear p50/p65 NF-κB.

Sumoylation of Peroxisome Proliferator-Activated Receptor γ by Apoptotic Cells Prevents Lipopolysaccharide-Induced NCoR Removal from κB Binding Sites Mediating Transrepression of Proinflammatory Cytokines

Efficient clearance of apoptotic cells (AC) by professional phagocytes is crucial for tissue homeostasis and resolution of inflammation. Macrophages respond to AC with an increase in antiinflammatory cytokine production but a diminished release of proinflammatory mediators. Mechanisms to explain attenuated proinflammatory cytokine formation remain elusive. We provide evidence that peroxisome proliferator-activated receptor gamma (PPARgamma) coordinates antiinflammatory responses following its activation by AC. Exposing murine RAW264.7 macrophages to AC before LPS stimulation reduced NF-kappaB transactivation and lowered target gene expression of, that is, TNF-alpha and IL-6 compared with controls. In macrophages overexpressing a dominant negative mutant of PPARgamma, NF-kappaB transactivation in response to LPS was restored, while macrophages from myeloid lineage-specific conditional PPARgamma knockout mice proved that PPARgamma transmitted an antiinflammatory response, which was delivered by AC. Expressing a PPARgamma-Delta aa32-250 deletion mutant, we observed no inhibition of NF-kappaB. Analyzing the PPARgamma domain structures within aa 32-250, we anticipated PPARgamma sumoylation in mediating the antiinflammatory effect in response to AC. Interfering with sumoylation of PPARgamma by mutating the predicted sumoylation site (K77R), or knockdown of the small ubiquitin-like modifier (SUMO) E3 ligase PIAS1 (protein inhibitor of activated STAT1), eliminated the ability of AC to suppress NF-kappaB. Chromatin immunoprecipitation analysis demonstrated that AC prevented the LPS-induced removal of nuclear receptor corepressor (NCoR) from the kappaB site within the TNF-alpha promoter. We conclude that AC induce PPARgamma sumoylation to attenuate the removal of NCoR, thereby blocking transactivation of NF-kappaB. This contributes to an antiinflammatory phenotype shift in macrophages responding to AC by lowering proinflammatory cytokine production.

NF-κB p50 promotes tumor cell invasion through negative regulation of invasion suppressor gene CRMP-1 in human lung adenocarcinoma cells

Lung adenocarcinoma Cl1-5 cells were selected from parental Cl1-0 cells based on their high metastatic potential. In a previous study, CRMP-1, an invasion suppressor gene, was shown to be suppressed in Cl1-5 cells. However, the regulation of CRMP-1 expression has not been explored. In this study, we showed nuclear factor-κB controls CRMP-1 expression. The electromobility shift assay showed that while Cl1-0 cells exhibited low NF-κB activity in response to TNF-α, an abundance of basal and TNF-α-induced NF-κB–DNA complex was detected in Cl1-5 cells. Supershift-coupled EMSA and Western blotting of nuclear proteins, however, revealed p50 protein, but not classic p65/p50 heterodimer in the complex. ChIP and EMSA demonstrated that p50 binds to a κB site residing between −1753 and −1743 of the CRMP-1 promoter region. Transfection of antisense p50 gene into Cl1-5 cells increased the CRMP-1 protein level and decreased the invasive activity of Cl1-5 cells.

Activation of Liver X Receptors Prevents Statin-induced Death of 3T3-L1 Preadipocytes

The biological functions of liver X receptors (LXRs) alpha and beta have primarily been linked to pathways involved in fatty acid and cholesterol homeostasis. Here we report a novel role of LXR activation in protecting cells from statin-induced death. When 3T3-L1 preadipocytes were induced to differentiate by standard isobutylmethylxanthine/dexamethasone/insulin treatment in the presence of statins, they failed to differentiate and underwent massive apoptosis. The simultaneous addition of selective LXR agonists prevented the statin-induced apoptosis. By using mouse embryo fibroblasts from wild-type (LXRalpha+/+/LXRbeta+/+), LXRalpha knock-out mice (LXRalpha(-/-)/LXRbeta+/+), LXRbeta knock-out mice (LXRalpha+/-/LXRbeta(-/-)), and LXR double knock-out mice (LXRalpha(-/-)/LXRbeta(-/-)) as well as 3T3-L1 cells transduced with retroviruses expressing either wild-type LXRalpha or a dominant negative version of LXRalpha, we demonstrate that the response to LXR agonists is LXR-dependent. Interestingly, LXR-mediated rescue of statin-induced apoptosis was not related to up-regulation of genes previously shown to be involved in the antiapoptotic action of LXR. Furthermore, forced expression of Bcl-2 did not prevent statin-induced apoptosis; nor did LXR action depend on protein kinase B, whose activation by insulin was impaired in statin-treated cells. Rather, LXR-dependent rescue of statin-induced apoptosis in 3T3-L1 preadipocytes required NF-kappaB activity, since expression of a dominant negative version of IkappaBalpha prevented LXR agonist-dependent rescue of statin-induced apoptosis. Thus, the results presented in this paper provide novel insight into the action of statins on and LXR-dependent inhibition of apoptosis.

