κB Binding Sites Research Articles

Epigenetic reprogramming driving successful and failed repair in acute kidney injury.

Acute kidney injury (AKI) causes epithelial damage followed by subsequent repair. While successful repair restores kidney function, this process is often incomplete and can lead to chronic kidney disease (CKD) in a process called failed repair. To better understand the epigenetic reprogramming driving this AKI-to-CKD transition, we generated a single-nucleus multiomic atlas for the full mouse AKI time course, consisting of ~280,000 single-nucleus transcriptomes and epigenomes. We reveal cell-specific dynamic alterations in gene regulatory landscapes reflecting, especially, activation of proinflammatory pathways. We further generated single-nucleus multiomic data from four human AKI samples including validation by genome-wide identification of nuclear factor κB binding sites. A regularized regression analysis identifies key regulators involved in both successful and failed repair cell fate, identifying the transcription factor CREB5 as a regulator of both successful and failed tubular repair that also drives proximal tubular cell proliferation after injury. Our interspecies multiomic approach provides a foundation to comprehensively understand cell states in AKI.

An additional NF-κB site allows HIV-1 subtype C to evade restriction by nuclear PYHIN proteins

SUMMARYSubtype C is the most prevalent clade of human immunodeficiency virus type 1 (HIV-1) worldwide. The reasons for this are poorly understood. Here, we demonstrate that a characteristic additional third nuclear factor κB (NF-κB) binding site in the long terminal repeat (LTR) promoter allows subtype C HIV-1 strains to evade restriction by nuclear PYHIN proteins, which sequester the transcription factor Sp1. Further, other LTR alterations are responsible for rare PYHIN resistance of subtype B viruses. Resistance-conferring mutations generally reduce the dependency of HIV-1 on Sp1 for virus production and render LTR transcription highly responsive to stimulation by NF-κB/p65. A third NF-κB binding site increases infectious virus yield in primary CD4+ T cells in an γ-interferon-inducible protein 16 (IFI16)-dependent manner. Comprehensive sequence analyses suggest that the frequency of circulating PYHIN-resistant HIV-1 strains is increasing. Our finding that an additional NF-κB binding site in the LTR confers resistance to nuclear PYHIN proteins helps to explain the dominance of clade C HIV-1 strains.

Toll-like Receptor (TLR)-induced Rasgef1b expression in macrophages is regulated by NF-κB through its proximal promoter

Ras Guanine Exchange Factor (RasGEF) domain family member 1b is encoded by a Toll-like receptor (TLR)-inducible gene expressed in macrophages, but transcriptional mechanisms that govern its expression are still unknown. Here, we have functionally characterized the 5' flanking Rasgef1b sequence and analyzed its transcriptional activation. We have identified that the inflammation-responsive promoter is contained within a short sequence (-183 to +119) surrounding the transcriptional start site. The promoter sequence is evolutionarily conserved and harbors a cluster of five NF-κB binding sites. Luciferase reporter gene assay showed that the promoter is responsive to TLR activation and RelA or cRel, but not RelB, transcription factors. Besides, site-directed mutagenesis showed that the κB binding sites are required for maximal promoter activation induced by LPS. Analysis by Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) revealed that the promoter is located in an accessible chromatin region. More important, Chromatin Immunoprecipitation sequencing (ChIP-seq) showed that RelA is recruited to the promoter region upon LPS stimulation of bone marrow-derived macrophages. Finally, studies with Rela-deficient macrophages or pharmacological inhibition by Bay11-7082 showed that NF-κB is required for optimal Rasgef1b expression induced by TLR agonists. Our data provide evidence of the regulatory mechanism mediated by NF-κB that facilitates Rasgef1b expression after TLR activation in macrophages.

Safe CRISPR-Cas9 Inhibition of HIV-1 with High Specificity and Broad-Spectrum Activity by Targeting LTR NF-κB Binding Sites

