NF-kappaB Site Research Articles

ONECUT1 variants beyond type 1 and type 2 diabetes: exploring clinical diversity and epigenetic associations in Arab cohorts.

ONECUT1 gene, encoding hepatocyte nuclear factor 6, is involved in pancreas and liver development. ONECUT1 mutations impair the function of pancreatic β-cells and control a transcriptional/epigenetic machinery regulating endocrine development. Homozygous nonsense and missense mutations at ONECUT1_p.E231 and a homozygous frameshift mutation at ONECUT1_p.M289 were reported in neonatal diabetes individuals of French, Turkish, and Indian ethnicity, respectively. Additionally, heterozygous variants were observed in Northern European T2D patients, and Italian patients with neonatal diabetes and early-/late-onset T2D. Examining diverse populations, such as Arabs known for consanguinity, can generalize the ONECUT1 involvement in diabetes. Upon screening the cohorts of Kuwaiti T1D and MODY families, and of Kuwaiti and Qatari T2D individuals, we observed two homozygous variants-the deleterious missense rs202151356_p.H33Q in one MODY, one T1D, and two T2D individuals, and the synonymous rs61735385_p.P94P in two T2D individuals. Heterozygous variants were also observed. Examination of GTEx, NephQTL, mQTLdb and HaploReg highlighted the rs61735385_p.P94P variant as eQTL influencing the tissue-specific expression of ONECUT1, as mQTL influencing methylation at CpG sites in and around ONECUT1 with the nearest site at 677-bases 3' to rs61735385_p.P94P; as overlapping predicted binding sites for NF-kappaB and EBF on ONECUT1. DNA methylation profiles of peripheral blood from 19 MODY-X patients versus eight healthy individuals revealed significant hypomethylation at two CpG sites-one located 617-bases 3' to the p.P94P variant and 8,102 bases away from transcription start; and the other located 14,999 bases away from transcription start. Our study generalizes the association of ONECUT1 with clinical diversity in diabetes.

Abstract 3549: DeltaNp63 overexpression induced cytokines and chemokines attract type2 suppressive inflammatory infiltrates through NF-kappaB activation in dysplastic and malignant epithelia

Abstract We and others have previously reported that overexpression of DeltaNp63, a TP53 family member and antagonist, is often accompanied by inflammation in pre-malignant as well as cancerous lesions. However, the molecular and cellular nature of the DeltaNp63-induced inflammation and whether it favors tumorigenesis is presently unclear. Here, we report that induced expression of DeltaNp63 in the basal epidermis of transgenic (Tg) mice leads to skin erythema with a microscopically hyperproliferative epidermis and heavy infiltration of inflammatory cells. In situ phenotypic analysis of the infiltrating cells revealed increased recruitment of F4/80+ and CD11b+ macrophages, Ly6B+ neutrophils, S-100+ dentritic cells, CD45+ leukocytes, and CD3+ lymphocytes. Macrophages of the suppressive M2 phenotype were identified as a CD68+ and F4/80+/CD206+ double positive subpopulation. Transcriptome analysis of the skin lesions from DeltaNp63 Tg mice revealed 683 up-regulated genes, ∼19% of which were specifically involved in inflammation/immune responses and related signaling pathways, including many cytokine and chemokine genes. These inflammatory cytokines and chemokines inferred predominant T helper 2 and M2 signatures, and formed network connections with NF-kappaB by Ingenuity Pathway Analysis. We annotated promoter regions of these cytokine and chemokine genes by Genomatix, most of which contained predicted NF-kappaB binding consensus motifs. In addition, in human squamous cell carcinoma (SCC) lines, we showed that DeltaNp63-mediated promoter activities of chemokines and cytokines were partially blocked by knockdown of DeltaNp63, and either NF-kappaB RELA/p65 or c-REL. The reporter activities were significantly diminished when NF-kappaB sites were mutated. Supporting DeltaNp63 activation of the NF-kappaB pathway in vivo, we observed increased expression of phosphorylated NF-κB RelA/p65 (pS276), c-Rel, IKKalpha and IKKbeta in the hyperproliferative skin of DeltaNp63 Tg mice. Consistent with the aforementioned data, we observed overexpression of DeltaNp63, increased infiltrating inflammatory cells, and elevated immunohistochemistry of NF-κB signaling molecules in human SCC tissues. Together, our data support a model in which the elevated level of DeltaNp63 in the epidermis promotes NF-kappaB activation and a skewed spectrum of cytokines and chemokines, which in turn trigger type 2 suppressive inflammatory responses with characteristics of those reported in pre- and malignant squamous epithelia. Our data suggest that the most frequently mutated and aberrantly expressed TP53 family members could trigger inflammatory and immune responses, which favor tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3549. doi:1538-7445.AM2012-3549