Cellular and molecular mechanisms that mediate basal and tumour necrosis factor‐α‐induced regulation of myosin light chain kinase gene activity

The patients with Crohn's disease (CD) have a ‘leaky gut’ manifested by an increase in intestinal epithelial tight junction (TJ) permeability. Tumour necrosis factor-α (TNF-α) is a proto-typical pro-inflammatory cytokine that plays a central role in intestinal inflammation of CD. An important pro-inflammatory action of TNF-α is to cause a functional opening of intestinal TJ barrier. Previous studies have shown that TNF-α increase in TJ permeability was regulated by an increase in myosin light chain kinase (MLCK) gene activity and protein expression. The major aim of this study was to elucidate the cellular and molecular mechanisms that mediate basal and TNF-α-induced increase in MLCK gene activity. By progressive 5′ deletion, minimal MLCK promoter was localized between −313 to +118 on MLCK promoter. A p53 binding site located within minimal promoter region was identified as an essential determinant for basal promoter activity. A 4 bp start site and a 5 bp downstream promoter element were required for MLCK gene activity. TNF-α-induced increase in MLCK promoter activity was mediated by NF-κB activation. There were eight κB binding sites on MLCK promoter. The NF-κB1 site at +48 to +57 mediated TNF-α-induced increase in MLCK promoter activity. The NF-κB2 site at −325 to −316 had a repressive role on promoter activity. The opposite effects on promoter activity were due to differences in the NF-κB dimer type binding to the κB sites. p50/p65 dimer preferentially binds to the NF-κB1 site and up-regulates promoter activity; while p50/p50 dimer preferentially binds to the NF-κB2 site and down-regulates promoter activity. In conclusion, we have identified the minimal MLCK promoter region, essential molecular determinants and molecular mechanisms that mediate basal and TNF-α-induced modulation of MLCK promoter activity in Caco-2 intestinal epithelial cells. These studies provide novel insight into the cellular and molecular mechanisms that regulate basal and TNF-α-induced modulation of MLCK gene activity.

Selection and characterization of anti-NF-κB p65 RNA aptamers

NF-kappaB transcription factors include a group of five mammalian proteins that form hetero- or homodimers and regulate hundreds of target genes involved in acute inflammation, HIV-1 transcription activation, and resistance to cancer therapy. We previously used in vitro selection to develop a small RNA aptamer (anti-p50) that binds the DNA-binding domain of NF-kappaB p50(2) with low nanomolar affinity but does not bind NF-kappaB p65(2). Here, we report the in vitro selection of anti-NF-kappaB p65 RNA aptamers using parallel in vitro selections with either a fully randomized RNA library or a degenerate RNA library based on the primary sequence of the 31-nucleotide anti-p50 RNA aptamer. We report the characterization of these aptamers with respect to NF-kappaB target specificity, affinity, minimal sequence requirements, secondary structure, and competition with DNA kappaB sites. These results expand opportunities for artificial inhibition of NF-kappaB transcription factor dimers containing p65 subunits.

IRF-1 Is Required for Full NF-κB Transcriptional Activity at the Human Immunodeficiency Virus Type 1 Long Terminal Repeat Enhancer

Human immunodeficiency virus type 1 (HIV-1) gene expression is controlled by a complex interplay between viral and host factors. We have previously shown that interferon-regulatory factor 1 (IRF-1) is stimulated early after HIV-1 infection and regulates promoter transcriptional activity even in the absence of the viral transactivator Tat. In this work we demonstrate that IRF-1 is also required for full NF-kappaB transcriptional activity. We provide evidence that IRF-1 and NF-kappaB form a functional complex at the long terminal repeat (LTR) kappaB sites, which is abolished by specific mutations in the two adjacent kappaB sites in the enhancer region. Silencing IRF-1 with small interfering RNA resulted in impaired NF-kappaB-mediated transcriptional activity and in repressed HIV-1 transcription early in de novo-infected T cells. These data indicate that in early phases of HIV-1 infection or during virus reactivation from latency, when the viral transactivator is absent or present at very low levels, IRF-1 is an additional component of the p50/p65 heterodimer binding the LTR enhancer, absolutely required for efficient HIV-1 replication.

