Subunit Of Nuclear Factor Research Articles

MtNF-YC6 and MtNF-YC11 are involved in regulating the transcriptional program of arbuscular mycorrhizal symbiosis.

Arbuscular mycorrhizal fungi are obligate symbionts that transfer mineral nutrients to host plants through arbuscules, a fungal structure specialized for exchange for photosynthetic products. MtNF-YC6 and MtNF-YC11, which encode the C subunits of nuclear factor Y (NF-Y) family in Medicago truncatula are induced specifically by arbuscular mycorrhizal symbiosis (AMS). A previous study showed that MtNF-YC6 and MtNF-YC11 are activated in cortical cells of mycorrhizal roots, but the gene functions were unknown. Herein, we identified both MtNF-YB17 and MtNF-YB12 as the interacting partners of MtNF-YC6 and MtNF-YC11 in yeast and plants. MtNF-YB17 was highly induced by AMS and activated in cortical cells only in mycorrhizal roots but MtNF-YB12 was not affected. The formation of B/C heterodimers led the protein complexes to transfer from the cytoplasm to the nucleus. Silencing MtNF-YC6 and C11 by RNA interference (RNAi) resulted in decreased colonization efficiency and arbuscule richness. Coincidently, genes associated with arbuscule development and degeneration in RNAi roots were also downregulated. In silico analysis showed CCAAT-binding motifs in the promoter regions of downregulated genes, further supporting the involvement of NF-Y complexes in transcriptional regulation of symbiosis. Taken together, this study identifies MtNF-YC6- or MtNF-YC11-containing protein complexes as novel transcriptional regulators of symbiotic program and provides a list of potential downstream target genes. These data will help to further dissect the AMS regulatory network.

Zinc Fingers and Homeobox Family in Cancer: A Double-Edged Sword.

The zinc fingers and homeobox (ZHX) family includes ZHX1, ZHX2, and ZHX3, and their proteins have similar unique structures, containing two C2H2-type zinc finger motifs and four or five HOX-like homeodomains. The members of the ZHX family can form homodimers or heterodimers with each other or with a subunit of nuclear factor Y. Previous studies have suggested that ZHXs can function as positive or negative transcriptional regulators. Recent studies have further revealed their biological functions and underlying mechanisms in cancers. This review summarized the advances of ZHX-mediated functions, including tumor-suppressive and oncogenic functions in cancer formation and progression, the molecular mechanisms, and regulatory functions, such as cancer cell proliferation, migration, invasion, and metastasis. Moreover, the differential expression levels and their association with good or poor outcomes in patients with various malignancies and differential responses to chemotherapy exert opposite functions of oncogene or tumor suppressors. Therefore, the ZHXs act as a double-edged sword in cancers.

Platelet-rich fibrin elicits an anti-inflammatory response in macrophages in vitro.

BackgroundPlatelet‐rich fibrin (PRF) serves as a reservoir of bioactive molecules to support wound healing and bone regeneration. The beneficial action of PRF might involve macrophage polarization from proinflammatory M1 toward pro‐resolving M2 phenotypes. This study aims to evaluate the effect of PRF on macrophage polarization.MethodsMurine primary macrophages and RAW 264.7 cells were exposed to saliva and lipopolysaccharides (LPS) with and without PRF lysates obtained by repeated freeze‐thawing or the secretome of PRF membranes, termed PRF conditioned medium. The expression of the M1 marker genes interleukin 1β (IL1β) and interleukin 6 (IL6) along with the M2 markers arginase‐1 and chitinase‐like 3 (Chil3 or YM1) were evaluated by real time polymerase chain reaction. Immunoassay and immunofluorescence staining were performed for IL6 and p65 translocation, a subunit nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐kB), respectively.ResultsWe report here that PRF lysates and PRF conditioned medium, the latter containing the secretome, greatly decreased the proinflammatory response of primary macrophages and RAW 264.7 cells as indicated by the expression of IL1β and IL6. The anti‐inflammatory activity of PRF lysates was further confirmed by IL6 immunoassay. Moreover, PRF lysates suppressed the translocation of p65 from the cytoplasm into the nucleus after incubation with saliva. In support of M2 polarization, PRF lysates and PRF conditioned medium enhanced the expression of arginase‐1 and YM1 in primary macrophages.ConclusionOur results indicate that PRF holds an anti‐inflammatory activity and shifts the macrophage polarization from an M1 toward an M2 phenotype.