Viral latency of human immunodeficiency virus type 1 (HIV-1) has become a major hurdle to a cure in the highly effective antiretroviral therapy (ART) era. The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system has successfully been demonstrated to excise or inactivate integrated HIV-1 provirus from infected cells by targeting the long terminal repeat (LTR) region. However, the guide RNAs (gRNAs) have classically avoided transcription factor binding sites (TFBSs) that are readily observed and known to be important in human promoters. Although conventionally thought unfavorable due to potential impact on human promoters, our computational pipeline identified gRNA sequences that were predicted to inactivate HIV-1 transcription by targeting the nuclear factor κB (NF-κB) binding sites (gNFKB0, gNFKB1) with a high safety profile (lack of predicted or observed human edits) and broad-spectrum activity (predicted coverage of known viral sequences). Genome-wide, unbiased identification of double strand breaks (DSBs) enabled by sequencing (GUIDE-seq) showed that the gRNAs targeting NF-κB binding sites had no detectable CRISPR-induced off-target edits in HeLa cells. 5′ LTR-driven HIV-1 transcription was significantly reduced in three HIV-1 reporter cell lines. These results demonstrate a working model to specifically target well-known TFBSs in the HIV-1 LTR that are readily observed in human promoters to reduce HIV-1 transcription with a high-level safety profile and broad-spectrum activity.

S100A4/non-muscle myosin II signaling regulates epithelial-mesenchymal transition and stemness in uterine carcinosarcoma

AbstractUterine carcinosarcoma (UCS) represents a true example of cancer associated with epithelial-mesenchymal transition (EMT), which exhibits cancer stem cell (CSC)-like traits. Although S100A4 is an inducer of EMT, little is known about its involvement in UCS tumorigenesis. Herein, we focused on the functional role of S100A4 during development of UCS. Expression of S100A4 and molecules associated with its function were also examined in 35 UCS cases. In endometrial carcinoma cell lines, S100A4 promoter activity and mRNA levels were significantly increased by the transfection of NF-κB/p65, independent of a putative κB-binding site in the promoter. Cells stably overexpressing S100A4 showed enhancement of CSC properties, along with decreased cell proliferation and acceleration of cell migration. These phenotypes were abrogated in S100A4-knockdown cells. A combination of S100A4 antibody-mediated co-immunoprecipitation and shotgun proteomics analysis revealed that S100A4 strongly interacted with non-muscle myosin II (NMII) heavy chains, including myosin 9 and myosin 14. Specific inhibition of NMII by blebbistatin phenocopied S100A4 overexpression and induced a fibroblast-like morphology. In clinical samples, S100A4 score was significantly higher in sarcomatous as compared with carcinomatous components of UCS, and was positively correlated with ALDH1, Slug, and vimentin scores, and inversely with Ki-67 labeling indices. These findings suggest that an S100A4/NMII-related signaling cascade may contribute to the establishment and maintenance of EMT/CSC properties, along with changes in cell proliferation and migration capability. These events may be initiated in carcinomatous components in UCS and lead to divergent sarcomatous differentiation.

Mammalian target of rapamycin and p70S6K mediate thrombin-induced nuclear factor-κB activation and IL-8/CXCL8 release in human lung epithelial cells

Thrombin plays a crucial role in lung inflammatory diseases such as asthma and chronic obstructive pulmonary disease (COPD). Thrombin induces the release of interleukin-8 (IL-8)/CXCL8 by lung epithelial cells, and this phenomenon plays a vital role in lung inflammation. Our previous studies have indicated that thrombin stimulates IL-8/CXCL8 expression through PI3K/Akt/IκB kinase (IKK)α/β/nuclear factor-κB (NF-κB) and p300 pathways in human lung epithelial cells. In the present study, we explored the roles of mammalian target of rapamycin (mTOR) and p70S6 kinase (p70S6K) in thrombin-induced NF-κB activation and IL-8/CXCL8 release in human lung epithelial cells. In this study, we found that rapamycin (an mTOR inhibitor) and p70S6K siRNA diminished thrombin-induced IL-8/CXCL8 release. Thrombin induced mTOR Ser2448 phosphorylation and p70S6K Thr389 phosphorylation in a time-dependent manner. Moreover, rapamycin attenuated thrombin-stimulated p70S6K phosphorylation. We also found that transfection of cells with the dominant negative mutant of Akt (Akt DN) reduced the thrombin-induced increase in mTOR phosphorylation and p70S6K phosphorylation. Moreover, thrombin-stimulated p300 phosphorylation was attenuated by Akt DN, rapamycin, and p70S6K siRNA. Thrombin triggered p70S6K translocation from the cytosol to the nucleus in a time-dependent manner. Thrombin induced the complex formation of p70S6K, p300, and p65; acetylation of p65 Lys310, and recruitment of p70S6K, p300, and p65 to the κB-binding site of the IL-8/CXCL8 promoter region. In conclusion, these results indicate that thrombin initiates the Akt-dependent mTOR/p70S6K signaling pathway to promote p300 phosphorylation and NF-κB activation and finally induces IL-8/CXCL8 release in human lung epithelial cells.

