NF kappaB Research Articles

Two-photon excitation STED microscopy

We report sub-diffraction resolution in two-photon excitation (TPE) fluorescence microscopy achieved by merging this technique with stimulated-emission depletion (STED). We demonstrate an easy-to-implement and promising laser combination based on a short-pulse laser source for two-photon excitation and a continuous-wave (CW) laser source for resolution enhancement. Images of fluorescent nanoparticles and the immunostained transcription regulator NF kappaB in mammalian cell nuclei exhibit resolutions of <50 nm and approximately 70 nm in the focal plane, respectively, corresponding to a 4-5.4-fold improvement over the diffraction barrier.

Mechanism of PP2A-mediated IKKβ dephosphorylation: a systems biological approach

BackgroundBiological effects of nuclear factor-κB (NFκB) can differ tremendously depending on the cellular context. For example, NFκB induced by interleukin-1 (IL-1) is converted from an inhibitor of death receptor induced apoptosis into a promoter of ultraviolet-B radiation (UVB)-induced apoptosis. This conversion requires prolonged NFκB activation and is facilitated by IL-1 + UVB-induced abrogation of the negative feedback loop for NFκB, involving a lack of inhibitor of κB (IκBα) protein reappearance. Permanent activation of the upstream kinase IKKβ results from UVB-induced inhibition of the catalytic subunit of Ser-Thr phosphatase PP2A (PP2Ac), leading to immediate phosphorylation and degradation of newly synthesized IκBα.ResultsTo investigate the mechanism underlying the general PP2A-mediated tuning of IKKβ phosphorylation upon IL-1 stimulation, we have developed a strictly reduced mathematical model based on ordinary differential equations which includes the essential processes concerning the IL-1 receptor, IKKβ and PP2A. Combining experimental and modelling approaches we demonstrate that constitutively active, but not post-stimulation activated PP2A, tunes out IKKβ phosphorylation thus allowing for IκBα resynthesis in response to IL-1. Identifiability analysis and determination of confidence intervals reveal that the model allows reliable predictions regarding the dynamics of PP2A deactivation and IKKβ phosphorylation. Additionally, scenario analysis is used to scrutinize several hypotheses regarding the mode of UVB-induced PP2Ac inhibition. The model suggests that down regulation of PP2Ac activity, which results in prevention of IκBα reappearance, is not a direct UVB action but requires instrumentality.ConclusionThe model developed here can be used as a reliable building block of larger NFκB models and offers comprehensive simplification potential for future modeling of NFκB signaling. It gives more insight into the newly discovered mechanisms for IKK deactivation and allows for substantiated predictions and investigation of different hypotheses. The evidence of constitutive activity of PP2Ac at the IKK complex provides new insights into the feedback regulation of NFκB, which is crucial for the development of new anti-cancer strategies.

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.

Regulation of cell survival by resveratrol involves inhibition of NFκB‐regulated gene expression in prostate cancer cells

Polyphenols have been proposed as antitumoral agents. We have shown that resveratrol (RES) induced cell cycle arrest and promoted apoptosis in prostate cancer cells by inhibition of the PI3K pathway. The RES effects on NF kappaB activity in LNCaP cells (inducible NF kappaB), and PC-3 cells (constitutive NF kappaB) are reported. Cells were treated with 1-150 microM of RES during 36 hr. NF kappaB subcellular localization was analyzed by western blot and immunofluorescence. I kappaB alpha was evaluated by immunoprecipitation followed by Western blot. Specific DNA binding of NF kappaB was determined by EMSA assays and NF kappaB-mediated transcriptional activity by transient transfection with a luciferase gene reporter system. RES induced a dose-dependent cytoplasmic retention of NF kappaB mediated by I kappaB alpha in PC-3 cells but not in LNCaP. RES-induced inhibition of NF kappaB specific binding to DNA was more significant in PC-3 cells. NF kappaB-mediated transcriptional activity induced by EGF and TNFalpha were inhibited by RES in both cell lines. LY294002 mimicked RES effects on NF kappaB activity. Antiproliferative and apoptotic effects of RES on human prostate cancer cells may be mediated by the inhibition of NF kappaB activity. This mechanism seems to be associated to RES-induced PI3K inhibition. RES could have therapeutic potential for prostate cancer treatment.