321 The role of abnormal cutaneous steroidogenesis in the pathogenesis of rosacea

Rosacea is a chronic inflammatory disorder usually affecting the central face. Chronic use of topical steroids on the face causes lesions similar to rosacea. Synthesis and function of glucocorticoid (GC) have been established in healthy skin and various cutaneous diseases but not in rosacea. In most skin disorders, increased inflammation is usually coupled with a decline in the production of cutaneous GC. We investigated the de novo GC synthesis in the skin and GC receptor expression in the lesional and the nonlesional skin of patients with erythematotelangiectatic rosacea (ETR). Both lesional and non-lesional facial skin samples from the same individuals were provided by 12 ETR patients (mean age, 40.1 years; range, 30 to 55 years). The key steroidogenic enzymes, CYP11A1, 3βHSD2 and CYP21A2, were significantly increased in the lesional skin at the gene and protein levels. CYP17A1 and steroidogenic acute regulatory protein (StAR) tended to be higher in the lesional skin. GC receptor was also significantly increased in the lesional skin. Progesterone was increased significantly in the lesional skin using liquid chromatography-mass spectrometry Simultaneously, p65/p50 subunits of nuclear factor kappa-light-chain enhancer of activated B cells (NFκB) and matrix metalloproteinases (MMP) gene expressions were significantly increased in the lesional skin. Furthermore, p65 and p105 protein levels were significantly increased. These results demonstrate that crosstalk between GC receptor and NFκB may be hampered in the lesional skin of ETR and may contribute to rosacea pathogenesis. We demonstrate that ETR is an inherently inflammatory disorder mediated by NFκB and that GC synthesis and GC receptor expression is increased in the lesional skin of ETR unlike in other cutaneous inflammatory diseases.

The NUCLEAR FACTOR-CONSTANS complex antagonizes Polycomb repression to de-repress FLOWERING LOCUS T expression in response to inductive long days in Arabidopsis.

Many plants sense the seasonal cues, day length or photoperiod changes, to align the timing of the developmental transition to flowering with changing seasons for reproductive success. Inductive day lengths through the photoperiod pathway induce the expression of FLOWERING LOCUS T (FT) or FT relatives that encode a major mobile florigen to promote flowering. In Arabidopsis thaliana, under inductive long days the photoperiod pathway output CONSTANS (CO) accumulates toward the end of the day, and associates with the B and C subunits of Nuclear Factor Y (NF-Y) to form the NF-CO complex that acts to promote FT expression near dusk, whereas Polycomb group (PcG) proteins function to silence FT expression. How NF-CO acts to antagonize the function of PcG proteins to regulate FT expression remains unclear. Here, we show that the NF-CO complex bound to the proximal FT promoter, through chromatin looping, acts in concert with an NF-Y complex bound to a distal enhancer to reduce the levels of PcG proteins, including both Polycomb repressive complex 1 (PRC1) and PRC2 at the FT promoter, leading to a relieving of Polycomb silencing and thus FT de-repression near dusk. Thus, our study provides molecular insights on how the 'active' photoperiod pathway and the 'repressive' Polycomb silencing system interact to control temporal FT expression, conferring the long-day induction of flowering in Arabidopsis.