Involvement of multiple transcription factors in regulation of IL-β-induced MCP-1 expression in alveolar type II epithelial cells

During acute lung injury, a large number of monocytes are recruited into the pulmonary tissue, which is mainly mediated by local production of monocyte chemotactic protein 1 (MCP-1). As an essential component of the lung tissues, alveolar type II epithelial cells are one of the major sources of MCP-1. Therefore, uncovering the mechanism whereby MCP-1 production is regulated in the alveolar type II cells will provide a pivotal theoretical basis for clinical intervention in acute lung injury. In the current study, we find that there is a κB binding site in the MCP-1 promoter region, and mutation of the site leads to reduced production of MCP-1 in alveolar type II epithelial cells. In contrast, overexpression of NF-κB p65 significantly increases MCP-1 expression. Furthermore, we elucidate that IKKα/β—NF-κB p65 signaling pathway and phosphorylation of serine 534 in NF-κB p65 are required for the maximal expression of MCP-1. Also, Activator protein 1 (AP-1) site in the promoter region and JNK1/2—c-Jun signaling are required for MCP-1 generation in alveolar type II epithelial cells. Moreover, a CCAAT/enhancer-binding protein (C/EBP) element is identified in the MCP-1 promoter region through the point mutation technique, and further experiments demonstrate that both C/EBPβ and C/EBPδ are involved in basic and IL-1β-mediated MCP-1 expression. Of note, specificity protein 1—Sp1 expression is not changed in alveolar type II epithelial cells incubated with IL-1β, but it still control MCP-1 production by binding to the consensus sequence in the promoter region. More importantly, we find that the results derived from the cell line—MLE-12 cells and primary cells are consistent. Taken together, our data provide insights into the molecular mechanism how MCP-1 expression in inflammatory alveolar type II epithelial cells is regulated at transcription level.

Molecular identification and transcriptional regulation of porcine IFIT2 gene.

IFN-induced protein with tetratricopeptide repeats 2 (IFIT2) plays important roles in host defense against viral infection as revealed by studies in humans and mice. However, little is known on porcine IFIT2 (pIFIT2). Here, we performed molecular cloning, expression profile, and transcriptional regulation analysis of pIFIT2. pIFIT2 gene, located on chromosome 14, is composed of two exons and have a complete coding sequence of 1407 bp. The encoded polypeptide, 468 aa in length, has three tetratricopeptide repeat motifs. pIFIT2 gene was unevenly distributed in all eleven tissues studied with the most abundance in spleen. Poly(I:C) treatment notably strongly upregulated the mRNA level and promoter activity of pIFIT2 gene. Upstream sequence of 1759 bp from the start codon which was assigned +1 here has promoter activity, and deltaEF1 acts as transcription repressor through binding to sequences at position − 1774 to − 1764. Minimal promoter region exists within nucleotide position − 162 and − 126. Two adjacent interferon-stimulated response elements (ISREs) and two nuclear factor (NF)-κB binding sites were identified within position − 310 and − 126. The ISRE elements act alone and in synergy with the one closer to start codon having more strength, so do the NF-κB binding sites. Synergistic effect was also found between the ISRE and NF-κB binding sites. Additionally, a third ISRE element was identified within position − 1661 to − 1579. These findings will contribute to clarifying the antiviral effect and underlying mechanisms of pIFIT2.

Brain-derived neurotrophic factor is regulated via MyD88/NF-\u03baB signaling in experimental Streptococcus pneumoniae meningitis

Streptococcus pneumoniae meningitis is an intractable disease of the central nervous system (CNS). Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophic family and found to participate in the immune inflammatory response. In this study, we investigated if activation of the classical inflammatory signaling pathway, myeloid differentiation factor 88 (MyD88)/nuclear factor-kappa B (NF-κB), regulates BDNF expression in experimental S. pneumoniae meningitis. MyD88 knockout (myd88−/−) mice and wild-type littermates were infected intracisternally with S. pneumoniae suspension. Twenty-four hours after inoculation, histopathology of brains was evaluated. Cytokine and chemokine in brains and spleens was analyzed using ELISA. NF-κB activation was evaluated using EMSA. Cortical and hippocampal BDNF was assessed using RT-PCR and ELISA, respectively. BDNF promoter activity was evaluated using ChIP-PCR. myd88−/− mice showed an obviously weakened inflammatory host response. This diminished inflammation was consistent with worse clinical parameters, neuron injury, and apoptosis. Deficiency in MyD88 was associated with decreased BDNF expression. Furthermore, we identified a valid κB-binding site in the BDNF promoter, consistent with activation of NF-κB induced by inflammation. To sum up, MyD88/NF-κB signaling has a crucial role in up-regulating BDNF, which might provide potential therapeutic targets for S. pneumoniae meningitis.