Phorbol ester-induced human cytomegalovirus major immediate-early (MIE) enhancer activation through PKC-delta, CREB, and NF-kappaB desilences MIE gene expression in quiescently infected human pluripotent NTera2 cells.

The ways in which human cytomegalovirus (HCMV) major immediate-early (MIE) gene expression breaks silence from latency to initiate the viral replicative cycle are poorly understood. A delineation of the signaling cascades that desilence the HCMV MIE genes during viral quiescence in the human pluripotent N-Tera2 (NT2) cell model provides insight into the molecular mechanisms underlying HCMV reactivation. In this model, we show that phorbol 12-myristate 13-acetate (PMA) immediately activates the expression of HCMV MIE RNA and protein and greatly increases the MIE-positive (MIE(+)) NT2 cell population density; levels of Oct4 (pluripotent cell marker) and HCMV genome penetration are unchanged. Decreasing PKC-delta activity (pharmacological, dominant-negative, or RNA interference [RNAi] method) attenuates PMA-activated MIE gene expression. MIE gene activation coincides with PKC-delta Thr505 phosphorylation. Mutations in MIE enhancer binding sites for either CREB (cyclic AMP [cAMP] response element [CRE]) or NF-kappaB (kappaB) partially block PMA-activated MIE gene expression; the ETS binding site is negligibly involved, and kappaB does not confer MIE gene activation by vasoactive intestinal peptide (VIP). The PMA response is also partially attenuated by the RNAi-mediated depletion of the CREB or NF-kappaB subunit RelA or p50; it is not diminished by TORC2 knockdown or accompanied by TORC2 dephosphorylation. Mutations in both CRE and kappaB fully abolish PMA-activated MIE gene expression. Thus, PMA stimulates a PKC-delta-dependent, TORC2-independent signaling cascade that acts through cellular CREB and NF-kappaB, as well as their cognate binding sites in the MIE enhancer, to immediately desilence HCMV MIE genes. This signaling cascade is distinctly different from that elicited by VIP.

Improvement and induction property of radiation-responsive promoter through DNA shuffling of 5′-flanking regions of the human p21 gene

A promoter that augments gene expression in response to stimulation of ionizing radiation would be a desired tool for radiogenetic therapy, a combination of radiotherapy and gene therapy. Although various promoters occurring naturally or artificially have been used for researches, one showing higher reactivity to ionizing radiation is desirable. In the present study, we attempted to improve a radiation-responsive promoter of the p21 through a technique called DNA shuffling. A library of DNA fragments was constructed by re-ligation of randomly digested promoter fragments and improved promoters were chosen out of the library. We repeated this process twice to obtain a promoter showing 2.6-fold better reactivity to ionizing radiation compared with its parent, p21 promoter after 10 Gy gamma-ray irradiation. Nucleotide sequence analyses revealed that the obtained promoter was densely packed with some of the cis-acting elements including binding sites for p53, NF-kappaB, NRF-2, AP-1 and NF-Y more than p21 promoter. In addition, it was shown that its induction by ionizing radiation was dependent upon p53 status of a cell line, suggesting that the promoter retained properties of the p21 promoter. This technique is simple and efficient to improve a promoter responsive to other stimulus of interest besides IR.