402 Abnormal glucocorticoid synthesis in the lesional skin of erythematotelangiectatic rosacea

Rosacea is a chronic inflammatory disorder usually affecting the central face. Chronic use of topical steroids on the face causes lesions similar to rosacea. Synthesis and function of glucocorticoid (GC) have been established in healthy skin and various cutaneous diseases but not in rosacea. In most skin disorders, increased inflammation is usually coupled with a decline in the production of cutaneous GC. We designed this study to investigate the de novo GC synthesis in the skin and GC receptor expression in the lesional and the nonlesional skin of patients with erythematotelangiectatic rosacea (ETR). The key steroidogenic enzymes, 11βHSD1, CYP11A1, 3βHSD2 and CYP21A2, were increased in the lesional skin at the gene and protein levels. CYP17A1 and steroidogenic acute regulatory protein (StAR) tended to be higher in the lesional skin. GC receptor was also significantly increased in the lesional skin. Simultaneously, p65/p50 subunits of nuclear factor kappa-light-chain enhancer of activated B cells (NF-κb) and matrix metalloproteinases (MMP) gene expressions were significantly increased in the lesional skin. These concurrent increments demonstrate that crosstalk between GC receptor and NF-κb may be hampered in the lesional skin of ETR and may contribute to rosacea pathogenesis. We demonstrate that ETR is an inherently inflammatory disorder mediated by NF-κb and that lesional GC synthesis and GC receptor expression is increased in ETR unlike in other cutaneous inflammatory diseases.

A novel arrangement of zinc finger nuclease system for in vivo targeted genome engineering: the tomato LEC1-LIKE4 gene case.

A selection-free, highly efficient targeted mutagenesis approach based on a novel ZFN monomer arrangement for genome engineering in tomato reveals plant trait modifications. How to achieve precise gene targeting in plants and especially in crops remains a long-sought goal for elucidating gene function and advancing molecular breeding. To address this issue, zinc finger nuclease (ZFN)-based technology was developed for the Solanum lycopersicum seed system. A ZFN architecture design with an intronic sequence between the two DNA recognition sites was evaluated for its efficiency in targeted gene mutagenesis. Custom engineered ZFNs for the developmental regulator LEAFY-COTYLEDON1-LIKE4 (L1L4) coding for the β subunit of nuclear factor Y, when transiently expressed in tomato seeds, cleaved the target site and stimulated imperfect repair driven by nonhomologous end-joining, thus, introducing mutations into the endogenous target site. The successful in planta application of the ZFN platform resulted in L1L4 mutations which conferred heterochronic phenotypes during development. Our results revealed that sequence changes upstream of the DNA binding domain of L1L4 can lead to phenotypic diversity including fruit organ. These results underscore the utility of engineered ZFN approach in targeted mutagenesis of tomato plant which may accelerate translational research and tomato breeding.

Divergent activity of the gonadotropin-releasing hormone receptor gene promoter among genetic lines of pigs is partially conferred by nuclear factor (NF)-kB, specificity protein (SP)1-like and GATA-4 binding sites