Tumor necrosis factor‑α and interleukin‑6 suppress microRNA‑1275 transcription in human adipocytes through nuclear factor‑κB.

Obesity is a confirmed risk factor for hyperlipidemia, type-II diabetes, hypertension, and cardiovascular disease. MicroRNAs (miRs) have emerged as an important field of study within energy metabolism and obesity. A previous study demonstrated miR-1275 to be markedly down-regulated during maturation of human preadipocytes. It has been reported that miR-1275 dysregulates expression in several types of cancer and infections. Little is currently known about the regulation of miR-1275 transcription. The aim of the current study was to explore the mechanism underlying the expression of miR-1275 in mature human adipocytes. After differentiation, human adipocytes were incubated with tumor necrosis factor (TNF)-α and interleukin-6. The results of reverse transcription-quantitative polymerase chain reaction demonstrated that miR-1275 can be down-regulated by TNF-α and IL-6, in human mature adipocytes. Bioinformatic analysis was used to predict nuclear factor (NF)-κB binding sites of miR-1275′s promoter region. Luciferase assay and rescue experiments were performed in HEK293T cells. NF-κB was involved in regulating miR-1275 transcription by binding to its promoter. In response to TNF-α, NF-κB was bound to the promoter of miR-1275 and inhibited its transcription. These results indicated that inflammatory factors could regulate miR-1275 transcription through NF-κB and influencing miR-1275 effects on obesity.

Up-Regulation of Transient Receptor Potential Melastatin 6 Channel Expression by Tumor Necrosis Factor-α in the Presence of Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor.

Anti-epidermal growth factor receptor (EGFR) drugs such as erlotinib and gefitinib cause a side effect of hypomagnesemia, but chemotherapy to treat this has not yet been developed. The transient receptor potential melastatin 6 (TRPM6) channel is involved in the reabsorption of Mg2+ in the renal tubule. We reported previously that the expression of TRPM6 is up-regulated by epidermal growth factor (EGF) in renal tubular epithelial NRK-52E and HEK293 cells. EGF-induced elevation of TRPM6 expression was inhibited by erlotinib, gefitinib, and lapatinib. We found that tumor necrosis factor-α (TNF-α) increases TRPM6 expression in the presence of erlotinib. Therefore, we investigated what molecules are involved in the up-regulation of TRPM6 expression by TNF-α. EGF increased the levels of phosphorylated extracellular signal-regulated kinase 1 and 2 (p-ERK1/2), which were inhibited by erlotinib. TNF-α did not change p-ERK1/2 levels, but increased the phosphorylation and nuclear localization of nuclear factor-κB (NF-κB), which were blocked by the NF-κB inhibitors BAY 11-7082 and pyrrolidinedithiocarbamate ammonium. Similarly, luciferase reporter activity of human TRPM6 was increased by TNF-α, which was blocked by NF-κB inhibitors, and was inhibited by a mutation in the κB-binding site in the proximal region of the TRPM6 promoter. A chromatin immunoprecipitation assay revealed that NF-κB binds to the κB-binding site, which was blocked by NF-κB inhibitors. In the presence of erlotinib, TNF-α increased Mg2+ influx, which was blocked by NF-κB inhibitors. These results suggest that TNF-α reverses the reduction in Mg2+ reabsorption caused by anti-EGFR drugs. J. Cell. Physiol. 232: 2841-2850, 2017. © 2016 Wiley Periodicals, Inc.

Vaccinia virus K1 ankyrin repeat protein inhibits NF-κB activation by preventing RelA acetylation.