TAp63 is a transcriptional target of NF‐κB

The p53 homologue p63 encodes multiple protein isoforms either with (TA) or without (DeltaN) the N-terminal transactivation domain. Accumulating evidence indicates that TAp63 plays an important role in various biological processes, including cell proliferation, differentiation, and apoptosis. However, how TAp63 is regulated remains largely unclear. In this study, we demonstrate that NF-kappaB induces TAp63 gene expression. The responsible elements for NF-kappaB-mediated TAp63 induction are located within the region from -784 to -296 bp in the TAp63 promoter, which contains two NF-kappaB binding sites. Ectopic expression of RelA stimulates TAp63 promoter-driven reporter activity and increases endogenous TAp63 mRNA levels. Inhibition of NF-kappaB by IkappaBalpha super-repressor or with a chemical inhibitor leads to down regulation of TAp63 mRNA expression and activity. In addition, mutations in the critical NF-kappaB-binding sites significantly abolish the effects of NF-kappaB on TAp63. Activation of NF-kappaB by TNFalpha enhances p50/RelA binding to the NF-kappaB binding sites. Furthermore, we show that an Sp1 site adjacent to the NF-kappaB sites plays a role in NF-kappaB-mediated upregulation of TAp63. Taken together, these data reveal that TAp63 is a transcriptional target of NF-kappaB, which may play a role in cell proliferation, differentiation and survival upon NF-kappaB activation by various stimuli.

Kaposi's sarcoma-associated herpesvirus-encoded viral FLICE inhibitory protein (vFLIP) K13 suppresses CXCR4 expression by upregulating miR-146a

Kaposi's sarcoma (KS) associated herpesvirus (KSHV)-encoded viral FLICE inhibitory protein (vFLIP) K13 is a potent activator of the NF-κB pathway. Here we demonstrate that infection with KHSV and ectopic expression of K13, but not its NF-κB-defective mutant, suppressed the expression of CXCR4. Suppression of CXCR4 by KSHV and K13 was associated with upregulated expression of miR-146a, a microRNA that is known to bind to the 3′ untranslated region of CXCR4 mRNA. Reporter studies identified two NF-κB sites in the promoter of miR-146a that were essential for its activation by K13. Accordingly, ectopic expression of K13, but not its NF-κB-defective mutant or other vFLIPs, strongly stimulated the miR-146a promoter activity, which could be blocked by specific genetic and pharmacological inhibitors of the NF-κB pathway. Finally, expression of CXCR4 was downregulated in clinical samples of KS and this was accompanied by increased expression of miR-146a. Our results demonstrate that K13-induced NF-κB activity suppresses CXCR4 via upregulation of miR-146a. Downregulation of CXCR4 expression by K13 may contribute to KS development by promoting premature release of KSHV-infected endothelial progenitors into the circulation.

PU.1 is regulated by NF-κB through a novel binding site in a 17 kb upstream enhancer element

The majority of patients with acute myeloid leukemia (AML) still die of their disease, and novel therapeutic concepts are needed. Timely expression of the hematopoietic master regulator PU.1 is crucial for normal development of myeloid and lymphoid cells. Targeted disruption of an upstream regulatory element (URE) located several kb upstream in the PU.1 promoter decreases PU.1 expression thereby inducing AML in mice. In addition, suppression of PU.1 has been observed in specific subtypes of human AML. Here, we identified nuclear factor-kappaB (NF-kappaB) to activate PU.1 expression through a novel site within the URE. We found sequence variations of this particular NF-kappaB site in 4 of 120 AML patients. These variant NF-kappaB sequences failed to mediate activation of PU.1. Moreover, the synergistic activation of PU.1 together with CEBPB through these variant sequences was also lost. Finally, AML patients with such variant sequences had suppressed PU.1 mRNA expression. This study suggests that changes of a single base pair in a distal element critically affect the regulation of the tumor suppressor gene PU.1 thereby contributing to the development of AML.