BackgroundBinding of gonadotropin-releasing hormone (GnRH) to its receptor (GnRHR) on gonadotropes within the anterior pituitary gland is essential to reproduction. In pigs, the GnRHR gene is also located near a genetic marker for ovulation rate, a primary determinant of prolificacy. We hypothesized that pituitary expression of the GnRHR gene is alternatively regulated in genetic strains with elevated ovulation rates (Chinese Meishan and Nebraska Index) vs. standard white crossbred swine (Control).MethodsLuciferase reporter vectors containing 5118 bp of GnRHR gene promoter from either the Control, Index or Meishan swine lines were generated. Transient transfection of line-specific, full length, deletion and mutation constructs into gonadotrope-derived αT3-1 cells were performed to compare promoter activity and identify regions necessary for divergent regulation of the porcine GnRHR gene. Additionally, transcription factors that bind the GnRHR promoter from each line were identified with electrophoretic mobility shift assays (EMSA).ResultsDramatic differences in luciferase activity among Control, Index and Meishan promoters (19-, 27- and 49-fold over promoterless control, respectively; P < 0.05) were established. A single bp substitution (−1690) within a previously identified upstream enhancer (−1779/−1667) bound GATA-4 in the Meishan promoter and the p52/p65 subunits of nuclear factor (NF)-κB in the homologous Control/Index promoters. Transient transfection of vectors containing block replacement mutations of either the GATA-4 or NF-κB binding sites within the context of their native promoters resulted in a 50 and 60 % reduction of luciferase activity, respectively (P < 0.05). Furthermore, two single-bp substitutions in the Meishan compared to Control/Index promoters resulted in binding of the p52 and p65 subunits of NF-κB and a specificity protein 1 (SP1)-like factor (−1235) as well as GATA-4 (−845). Vectors containing the full-length Meishan promoter harboring individual mutations spanning these regions reduced luciferase activity by 25 and 20 %, respectively, compared to native sequence (P < 0.05).ConclusionsElevated activity of the Meishan GnRHR gene promoter over Control/Index promoters in αT3-1 cells is partially due to three single nucleotide polymorphisms resulting in the unique binding of GATA-4 (−1690), the p52/p65 subunits of NF-kB in combination with a SP1-like factor (−1235), and GATA-4 (−845).Electronic supplementary materialThe online version of this article (doi:10.1186/s12958-016-0170-0) contains supplementary material, which is available to authorized users.

LECT2 association with macrophage-mediated killing of Helicobacter pylori by activating NF-κB and nitric oxide production.

Helicobacter pylori employs unique methods to colonize the stomach, which induces chronic inflammation. It is also able to avoid eradication by macrophages and other immune cells. Leukocyte cell-derived chemotaxin 2 (LECT2), a multi-functional cytokine involved in many pathological conditions, has recently been shown to activate macrophages via the CD209a receptor. Therefore, we aimed to investigate the effects of LECT2 on H. pylori-infected macrophages. Macrophages were treated with recombinant LECT2, and both their ability to kill H. pylori and produce nitric oxide were analyzed. Western blot was performed to determine nuclear translocation and protein phosphorylation of p65, a subunit of nuclear factor (NF)-κB. Transfection experiments were performed to analyze the signaling pathway of LECT2 in macrophages. We found that treatment with LECT2 enhanced H. pylori killing and nitric oxide production in macrophages. In addition, DNA-binding activity and nuclear translocation of p65 were up-regulated by LECT2 treatment. Furthermore, we found that NF-κB activation by LECT2 was mediated by Raf-1 in macrophages, and Raf-1 phosphorylation was specifically altered in response to LECT2. Moreover, LECT2 induced Ser28 phosphorylation in the intracellular domain of CD209a. CD209a Ser28 phosphorylation was required for LECT2-induced Raf-1 and NF-κB activation in RAW264.7 macrophages. Our study showed that the effects of LECT2 on H. pylori killing and nitric oxide production were dependent on CD209a phosphorylation, Raf-1, and NF-κB activation. Together, these results demonstrate for the first time that exposure to LECT2 can modulate specific intracellular mechanisms downstream of CD209a to enhance H. pylori killing and nitric oxide production in macrophages.

Cloning and Characterization of 5' Flanking Regulatory Sequences of AhLEC1B Gene from Arachis Hypogaea L.

LEAFY COTYLEDON1 (LEC1) is a B subunit of Nuclear Factor Y (NF-YB) transcription factor that mainly accumulates during embryo development. We cloned the 5′ flanking regulatory sequence of AhLEC1B gene, a homolog of Arabidopsis LEC1, and analyzed its regulatory elements using online software. To identify the crucial regulatory region, we generated a series of GUS expression frameworks driven by different length promoters with 5′ terminal and/or 3′ terminal deletion. We further characterized the GUS expression patterns in the transgenic Arabidopsis lines. Our results show that both the 65bp proximal promoter region and the 52bp 5′ UTR of AhLEC1B contain the key motifs required for the essential promoting activity. Moreover, AhLEC1B is preferentially expressed in the embryo and is co-regulated by binding of its upstream genes with both positive and negative corresponding cis-regulatory elements.