The vaccinia virus (VACV) K1 protein inhibits dsRNA-dependent protein kinase (PKR) activation. A consequence of this function is that K1 inhibits PKR-induced NF-κB activation during VACV infection. However, transient expression of K1 also inhibits Toll-like receptor (TLR)-induced NF-κB activation. This suggests that K1 has a second NF-κB inhibitory mechanism that is PKR-independent. This possibility was explored by expressing K1 independently of infection and stimulating NF-κB under conditions that minimized or excluded PKR activation. K1 inhibited both TNF- and phorbol 12-myristate 13-acetate (PMA)-induced NF-κB activation, as detected by transcription of synthetic (e.g. luciferase) and natural (e.g. CXCL8) genes controlled by NF-κB. K1 also inhibited NF-κB activity in PKRkd cells, cells that have greatly decreased amounts of PKR. K1 no longer prevented IκBα degradation or NF-κB nuclear translocation in the absence of PKR, suggesting that K1 acted on a nuclear event. Indeed, K1 was present in the nucleus and cytoplasm of stimulated and unstimulated cells. K1 inhibited acetylation of the RelA (p65) subunit of NF-κB, a nuclear event known to be required for NF-κB activation. Moreover, p65-CBP (CREB-binding protein) interactions were blocked in the presence of K1. However, K1 did not preclude NF-κB binding to oligonucleotides containing κB-binding sites. The current interpretation of these data is that NF-κB-promoter interactions still occur in the presence of K1, but NF-κB cannot properly trigger transcriptional activation because K1 antagonizes acetylation of RelA. Thus, in comparison to all known VACV NF-κB inhibitory proteins, K1 acts at one of the most downstream events of NF-κB activation.

Transcriptional and posttranscriptional regulation of CXCL8/IL-8 gene expression induced by connective tissue growth factor.

Connective tissue growth factor (CTGF), a CCN family member, is a secreted protein regulating cellular functions, including fibrosis, apoptosis, adhesion, migration, differentiation, proliferation, angiogenesis, and chondrogenesis. CTGF increases proinflammatory factor production; however, inflammatory cytokine regulation by CTGF is poorly understood. The aim of this study was to identify novel biological functions and elucidate the functional mechanisms of CTGF. Specifically, the study focused on the ability of CTGF-primed monocytes to secrete interleukin 8 (CXCL8/IL-8) and determined the signaling pathways involved in CTGF-induced CXCL8/IL-8 gene regulation during inflammation. We transfected wild-type or mutant CXCL8/IL-8 promoter-derived luciferase reporter constructs into 293T cells to examine the effect of CTGF on the CXCL8/IL-8 promoter. The results showed that the activator protein-1 and nuclear factor κB binding sites of the CXCL8/IL-8 promoter are essential for CTGF-induced CXCL8/IL-8 transcription. Moreover, the CTGF-induced activation of p38 mitogen-activated protein kinase (MAPK), c-Jun-N-terminal kinase, and extracellular signal-regulated kinase (ERK) is involved in this process. In addition, adenosine-uridine-rich elements (AREs) of the CXCL8/IL-8 3'-untranslated region (3'-UTR) reduce CXCL8/IL-8 mRNA stability. To investigate whether CTGF regulates CXCL8/IL-8 gene expression at the posttranscriptional level, we transfected 293 cells with serial luciferase constructs containing different segments of the CXCL8/IL-8 3'-UTR and then stimulated the cells with CTGF. The results suggested that CTGF stabilized luciferase mRNA and increased luciferase activity by regulating the CXCL8/IL-8 3'-UTR. Moreover, the p38 MAPK pathway may contribute to CTGF-induced CXCL8/IL-8 mRNA stabilization.

A genome-wide analysis of antimicrobial effector genes and their transcription patterns in Manduca sexta

Antimicrobial proteins/peptides (AMPs) are effectors of innate immune systems against pathogen infection in multicellular organisms. Over half of the AMPs reported so far come from insects, and these effectors act in concert to suppress or kill bacteria, fungi, viruses, and parasites. In this work, we have identified 86 AMP genes in the Manduca sexta genome, most of which seem likely to be functional. They encode 15 cecropins, 6 moricins, 6 defensins, 3 gallerimycins, 4 X-tox splicing variants, 14 diapausins, 15 whey acidic protein homologs, 11 attacins, 1 gloverin, 4 lebocins, 6 lysozyme-related proteins, and 4 transferrins. Some of these genes (e.g. attacins, cecropins) constitute large clusters, likely arising after rounds of gene duplication. We compared the amino acid sequences of M. sexta AMPs with their homologs in other insects to reveal conserved structural features and phylogenetic relationships. Expression data showed that many of them are synthesized in fat body and midgut during the larval-pupal molt. Certain genes contain one or more predicted κB binding sites and other regulatory elements in their promoter regions, which may account for the dramatic mRNA level increases in fat body and hemocytes after an immune challenge. Consistent with these strong mRNA increases, many AMPs become highly abundant in the larval plasma at 24 h after the challenge, as demonstrated in our previous peptidomic study. Taken together, these data suggest the existence of a large repertoire of AMPs in M. sexta, whose expression is up-regulated via immune signaling pathways to fight off invading pathogens in a coordinated manner.