Human Rhinovirus Infection Up-Regulates MMP-9 Production in Airway Epithelial Cells via NF-κB

Human rhinovirus (HRV) infections up-regulate proinflammatory mediators and growth factors that are associated with exacerbations of inflammatory airway diseases, such as asthma and chronic obstructive pulmonary disease (COPD). Matrix metalloproteinase (MMP)-9 was shown to be increased in the airways of patients with asthma and COPD. We sought to determine whether HRV infection modulated the expression of MMP-9 and its highest-affinity inhibitor, the tissue inhibitor of metalloproteinase (TIMP)-1, and we explored the mechanism by which this modulation occurs. In vitro studies, using RT-PCR, ELISA, zymography, and a fluorescent activity assay, demonstrated that MMP-9 mRNA, protein, and activity were increased upon infection with HRV, whereas TIMP-1 mRNA and protein remained unchanged. These results were verified in vivo, using nasal lavage samples obtained from subjects with confirmed rhinovirus infections. Human rhinovirus infections were shown to up-regulate NF-kappaB, and NF-kappaB has also been reported to play a role in the expression of MMP-9. We therefore investigated the role of NF-kappaB in HRV-induced MMP-9 expression. Using two inhibitors of IkappaBalpha kinase beta, we observed a concentration-dependent decrease in HRV-induced MMP-9 expression. The role of NF-kappaB in HRV-induced MMP-9 expression was further confirmed using MMP-9 promoter luciferase constructs, which demonstrated that an NF-kappaB site at -620/-607 base pairs was necessary for HRV-induced MMP-9 expression. Electrophoretic mobility shift assays and supershift assays confirmed the nuclear translocation and binding of p50/p65 NF-kappaB subunits to an MMP-9-specific NF-kappaB oligonucleotide. This increase in MMP-9 may be a mechanism by which rhinovirus infections contribute to airway inflammation and, potentially, to airway remodeling.

CXCR7 is inducible by HTLV‐1 Tax and promotes growth and survival of HTLV‐1‐infected T cells

Human T-lymphotropic virus type 1 (HTLV-1), the etiological agent of adult T-cell leukemia (ATL), encodes the potent transcriptional activator Tax, which is required for HTLV-1-induced immortalization of T cells. CXCR7 is an atypical chemokine receptor frequently expressed by tumor cells and known to promote cell growth and survival. We found that HTLV-1-immortalized T cells expressing Tax consistently expressed CXCR7. Induction of Tax in JPX-9 upregulated CXCR7. Wild-type Tax efficiently activated the CXCR7 promoter via a proximal NF-kappaB site, while a mutant Tax selectively defective in NF-kappaB activation did not. CCX754, a synthetic CXCR7 antagonist, inhibited cell growth and increased apoptosis of HTLV-1-immortalized T cells. Knockdown of CXCR7 by small interfering RNA also reduced cell growth. Stable expression of CXCR7 in a CXCR7-negative ATL cell line promoted cell growth and survival. Taken together, CXCR7 is inducible by Tax and may play an important role in HTLV-1-induced immortalization of T cells by promoting growth and survival of HTLV-1-infected T cells.

The Met protooncogene is a transcriptional target of NFkappaB: Implications for cell survival

NF kappaB transcription factor regulates gene expression in response to extracellular stimuli such as TNF alpha. The genes regulated by NF kappaB encode for proteins which control cell growth and survival. Met is the tyrosine kinase receptor for hepatocyte growth factor, and it too promotes cell mitogenesis and survival. Previously, we showed that Met gene expression is regulated by TNF alpha. In this report, we identify and characterize a TNF alpha response element in the Met promoter. This element contains tandem C/EBP sites adjacent to an NF kappaB site. Binding of the NF kappaB p65 subunit and C/EBP beta to this element is induced by TNF alpha. To examine the interplay of NF kappaB and Met in vivo, we determined that Met mRNA and protein levels are reduced in the livers of p65-/- mice as compared to controls. In p65-/- mouse embryonic fibroblasts (MEFs), Met induction by TNF alpha is abrogated while Met's basal gene expression is reduced by half as compared to controls. When overexpressed in p65-/- MEFs, Met confers resistance to TNF-alpha-mediated cell death. Conversely, expression of dominant negative Met in wild-type MEFs renders them sensitive to cell death induced by TNF alpha. A similar response following TNF alpha challenge was observed in hepatocytic cells treated with siRNA to knockdown endogenous Met. Together, these results indicate that the Met gene is a direct target of NF kappaB and that Met participates in NF kappaB-mediated cell survival.