Ingenane-type diterpene compounds from Euphorbia kansui modulate IFN-γ production through NF-κB activation.

Euphorbia kansui, a traditional medical herb, has been shown to have anti-tumour and anti-viral activities. Previously, we have reported that E. kansui increases interferon-gamma (IFN-γ) production in natural killer (NK) cells. However, it is not clear how E. kansui regulates IFN-γ secretion by NK cells. In this study, E. kansui was separated into six individual compounds from the same chloroform fraction so that the activity of each compound could be compared. E. kansui compounds induced IFN-γ secretion through the phosphorylation of protein kinase D and IκB kinase pathways. Furthermore, E. kansui compounds activated the translocation of p65, a sub-unit of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), to the nucleus and induced NF-κB at the transcriptional level. These findings suggest that E. kansui enhances IFN-γ secretion through the NF-κB pathway in NK cells. © 2015 Society of Chemical Industry.

A wheat CCAAT box-binding transcription factor increases the grain yield of wheat with less fertilizer input.

Increasing fertilizer consumption has led to low fertilizer use efficiency and environmental problems. Identifying nutrient-efficient genes will facilitate the breeding of crops with improved fertilizer use efficiency. This research performed a genome-wide sequence analysis of the A (NFYA), B (NFYB), and C (NFYC) subunits of Nuclear Factor Y (NF-Y) in wheat (Triticum aestivum) and further investigated their responses to nitrogen and phosphorus availability in wheat seedlings. Sequence mining together with gene cloning identified 18 NFYAs, 34 NFYBs, and 28 NFYCs. The expression of most NFYAs positively responded to low nitrogen and phosphorus availability. In contrast, microRNA169 negatively responded to low nitrogen and phosphorus availability and degraded NFYAs. Overexpressing TaNFYA-B1, a low-nitrogen- and low-phosphorus-inducible NFYA transcript factor on chromosome 6B, significantly increased both nitrogen and phosphorus uptake and grain yield under differing nitrogen and phosphorus supply levels in a field experiment. The increased nitrogen and phosphorus uptake may have resulted from the fact that that overexpressing TaNFYA-B1 stimulated root development and up-regulated the expression of both nitrate and phosphate transporters in roots. Our results suggest that TaNFYA-B1 plays essential roles in root development and in nitrogen and phosphorus usage in wheat. Furthermore, our results provide new knowledge and valuable gene resources that should be useful in efforts to breed crops targeting high yield with less fertilizer input.

Expression of nuclear factor Y, subunit C (NFY-C), and retinoic acid receptor-related orphan receptor alpha (RORA) in colorectal adenocarcinoma.

e14085 Background: NFY-C gene is one of the three subunits of nuclear factor Y, a highly conserved transcription factor, which binds with high specificity to CCAAT motifs in the promoters of various genes. RORA is a member of the NR1 subfamily of nuclear hormone receptors. It can bind as a monomer or a homodimer to hormone response elements upstream of several genes to enhance their expression. Methods: mRNA levels of NFY-C and RORA were evaluated by quantitative RT-PCR in 81 neoplastic colorectal tissue specimens and 51 normal tissue specimens from patients with colorectal adenocarcinoma. All patients had undergone curative resections at the University Hospital of Patras, between 1995 and 2005. mRNA levels were normalised to the Alu-Sq levels and were analysed in relation to clinicopathological parameters. Protein expression of NFY-C was assessed by immunochemistry in 60 malignant and 20 normal samples from patients with colorectal adenocarcinoma. Results: There was a significant difference in the mRNA expression levels of NFY-C and RORA between normal and malignant tissues (p < 0.001 and p = 0.015, respectively). The mRNA levels of NFY- C and RORA were also related to the primary site of the tumor (p = 0.05 and p = 0.03, respectively). A 3-year survival benefit was also observed in patients with high expression levels of NFY-C (p = 0.023). There was no correlation between the mRNA levels of NFY-C or RORA and age, gender, grade, stage and relapse of the disease. However, mRNA levels of NFYC of stage B patients were significantly correlated with time to disease progression (p = 0.035). NFY-C protein was detected only in the cytoplasm both in malignant and normal tissues, with strong and weak intensity respectively. NFYC protein expression levels were correlated with mRNA expression levels of NFYC in malignant tissues (p = 0.011) and with the primary site of the tumour (p < 0.001). Conclusions: NFY-C and RORA exhibited elevated levels in colon carcinomas compared to normal tissue samples indicating a possible role for these molecules in colon carcinogenesis. The role of NFY-C and RORA in colorectal cancer warrants further investigation. No significant financial relationships to disclose.