G-Protein coupled receptor kinases mediate TNF alpha-induced NF kappaB signaling via direct interaction with and phosphorylation of I kappa B alpha (38.15)

Abstract Tumor necrosis factor-α (TNFα) is a multifunctional cytokine involved in the pathophysiology of many chronic inflammatory diseases. TNFα activation of the nuclear factor κB (NFκB) signaling pathway particularly in macrophages has been implicated in many diseases. We demonstrate here that G-protein coupled receptor kinase-2 and 5 (GRK2 and 5) regulate TNFα-induced NFκB signaling in macrophages. RNAi knockdown of GRK2 or 5 in macrophages significantly inhibited TNFα-induced IκBα phosphorylation and degradation, NFκB activation, and expression of the NFκB-regulated gene macrophage inflammatory protein-1β. Consistent with these results, over-expression of catalytically active GRK2 or 5 enhanced TNFα-induced NFκB activity. In addition, we show that GRK2 and 5 interact with IκBα in intact cells as well as in vitro via the N-terminal domain of IκBα and that IκBα is a substrate for GRK2 and 5 in vitro. Taken together, these results demonstrate that GRK2 and 5 are important mediators of TNFα-induced NFκB activity and suggest that GRKs could be potential therapeutic targets for treating TNFα-mediated inflammatory diseases.

An optimized extended DNA kappa B site that enhances plasmid DNA nuclear import and gene expression

The nuclear factor kappa B (NF kappaB) transcription factor, which shuttles between the cytoplasm and the nucleus under specific conditions, is a suitable intracellular target to increase the nuclear import of plasmid DNA. We report the design of an optimized and extended NF kappaB DNA binding sequence that promotes an efficient plasmid nuclear import. On the basis of structural studies, the 5'-CTGGGGACTTTCCAGCTGGGGACTTTCCAGCTGGGGACTTTCCAGG-3' segment (termed 3NF) comprising three 10-bp kappaB sites (GGGACTTTCC) separated by a 5-bp optimized spacer (AGCTG) was selected for its capacity to ensure the best structural fit with NF kappaB and to fix simultaneously three proteins. Plasmids encoding luciferase and bearing this sequence (3NF-plasmids) were constructed and their nuclear import and gene expression efficiencies compared with that of plasmids containing classical kappaB motifs. A high luciferase expression was associated with plasmids containing one (p3NF-luc) or two (p3NF-luc-3NF) 3NF sequences. In situ hybridization experiments and quantitative measurement of the number of plasmid copies demonstrated that the nuclear delivery of 3NF-plasmids was more efficient than that of 3NF-free plasmids. Cross-linked immunoprecipitation showed that 3NF-plasmids were recognized by NF kappaB inside cells upon transfection. The nuclear delivery was inhibited with BAY 11-7085, an inhibitor of NF kappaB activation. Finally, p3NF-luc-3NF, the most efficient construct for in vitro transfection, had a long-lived luciferase expression in vivo. The results obtained in the present study demonstrate the NF kappaB-mediated nuclear delivery of 3NF-plasmids. Due to its high affinity for fixing several NF kappaB, the 3NF sequence is a very promising helper for a nonviral gene delivery system.