Convergence of IL-1β and VDR Activation Pathways in Human TLR2/1-Induced Antimicrobial Responses

Antimicrobial effector mechanisms are central to the function of the innate immune response in host defense against microbial pathogens. In humans, activation of Toll-like receptor 2/1 (TLR2/1) on monocytes induces a vitamin D dependent antimicrobial activity against intracellular mycobacteria. Here, we report that TLR activation of monocytes triggers induction of the defensin beta 4 gene (DEFB4), requiring convergence of the IL-1β and vitamin D receptor (VDR) pathways. TLR2/1 activation triggered IL-1β activity, involving the upregulation of both IL-1β and IL-1 receptor, and downregulation of the IL-1 receptor antagonist. TLR2/1L induction of IL-1β was required for upregulation of DEFB4, but not cathelicidin, whereas VDR activation was required for expression of both antimicrobial genes. The differential requirements for induction of DEFB4 and cathelicidin were reflected by differences in their respective promoter regions; the DEFB4 promoter had one vitamin D response element (VDRE) and two NF-κB sites, whereas the cathelicidin promoter had three VDREs and no NF-κB sites. Transfection of NF-κB into primary monocytes synergized with 1,25D3 in the induction of DEFB4 expression. Knockdown of either DEFB4 or cathelicidin in primary monocytes resulted in the loss of TLR2/1-mediated antimicrobial activity against intracellular mycobacteria. Therefore, these data identify a novel mechanism of host defense requiring the induction of IL-1β in synergy with vitamin D activation, for the TLR-induced antimicrobial pathway against an intracellular pathogen.

NF-κB Mediates Tumor Necrosis Factor α-Induced Expression of Optineurin, a Negative Regulator of NF-κB

Optineurin is a ubiquitously expressed multifunctional cytoplasmic protein encoded by OPTN gene. The expression of optineurin is induced by various cytokines. Here we have investigated the molecular mechanisms which regulate optineurin gene expression and the relationship between optineurin and nuclear factor κB (NF-κB). We cloned and characterized human optineurin promoter. Optineurin promoter was activated upon treatment of HeLa and A549 cells with tumor necrosis factor α (TNFα). Mutation of a putative NF-κB-binding site present in the core promoter resulted in loss of basal as well as TNFα-induced activity. Overexpression of p65 subunit of NF-κB activated this promoter through NF-κB site. Oligonucleotides corresponding to this putative NF-κB-binding site showed binding to NF-κB. TNFα-induced optineurin promoter activity was inhibited by expression of inhibitor of NF-κB (IκBα) super-repressor. Blocking of NF-κB activation resulted in inhibition of TNFα-induced optineurin gene expression. Overexpressed optineurin partly inhibited TNFα-induced NF-κB activation in Hela cells. Downregulation of optineurin by shRNA resulted in an increase in TNFα-induced as well as basal NF-κB activity. These results show that optineurin promoter activity and gene expression are regulated by NF-κB pathway in response to TNFα. In addition these results suggest that there is a negative feedback loop in which TNFα-induced NF-κB activity mediates expression of optineurin, which itself functions as a negative regulator of NF-κB.

Genomic structure, expression pattern and functional characterization of crustinPm5, a unique isoform of crustin from Penaeus monodon

A unique isoform of crustin, crustinPm5, was identified from a gill-epipodite cDNA library of the tiger shrimp, Penaeus monodon. The crustinPm5 cDNA contains an open reading frame (ORF) of 510 bp encoding a 169 amino acid protein. The deduced amino acid sequence of crustinPm5 showed 38% and 37% overall sequence identity with those of crustinPm1 and crustin-likePm, respectively, two crustin isoforms previously reported. The crustinPm5 gene contained four exons interrupted by three introns whilst the upstream sequence contains a putative promoter with two potential binding sites for NF-kappaB, one complete heat-shock regulatory element (HSE) and five putative GATA factor binding sites. The transcripts of crustinPm5 were primarily observed in the epipodite and eyestalk and not in hemocytes. Expression analysis revealed that the transcript levels of crustinPm5, crustinPm1 and crustin-likePm in epipodite were up-regulated upon heat treatment and hyperosmotic salinity stress. The recombinant crustinPm5 exhibited antimicrobial activity against some Gram-positive bacteria in vitro, but did not inhibit the growth of any Gram-negative bacteria tested. These results, together with the transcript expression pattern, indicate a diverse function of the proteins in the crustin family particularly crustinPm5 that might function as a stress mediator in addition to its antibacterial action.