Tumor necrosis factor-α promotes the lymphangiogenesis of gallbladder carcinoma through nuclear factor-κB-mediated upregulation of vascular endothelial growth factor-C.

Vascular endothelial growth factor (VEGF)-C is an important lymphangiogenic factor involved in the lymphangiogenesis of gallbladder carcinoma (GBC) and the lymph node metastasis of the tumor. Tumor necrosis factor (TNF)-α, a key inflammatory cytokine responding to chronic inflammation of GBC, has been reported to stimulate the expression of VEGF-C in some nonneoplastic cells. But whether TNF-α promotes the expression of VEGF-C in GBC has yet to be determined. Therefore, in the present study, the concentration of TNF-α and VEGF-C and the lymphatic vessel density (LVD) in the clinical GBC specimens were analyzed, and a linear correlation was found between the concentration of TNF-α and that of VEGF-C, the lymphatic vessel density (LVD); The transcription and protein level of VEGF-C in NOZ cell line were detected by real-time polymerase chain reaction (PCR) and enzyme linked immunosorbent assay (ELISA), and TNF-α enhanced the expression of VEGF-C in NOZ cell lines in a dose and time-dependent manner. Lymphatic tube formation in vitro was observed in a three-dimensional coculture system consisting of HDLECs and NOZ cell lines, and lymphatic vessels of GBC in nude mice model was detected by immunohistochemistry. TNF-α promoted the tube formation of lymphatic endothelial cells in vitro and the lymphangiogenesis of GBC in nude mice; The nuclear factor (NF)-κB binding site on the VEGF-C promoter was identified using Site-directed mutagenesis, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation assay (ChIP). Taken together, TNF-α can upregulate the expression of VEGF-C and promote the lymphangiogenesis of GBC via NF-κB combining with the promoter of VEGF-C.

Transcriptional regulation of CCL20 expression

Chemokines are key mediators of leukocyte recruitment during immunoregulatory and proinflammatory responses. CCL20 is a cysteine–cysteine chemokine that was originally shown to be chemotactic for immature dendritic cells, effector or memory CD4+ T lymphocytes, and B lymphocytes. Additionally, CCL20 and its only receptor (CCR6) are exploited by cancer cells for migration and metastatic spread and play important roles in the development and progression of cancer. However, it still remains unclear how the activity of the CCL20/CCR6 axis is controlled and regulated at the transcriptional level. The CCL20 promoter region contains a transcription start site, a nuclear factor (NF)-κB binding site, a CCAAT/enhancer-binding proteins binding site, an activator protein-1 binding sites, and a specificity protein 1 (Sp1)-binding site. In this review, we outline recent advances in our understanding of the structure of the CCL20 promoter region and discuss the transcriptional regulation of the CCL20 promoter.

Transcriptional Repression of the Transforming Growth Factor β (TGF-β) Pseudoreceptor BMP and Activin Membrane-bound Inhibitor (BAMBI) by Nuclear Factor κB (NF-κB) p50 Enhances TGF-β Signaling in Hepatic Stellate Cells