IL-10 attenuates TNF- -induced NF B pathway activation and cardiomyocyte apoptosis

We have recently reported that tumour necrosis factor-alpha (TNF-alpha) increases oxidative stress and apoptosis in cardiomyocytes by upregulating p38 mitogen-activated protein (MAP) kinase (MAPK) phosphorylation. Interleukin-10 (IL-10) blocked these effects of TNF-alpha by upregulating extracellular signal-regulated kinase 1/2 (ERK 1/2) MAPK phosphorylation. However, the precise site of this IL-10 action is still unknown, and this is investigated in the present study. Cardiomyocytes isolated from adult Sprague-Dawley rats were exposed to TNF-alpha (10 ng/mL), IL-10 (10 ng/mL), and IL-10+TNF-alpha (ratio 1) for 4 h. Hydrogen peroxide and antioxidant trolox were used as positive controls. Exposure to TNF-alpha resulted in an increase in the production of reactive oxygen species, the number of apoptotic cells, caspase-3 activation, and poly-ADP ribose polymerase (PARP) cleavage. Increased oxidative stress by using hydrogen peroxide also caused apoptosis. The changes due to TNF-alpha were associated with an increase in the inhibitor of kappaB kinase (IKK) and nuclear factor kappa-B (NF kappaB) phosphorylation. IL-10 by itself had no effect, but it prevented the above mentioned TNF-alpha-induced changes. Trolox also mitigated TNF-alpha induced changes. Pre-exposure of cells to an IKK inhibitor (PS-1145) prevented TNF-alpha-induced caspase-3 and PARP cleavage. Inhibition of ERK 1/2 MAPK with PD98059 attenuated the protective role of IL-10 against TNF-alpha-induced activation of IKK and NF kappaB as well as cardiomyocyte apoptosis. The present study shows that TNF-alpha-induced activation of the NF kappaB pathway plays a critical role in cardiomyocyte apoptosis. IL-10 prevents TNF-alpha-induced NF kappaB activation and pro-apoptotic changes in cardiomyocytes by inhibiting IKK phosphorylation through the activation of ERK 1/2 MAPK.

6‐Shogaol suppressed lipopolysaccharide‐induced up‐expression of iNOS and COX‐2 in murine macrophages

Ginger, the rhizome of Zingiber officinale, is a traditional medicine with carminative effect, antinausea, anti-inflammatory, and anticarcinogenic properties. In this study, we investigated the inhibitory effects of 6-shogaol and a related compound, 6-gingerol, on the induction of nitric oxide synthase (NOS) and cyclooxygenase-2 (COX-2) in murine RAW 264.7 cells activated with LPS. Western blotting and reverse transcription-PCR analyses demonstrated that 6-shogaol significantly blocked protein and mRNA expression of inducible NOS (iNOS) and COX-2 in LPS-induced macrophages. The in vivo anti-inflammatory activity was evaluated by a topical 12-O-tetradecanoylphorbol 13-acetate (TPA) application to mouse skin. When applied topically onto the shaven backs of mice prior to TPA, 6-shogaol markedly inhibited the expression of iNOS and COX-2 proteins. Treatment with 6-shogaol resulted in the reduction of LPS-induced nuclear translocation of nuclear factor-kappaB (NF kappaB) subunit and the dependent transcriptional activity of NF kappaB by blocking phosphorylation of inhibitor kappaB (I kappaB)alpha and p65 and subsequent degradation of I kappaB alpha. Transient transfection experiments using NF kappaB reporter constructs indicated that 6-shogaol inhibits the transcriptional activity of NF kappaB in LPS-stimulated mouse macrophages. We found that 6-shogaol also inhibited LPS-induced activation of PI3K/Akt and extracellular signal-regulated kinase 1/2, but not p38 mitogen-activated protein kinase (MAPK). Taken together, these results show that 6-shogaol downregulates inflammatory iNOS and COX-2 gene expression in macrophages by inhibiting the activation of NF kappaB by interfering with the activation PI3K/Akt/I kappaB kinases IKK and MAPK.