NF-κB Transcription Factor p50 Critically Regulates Tissue Factor in Deep Vein Thrombosis

NF-kappaB transcription factors regulate the expression of tissue factor (TF), a principal initiator of the coagulation cascade. Dominant among them is the p50/p65 heterodimer. Here we report that Andrographolide (Andro; a p50 inhibitor) and genetic deletion of p50 attenuated TF activity in stimulated endothelial cells and monocytes/macrophages. Results of the electrophoretic mobility "supershift" assay and chromatin immunoprecipitation demonstrated the direct interaction of the p50/p65 heterodimer with the NF-kappaB site of the human TF promoter. Andro-treated and p50 null mice both exhibited blunted TF expression and reduced venous thrombosis, which were recapitulated by an anti-murine TF antibody in vivo. Our findings thus indicate that regulation of TF by NF-kappaB transcription factor p50 is essential for the pathogenesis of deep vein thrombosis and suggest that specific inhibitors of p50, such as Andro, may be therapeutically valuable for preventing and perhaps treating venous thrombosis.

Control of Stochastic Gene Expression by Host Factors at the HIV Promoter

The HIV promoter within the viral long terminal repeat (LTR) orchestrates many aspects of the viral life cycle, from the dynamics of viral gene expression and replication to the establishment of a latent state. In particular, after viral integration into the host genome, stochastic fluctuations in viral gene expression amplified by the Tat positive feedback loop can contribute to the formation of either a productive, transactivated state or an inactive state. In a significant fraction of cells harboring an integrated copy of the HIV-1 model provirus (LTR-GFP-IRES-Tat), this bimodal gene expression profile is dynamic, as cells spontaneously and continuously flip between active (Bright) and inactive (Off) expression modes. Furthermore, these switching dynamics may contribute to the establishment and maintenance of proviral latency, because after viral integration long delays in gene expression can occur before viral transactivation. The HIV-1 promoter contains cis-acting Sp1 and NF-κB elements that regulate gene expression via the recruitment of both activating and repressing complexes. We hypothesized that interplay in the recruitment of such positive and negative factors could modulate the stability of the Bright and Off modes and thereby alter the sensitivity of viral gene expression to stochastic fluctuations in the Tat feedback loop. Using model lentivirus variants with mutations introduced in the Sp1 and NF-κB elements, we employed flow cytometry, mRNA quantification, pharmacological perturbations, and chromatin immunoprecipitation to reveal significant functional differences in contributions of each site to viral gene regulation. Specifically, the Sp1 sites apparently stabilize both the Bright and the Off states, such that their mutation promotes noisy gene expression and reduction in the regulation of histone acetylation and deacetylation. Furthermore, the NF-κB sites exhibit distinct properties, with κB site I serving a stronger activating role than κB site II. Moreover, Sp1 site III plays a particularly important role in the recruitment of both p300 and RelA to the promoter. Finally, analysis of 362 clonal cell populations infected with the viral variants revealed that mutations in any of the Sp1 sites yield a 6-fold higher frequency of clonal bifurcation compared to that of the wild-type promoter. Thus, each Sp1 and NF-κB site differentially contributes to the regulation of viral gene expression, and Sp1 sites functionally “dampen” transcriptional noise and thereby modulate the frequency and maintenance of this model of viral latency. These results may have biomedical implications for the treatment of HIV latency.