Bacterial Effector Binding to Ribosomal Protein S3 Subverts NF-κB Function

Enteric bacterial pathogens cause food borne disease, which constitutes an enormous economic and health burden. Enterohemorrhagic Escherichia coli (EHEC) causes a severe bloody diarrhea following transmission to humans through various means, including contaminated beef and vegetable products, water, or through contact with animals. EHEC also causes a potentially fatal kidney disease (hemolytic uremic syndrome) for which there is no effective treatment or prophylaxis. EHEC and other enteric pathogens (e.g., enteropathogenic E. coli (EPEC), Salmonella, Shigella, Yersinia) utilize a type III secretion system (T3SS) to inject virulence proteins (effectors) into host cells. While it is known that T3SS effectors subvert host cell function to promote diarrheal disease and bacterial transmission, in many cases, the mechanisms by which these effectors bind to host proteins and disrupt the normal function of intestinal epithelial cells have not been completely characterized. In this study, we present evidence that the E. coli O157:H7 nleH1 and nleH2 genes encode T3SS effectors that bind to the human ribosomal protein S3 (RPS3), a subunit of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) transcriptional complexes. NleH1 and NleH2 co-localized with RPS3 in the cytoplasm, but not in cell nuclei. The N-terminal region of both NleH1 and NleH2 was required for binding to the N-terminus of RPS3. NleH1 and NleH2 are autophosphorylated Ser/Thr protein kinases, but their binding to RPS3 is independent of kinase activity. NleH1, but not NleH2, reduced the nuclear abundance of RPS3 without altering the p50 or p65 NF-κB subunits or affecting the phosphorylation state or abundance of the inhibitory NF-κB chaperone IκBα NleH1 repressed the transcription of a RPS3/NF-κB-dependent reporter plasmid, but did not inhibit the transcription of RPS3-independent reporters. In contrast, NleH2 stimulated RPS3-dependent transcription, as well as an AP-1-dependent reporter. We identified a region of NleH1 (N40-K45) that is at least partially responsible for the inhibitory activity of NleH1 toward RPS3. Deleting nleH1 from E. coli O157:H7 produced a hypervirulent phenotype in a gnotobiotic piglet model of Shiga toxin-producing E. coli infection. We suggest that NleH may disrupt host innate immune responses by binding to a cofactor of host transcriptional complexes.