TLR4 signaling induces down-regulation of the bone morphogenic protein (BMP) and activin membrane-bound inhibitor (BAMBI), which enhances TGF-β signaling during hepatic stellate cell (HSC) activation. We investigated the mechanism by which TLR4 signaling down-regulates BAMBI expression in HSCs and found that TLR4- and TNF-α-mediated BAMBI down-regulation is dependent on regulation of BAMBI promoter activity through the interaction with NF-κBp50 and HDAC1 in HSCs. Bambi was predominantly expressed in HSCs, at high levels in quiescent HSCs but at low levels in in vivo-activated and LPS-stimulated HSCs. In human HSCs, BAMBI expression was down-regulated in response to LPS and TNF-α. A BAMBI reporter assay demonstrated that the regulatory element to repress BAMBI transcription is located between 3384 and 1560 bp upstream from the transcription start site. LPS stimulation down-regulated BAMBI expression in cells with NF-κBp65 knockdown. However, it failed to down-regulate BAMBI in cells with inactivation of NF-κB or NF-κBp50 silencing, indicating that NF-κBp50 is a factor for BAMBI down-regulation. ChIP analysis revealed that LPS and TNF-α induced binding of the NF-κBp50/p50 homodimer to the BAMBI promoter region. We also found that HDAC1 is bound to this region as part of the NF-κBp50-HDAC1 complex, repressing transcriptional activity of the BAMBI promoter. Finally, we confirmed that LPS does not repress BAMBI reporter activity using a BAMBI reporter construct with a mutation at 3166 bp upstream of the coding region. In summary, our study demonstrates that LPS- and TNF-α-induced NF-κBp50-HDAC1 interaction represses BAMBI transcriptional activity, which contributes to TLR4-mediated enhancement of TGF-β signaling in HSCs during liver fibrosis.

Regulation of c-Fos Gene Expression by NF-κB: A p65 Homodimer Binding Site in Mouse Embryonic Fibroblasts but Not Human HEK293 Cells

The immediate early gene c-Fos is reported to be regulated by Elk-1 and cAMP response element-binding protein (CREB), but whether nuclear factor (NF)-κB is also required for controlling c-Fos expression is unclear. In this study, we determined how NF-κB’s coordination with Elk/serum response factor (SRF) regulates c-fos transcription. We report that PMA strongly induced c-Fos expression, but tumor necrosis factor (TNF)-α did not. In mouse embryonic fibroblasts, the PMA induction of c-Fos was suppressed by a deficiency in IKKα, IKKβ, IKKγ, or p65. By contrast, in human embryonic kidney 293 cells, PMA induced c-Fos independently of p65. In accordance with these results, we identified an NF-κB binding site in the mouse but not human c-fos promoter. Under PMA stimulation, IKKα/β mediated p65 phosphorylation and the binding of the p65 homodimer to the NF-κB site in the mouse c-fos promoter. Furthermore, our studies demonstrated independent but coordinated functions of the IKKα/β-p65 and extracellular signal-regulated kinase (ERK)-Elk-1 pathways in the PMA induction of c-Fos. Collectively, these results reveal the distinct requirement of NF-κB for mouse and human c-fos regulation. Binding of the p65 homodimer to the κB site was indispensable for mouse c-fos expression, whereas the κB binding site was not present in the human c-fos promoter. Because of an inability to evoke sufficient ERK activation and Elk-1 phosphorylation, TNF-α induces c-Fos more weakly than PMA does in both mouse and human cells.

Basic characterization of avian NK‐lysin (NKL) from the Japanese quail, Coturnix japonica

We identified an antimicrobial cationic peptide that was expressed in the natural killer cells and cytotoxic T-lymphocytes of Japanese quail. The gene, designated CjNKL, was located downstream of AEBP1L and POLD2 in a region syntenic with the chicken genome. CjNKL comprised four exons, as does chicken GgNKL. The coding sequence in CjNKL was 411 bp long and exon 3 of CjNKL lacked 9 bp when compared to chicken GgNKL, but CjNKL and GgNKL were 81% identical at the nucleic acid level. The saposin like type-B domain of CjNKL contained the six essential cysteines, one proline, 15 cationic amino acids residues, and an antibacterial region that are characteristic of NKL proteins. The 5' flanking region of CjNKL contained positive regulatory elements, an activator protein-1 binding site and two nuclear factor (NF)-κB binding sites, and a negative regulatory element, CAAT/enhancer binding protein β (C/EBPβ) binding site. However, the number of NF-κB sites and C/EBPβ sites within CjNKL are fewer than the number within GgNKL. Additionally, we confirmed that CjNKL was transcribed in at least 18 tissues, including immune and digestive tissues. These data indicated that transcriptional activation of CjNKL differed slightly from those of GgNKL.

PTU-154 Sirt2 Modulates the Inflammatory Response in Oesophageal Adenocarcinoma

IntroductionGastro-oesophageal reflux disease is the main risk factor for Barrett’s oesophagus (BE), the precursor lesion to oesophageal adenocarcinoma. In BE, GORD leads to chronic inflammation and to NF-κB pathway activation.SIRT2...