Probiotic lactobacilli and VSL#3 induce enterocyte β-defensin 2

Recent evidence suggests that probiotic bacteria may stabilize gut barrier function via induction of anti-microbial peptides such as defensins. This study aimed to elucidate the induction mechanism of the human beta defensin-2 (hBD-2) gene by different probiotic lactobacillus strains. The expression of hBD-2 mRNA peaked at 6 h of incubation upon treatment of Caco-2 cells and increased with higher dosage of various probiotic bacteria. Deletion of nuclear factor (NF)-kappaB and activator protein-1 (AP-1) binding sites on the hBD-2 promoter resulted in a complete abrogation of promoter activation by probiotics. As revealed by the use of specific mitogen-activated protein kinase (MAPK) inhibitors the hBD-2 induction was dependent on the MAPK extracellular regulated kinase (ERK 1/2), p38 and c-Jun N-terminal kinase (JNK), although to varying degrees. Several Lactobacillus strains and VSL#3, a probiotic cocktail of four lactobacilli, three bifidum and one streptococcus species, induced the secretion of the hBD-2 peptide into the culture media as shown by enzyme-linked immunosorbent assay (ELISA). Thus, the present study suggests that lactobacilli and the VSL#3 bacterial mixture strengthen intestinal barrier functions through the up-regulation of hBD-2 via induction of proinflammatory pathways including NF-kappaB and AP-1 as well as MAPKs.

Hypoxia transcriptionally induces macrophage-inflammatory protein-3α/CCL-20 in primary human mononuclear phagocytes through nuclear factor (NF)-κB

Hypoxia, a condition of low oxygen tension, occurring in many pathological processes, modifies the mononuclear phagocyte transcriptional profile. Here, we demonstrate hypoxic up-regulation of the CCL20 chemokine in primary human monocytes (Mn) and macrophages. mRNA induction was paralleled by protein secretion and dependent on gene transcription activation. Functional studies of the CCL20 promoter using a series of 5'-deleted and mutated reporter constructs demonstrated the requirement for the NF-kappaB-binding site located at position -92/-82 for gene transactivation by hypoxia, as 1) transcription was abrogated by a 3-bp mutation of the NF-kappaB motif; 2) three copies of the wild-type NF-kappaB-binding site conferred hypoxia responsiveness to a minimal heterologous promoter; and 3) hypoxia increased specific NF-kappaB binding to this sequence. Furthermore, we provide evidence of the specific role of a single NF-kappaB family member, p50, in mediating CCL20 gene transcription in hypoxic Mn. p50 homodimers were the only detectable NF-kappaB complexes binding the cognate kappaB site on the CCL20 promoter upon hypoxia exposure, and NF-kappaBp50 knockdown by lentiviral-mediated short hairpin RNA interference resulted in complete binding inhibition. NF-kappaBp50 overexpression in transient cotransfection studies promoted CCL20 gene transactivation, which was abrogated by mutation of the -92/-82 kappaB site. Moreover, nuclear expression of the other NF-kappaB family members was inhibited in hypoxic Mn. In conclusion, this study characterizes a previously unrecognized role for hypoxia as a transcriptional inducer of CCL20 in human mononuclear phagocytes and highlights the importance of the NF-kappaB pathway in mediating this response, with potential implications for inflammatory disease and cancer pathogenesis.

Nuclear factor of activated T-cells (NFAT) plays a role in SV40 infection

Recent evidence highlighted a role for the transcription factor, nuclear factor of activated T-cells (NFAT), in the transcription of the human polyomavirus JCV. Here we show that NFAT is also important in the transcriptional control of the related polyomavirus, Simian Virus 40 (SV40). Inhibition of NFAT activity reduced SV40 infection of Vero, 293A, and HeLa cells, and this block occurred at the stage of viral transcription. Both NFAT3 and NFAT4 bound to the SV40 promoter through κB sites located within the 72 bp repeated enhancer region. In Vero cells, NFAT was involved in late transcription, but in HeLa and 293A cells both early and late viral transcription required NFAT activity. SV40 large T-Ag was found to increase NFAT activity and provided a positive feedback loop to transactivate the SV40 promoter.