Nfy-c expression in colorectal cancer

e15086 Background: Nuclear factor Y- C (NFY-C) gene encodes one of the three subunits of nuclear factor Y, a highly conserved transcription factor which binds to the promoters of a variety of genes, which are implicated in cell cycle progression, drug metabolism and antigen presentation. The purpose of the current study was to investigate the role of NFY-C in colorectal cancer by evaluating its mRNA expression in both malignant and normal colonic tissue from patients with colorectal carcinomas with and without disease relapse. Methods: Publicly available expression microarray data were analyzed to reveal elevated levels of NFY-C in patients with colorectal carcinoma who relapsed compared to patients who remained disease free. The mRNA levels of NFY-C were evaluated by quantitative RT-PCR in 81 neoplastic colorectal tissue specimens and 23 normal tissue specimens from patients with colorectal cancer who had undergone curative resections at the University Hospital of Patras, Greece, between 1995 and 2005. The mRNA levels were analysed in relation to clinicopathological parameters. Results: No significant difference was found in the expression levels of NFY-C between normal and malignant tissues. The expression levels of NFY-C were not related to age, gender, grade, stage or primary site of the disease. However, a statistically significant difference (p=0.02) was observed in NFY-C levels between patients with and without disease recurrence. More specifically, patients with low NFY-C levels relapsed more often than patients who overexpressed NFY-C. Nevertheless, the expression was not related to time to disease progression. Finally, patients with higher NFY-C expression levels seem to have improved survival, compared to patients with low NFY-C expression levels although the difference was not statistically significant. Conclusions: Expression levels of NFY-C seem to be associated with disease recurrence. The role of NFY-C in colorectal cancer warrants further investigation. No significant financial relationships to disclose.

EFFECT OF ANTISENSE OLIGONUCLEOTIDES TO NUCLEAR FACTOR-κB ON THE SURVIVAL OF LPS-INDUCED ARDS IN MOUSE

Because nuclear factor (NF)- κ B-regulated cytokines, including tumor necrosis factor- α (TNF- α) , from monocytes and macrophages have been implicated in the pathogenesis and development of septic shock and acute respiratory distress syndrome (ARDS), the effect of the antisense oligonucleotide to the p65 subunit of NF- κ B on the survival of lipopolysaccharide (LPS)-induced ARDS in BALB/c mice was examined. None and 70% of the animals died of diffuse hemorrhagic lung edema 1 to 2.5 days after intraperitoneal administration of 10 and 20 mg/kg LPS alone, respectively. Intravenously administered antisense oligonucleotide alone did not produce any significant changes in the behavior or lung histology. After intravenous administration of the anti-sense oligonucleotide, both peripheral blood monocytes and alveolar macrophages in bronchoalveolar lavage fluid were confirmed to contain sufficiently large amounts of intracellular antisense oligonucleotides for their function using fluorescein isothiocyanate (FITC)-labeled microscopy. The antisense oligonucleotide administered 6 hours before the intraperitoneal administration of LPS significantly decreased the survival rate with the progress of hemorrhagic edema in lung histology; 90% and 100% of animals treated with the antisense oligonuleotide died 0.5 to 1.5 days after the administration of 10 and 20 mg/kg LPS, respectively. These findings suggest that the suppression of cytokines and mediators in monocytes and alveolar macrophages by the antisense oligonucleotide to the p65 subunit of NF- κ B worsens the survival of LPS-induced ARDS in mice with the progress of hemorrhagic lung edema.

Chronic inflammation and susceptibility to bacterial infections in mice lacking the polypeptide (p)105 precursor (NF-kappaB1) but expressing p50.

The polypeptide (p)50 molecule, a subunit of nuclear factor (NF)-kappaB, is produced after proteolytic processing of the p105 precursor (NF-kappaB1). Although the p105 precursor has been postulated to play a role in the regulation of the Rel/NF-kappaB activity, its physiological relevance remains unclear. To investigate that, we generated mutant mice lacking the COOH terminal half of the p105 precursor, but expressing the p50 product (p105-/-). These mutant mice displayed an inflammatory phenotype composed of lymphocytic infiltration in lungs and liver, and an increased susceptibility to opportunistic infections. Enlargement of multiple lymph nodes, splenomegaly due to erythrocytic extramedullary hematopoiesis, and lymphoid hyperplasia were also observed in p105-/- mice. Cytokine production in p105-/- macrophages was severely impaired, whereas proliferative responses of p105-/- B cells were increased. T cell functions were only moderately impaired in mutant mice. Loss of p105 also led to enhanced constitutive p50 homodimer and inducible NF-kappaB activities in unstimulated and stimulated cells, respectively. As several genes regulated by Rel/NF-kappaB were upregulated in p105-/- thymus but downregulated in p105-/- macrophages, the enhanced p50 homodimers appear to function as transcriptional activators or repressors, depending on the cell type. Thus, the p105 precursor is indispensable in the control of p50 activity, and lack of the precursor has distinct effects on different cells.