AMP-Activated Protein Kinase-Regulated Activation of the PGC-1α Promoter in Skeletal Muscle Cells

The mechanisms by which PGC-1α gene expression is controlled in skeletal muscle remains largely undefined. Thus, we sought to investigate the transcriptional regulation of PGC-1α using AICAR, an activator of AMPK, that is known to increase PGC-1α expression. A 2.2 kb fragment of the human PGC-1α promoter was cloned and sequence analysis revealed that this TATA-less sequence houses putative consensus sites including a GC-box, a CRE, several IRSs, a SRE, binding sites for GATA, MEF2, p 53, NF-κB, and EBox binding proteins. AMPK activation for 24 hours increased PGC-1α promoter activity with concomitant increases in mRNA expression. The effect of AICAR on transcriptional activation was mediated by an overlapping GATA/EBox binding site at −495 within the PGC-1α promoter based on gel shift analyses that revealed increases in GATA/EBox DNA binding. Mutation of the EBox within the GATA/EBox binding site in the promoter reduced basal promoter activity and completely abolished the AICAR effect. Supershift analyses identified USF-1 as a DNA binding transcription factor potentially involved in regulating PGC-1α promoter activity, which was confirmed in vivo by ChIP. Overexpression of either GATA-4 or USF-1 alone increased the p851 PGC-1α promoter activity by 1.7- and 2.0-fold respectively, while co-expression of GATA-4 and USF-1 led to an additive increase in PGC-1α promoter activity. The USF-1-mediated increase in PGC-1α promoter activation led to similar increases at the mRNA level. Our data identify a novel AMPK-mediated regulatory pathway that regulates PGC-1α gene expression. This could represent a potential therapeutic target to control PGC-1α expression in skeletal muscle.

Abstract 403: BMPER Is an Angiogenic Modulator that is Regulated by KLF-15 and FoxO3A and Controls Bone Morphogentic Protein Activity

BMPER (bone morphogenetic protein [BMP] endothelial precursor cell derived regulator) is an extracellular protein, that interacts with BMPs and thereby modulates BMP dependent vasculogenesis and angiogenesis. Our previous observations suggest a complex regulation of BMPER expression. During embryogenesis BMPER is expressed at the time and at sites of vasculogenesis, whereas in the adult organism it is expressed in heart, lung and skin. Methods and Results: We have cloned the mouse BMPER promoter and appropriate deletion constructs into pGL3 to regulate luciferase expression. As predicted in silicio, we found that Sp1 and Sp1-like transcription factors such as the krueppel-like factors (KLFs) regulate BMPER transcription. KLF-15 resulted in a 4.5 fold upregulation. Accordingly, BMPER expression was inhibited by the Sp-1/SP-1 like inhibitor mitramycin A. Site specific mutation of a proximal KLF-15 binding site reduced the effect of KLF-15 on BMPER expression. Along the same lines, knock down of KLF-15 in HUVEC by siRNA reduced BMPER expression. The transactivating effect of KLF-15 could be competed away by coexpression of Sp-1 suggesting that both factors may compete for the same binding site in the BMPER promoter. In EMSA, an oligo representing a well characterized KLF-15 binding site in the AceCs2 promoter but not an oligo encoding for a NFkappa-B site competed with the oligo coding for the KLF-15 site in the BMPER promoter. In contrast FoxO3A, a member of the FoxO family of transcription factors, serves as an inhibitor of BMPER expression, as shown by gain and lack of FoxO3A experiments. Additionally, we found that BMPER stimulates angiogenesis in a BMP-4 dependent manner in several in vitro and in vivo assays. Vice versa, BMPER is necessary for BMP-4 to exert is angiogenic activity on endothelial cells. Conclusion: BMPER is upregulated by KLF-15 and inhibited by FoxO3a. BMPER has angiogenic activity and is a key modulator of the BMP pathway.

Novel HIV-1 long terminal repeat (LTR) sequences of subtype B and mosaic intersubtype B/C recombinants in North India

Although the HIV-1 epidemic in India is mainly due to subtype C, other subtypes have also been reported from different parts of India. HIV-1 LTR sequence analysis from six HIV-1 infected individuals from North India was carried out to determine the nature and extent of variations. Four out of six samples formed a unique phylogenetic cluster which was close to subtype B. The other two samples (A3 and S3) turned out to be novel mosaic recombinants showing resemblance to subtypes B, B/C-India and B/C-Myanmar gene segments. All four subtype B LTR samples and the two B/C recombinants showed conserved as well as unique polymorphisms in all of the putative transcription factor binding sites (TFBS). These changes may potentially alter basal as well as Tat-mediated HIV-1 LTR promoter activation. The two recombinants possessed three copies of the NF-kappaB TFBS as seen with the majority of subtype C and recombinant B/C isolates reported earlier, but the other four non-recombinant B-LTRs showed only two copies of the NF-kappaB site. This is the first study to show a dominance of unique subtype B-LTRs and strongly suggests that this region could also be a hot spot for the formation of highly complex inter subtype B/C recombinants.

Characterization of Short Range DNA Looping in Endotoxin-mediated Transcription of the Murine Inducible Nitric-oxide Synthase (iNOS) Gene

The local structural properties and spatial conformations of chromosomes are intimately associated with gene expression. The spatial associations of critical genomic elements in inducible nitric-oxide synthase (iNOS) transcription have not been previously examined. In this regard, the murine iNOS promoter contains 2 NF-kappaB binding sites (nt -86 and nt -972) that are essential for maximal transactivation of iNOS by LPS. Although AP-1 is commonly listed as an essential transcription factor for LPS-mediated iNOS transactivation, the relationship between AP-1 and NF-kappaB in this setting is not well studied. In this study using a model of LPS-stimulated ANA-1 murine macrophages, we demonstrate that short range DNA looping occurs at the iNOS promoter. This looping requires the presence of AP-1, c-Jun, NF-kappaB p65, and p300-associated acetyltransferase activity. The distal AP-1 binding site interacts via p300 with the proximal NF-kappaB binding site to create this DNA loop to participate in iNOS transcription. Other geographically distant AP-1 and NF-kappaB sites are certainly occupied, but selected sites are critical for iNOS transcription and the formation of the c-Jun, p65, and p300 transcriptional complex. In this "simplified" model of murine iNOS promoter, numerous transcription factors recognize and bind to various response elements, but these locales do not equally contribute to iNOS gene transcription.

NF-κB Inhibits Sodium Transport via Down-regulation of SGK1 in Renal Collecting Duct Principal Cells

Tubulointerstitial inflammation is a common feature of renal diseases. We have investigated the relationship between inflammation and Na(+) transport in the collecting duct (CD) using the mCCD(cl1) and mpkCDD(cl4) principal cell models. Lipopolysaccharide (LPS) decreased basal and aldosterone-stimulated amiloride-sensitive transepithelial current in a time-dependent manner. This effect was associated with a decrease in serum and glucocorticoid-regulated kinase 1 (SGK1) mRNA and protein levels followed by a decrease in epithelial sodium channel (ENaC) alpha-subunit mRNA levels. The LPS-induced decrease in SGK1 expression was confirmed in isolated rat CD. This decreased expression of either SGK1 or the ENaC alpha-subunit was not due to enhanced degradation of mRNA. In contrast, LPS inhibited transcriptional activity of the SGK1 promoter measured by luciferase-reporter gene assay. The effect of LPS was not mediated by inhibition of mineralocorticoid or glucocorticoid receptor, because expression of both receptors was unchanged and blockade of either receptor by spironolactone or RU486, respectively, did not prevent the down-regulation of SGK1. The effect of LPS was mediated by the canonical NF-kappaB pathway, as overexpression of a constitutively active mutant, IKKbeta (inhibitor of nuclear factor kappaB kinase-beta) decreased SGK1 mRNA levels, and knockdown of p65 NF-kappaB subunit by small interfering RNA increased SGK1 mRNA levels. Chromatin immunoprecipitation showed that LPS increased p65 binding to two NF-kappaB sites along the SGK1 promoter. In conclusion, we show that activation of the NF-kappaB pathway down-regulates SGK1 expression, which might lead to decreased ENaC alpha-subunit expression, ultimately resulting in decreased Na(+) transport.