Mice deficient in nuclear factor (NF)-kappa B/p52 present with defects in humoral responses, germinal center reactions, and splenic microarchitecture.

p52 is a subunit of nuclear factor (NF)-kappa B transcription factors, most closely related to p50. Previously, we have shown that p52, but not p50 homodimers can form transactivating complexes when associated with Bcl-3, an unusual member of the I kappa B family. To determine nonredundant physiologic roles of p52, we generated mice deficient in p52. Null mutant mice were impaired in their ability to generate antibodies to T-dependent antigens, consistent with an absence of B cell follicles and follicular dendritic cell networks in secondary lymphoid organs, and an inability to form germinal centers. Furthermore, the splenic marginal zone was disrupted. These phenotypes are largely overlapping with those observed in Bcl-3 knockout animals, but distinct from those of p50 knockouts, supporting the notion of a physiologically relevant complex of p52 homodimers and Bcl-3. Adoptive transfer experiments further suggest that such a complex may be critical in accessory cell functions during antigen-specific immune reactions. Possible roles of p52 and Bcl-3 are discussed that may underlie the oncogenic potential of these proteins, as evidenced by recurrent chromosomal translocations of their genes in lymphoid tumors.

Interleukin-10 gene expression in adult T-cell leukemia

We studied the serum levels of interleukin-10 (IL-10), in patients with adult T-cell leukemia (ATL) caused by human T-cell leukemia virus type I (HTLV-I) infection. Elevated IL-10 levels were observed in 33 of 45 patients with ATL. Fresh leukemic cells from ATL patients as well as HTLV-I-infected T-cell lines MT-2, SLB-1, and C10/MJ expressed IL-10 mRNA by reverse transcription-polymerase chain reaction analysis, whereas IL-10 mRNA was not detected in normal peripheral mononuclear cells and an uninfected T-cell line Jurkat. IL-10 protein was also detected in the culture medium of leukemic cells from ATL patients as well as these HTLV-I-infected cell lines, and in the extracellular fluids of ATL patients. Interestingly, MT-4 cells, which did not express Tax although transformed by HTLV-I, did not express IL-10 at either the mRNA or protein level. To elucidate the role of the HTLV-I encoded transactivator Tax in IL-10 gene expression, Jurkat cells were transfected with a Tax expression plasmid. In transiently transfected Jurkat cells, endogenous IL-10 mRNA expression was induced by Tax. Stably transfected Jurkat cell lines expressed IL-10 mRNA and secreted IL-10 protein into the culture medium. The nuclear factor (NF)-kappa B pathway is a target for Tax transactivation. We treated MT-2 cells with phosphorothioate antisense oligonucleotides to the p65 subunit of NF- kappa B. A reduction in the expression of p65 was accompanied by a reduction in IL-10 gene expression and IL-10 production. We showed that the IL-10 kappa B-like sites ( kappa B1,-2,034 to -2,025; kappa B2, - 1,961 to -1,952; kappa B3, -452 to -443) specifically formed a complex with NF-kappa B-containing nuclear extract from MT-2 cells and that NF- kappa B bound with the highest affinity to the kappa B2 element (kappa B2 &gt; kappa B3 &gt; kappa B1). These data suggest a general role for NF- kappa B activation in the induction of IL-10 gene transcription. Activation of IL-10 in HTLV-I-infected cells may contribute to the pathology associated with HTLV-I infection.