TNF‐α stimulates Akt by a distinct aPKC‐dependent pathway in premalignant keratinocytes

Tumor necrosis factor-alpha (TNF-alpha) is an important proinflammatory cytokine involved in the pathogenesis of inflammatory skin diseases and cutaneous squamous cell carcinoma. Some of these effects are mediated by the stimulatory effect of this cytokine on the Akt signalling pathway, which renders keratinocytes less susceptible to proapoptotic stimuli and enhances cell growth. We have recently shown that TNF-alpha-induced Akt activation may promote the early stages of skin cancer. In this work, we demonstrate that in the premalignant keratinocyte cell line HaCaT, TNF-alpha activates Akt, ERK1/2 and p38. The specific peptide blocking the activity of the atypical protein kinase C (aPKC) species zeta and iota/lambda abrogated the effects of TNF-alpha on Akt and ERK1/2 but increased the activation of p38. The TNF-alpha-dependent phosphorylation of Akt-ERK1/2 was slightly decreased by NF kappaB inhibition and in the presence of p38 blockers. Akt/ERK signalling but not p38 activation was abolished in the presence of the iron chelator desferroxamine that blocks formation of hydroxyl ( OH) radicals. Thus, the TNF-alpha signalling in keratinocytes seems to bifurcate into an aPKC-, NFkB- and OH-dependent pathway resulting in the activation of survival and mitogenic pathways mediated by Akt and ERK1/2, and a signalling pathway conveyed by p38 that contributes to Akt activation but is suppressed by aPKC. Our data may be utilized in the development of more selective anti-TNF-alpha therapeutic strategies.

Studies of Nuclear Factor-κB in peripheral mononuclear cell of type 2 diabetes patients with macrovascular complications

Objective To study the relationship between NF kappa-B (NF-κB) and the development of diabetic macroangiopathy in patients with type 2 diabetes, the expression level of Nuclear Faetor-κB in peripheral mononuclear cell of type 2 diabetes patients with and without macroangiopathy were compared, Methods Enzyme-linked immunosorbent assay was used to measure soluble intercellular adhesion molecule-1 (slCAM-1) in plasma and immunohistochemical method was used to measure the activity of NF-κB in peripheral blood mononu-clear cell in 50 type 2 diabetes patients with macroangiopathy(G3) , 41 type 2 diabetes patients without maeroangiopathy(G2) and 30 nor-real control subjects(G1). Linear correlation model was used to analyze the correlation between the level of sICAM-1 and NF-κB. Results The expression level of Nuclear Factor-κB in peripheral mononuclear cell of G3 was significantly higher than that in G2 and G1(P <0.01). The concentration of plasma sICAM-1 in G3 was significantly higher than that in G2 and G1. The expression level of Nuclear Factor-κB in peripheral mononuclear cell of G2 was significantly higher than that in GI (P < 0.01). The concentration of plasma sICAM-1 in G2 was significantly higher than that in G1. The levels of plasma sICAM-1 in three groups were positive correlated with activity of NF-κB in peripher-al blood mononuclear cells(r = 0.376, 0.283, 0.302, P < 0.05). Conclusion Increased level of NF kappa-B in peripheral blood mono-nuclear might be risk factor for peripheral macroangiopathy in patients with diabetes. It may induce macroangiopathy via up-regulating sI- CAM-1 expression. Key words: Diabetic angiopathies; NF-kappa B/BL

Association of genetic profiles to Crohn’s disease by linear combinations of single nucleotide polymorphisms

A large number of single nucleotide polymorphisms (SNPs) are supposed to be involved in onset, differentiation and development of complex diseases. Univariate analysis is limited in studying complex traits since does not take into account gene-gene interaction, and the correlation of multiple SNPs with a specific phenotype. Moreover it might underestimate gene variants with weaker genetic contribution. Therefore more sophisticated techniques should be adopted when investigating the role of a panel of genetic markers in disease predisposition. In this paper we describe a general method to simultaneously investigate the association between SNPs profile and Crohn's disease (CD), by evaluating the susceptibility or protective role of single or groups of markers. As an association measure we adopted a weighted linear combination of SNPs in which suitable weighting vectors belonged to predefined and over-complete vocabularies of vectors (frames), or were determined by the data. The proposed method found a weighted linear combination of SNPs statistically associated to CD (p=3.81 x 10(-10)) describing the role of the markers in the pathology. In particular, MCP1-A2518G gave the major contribution as protective locus, similarly to TNF-alpha-C857T, DLG5 rs124869, PTPN22 C1858T variants. The NF kappaB -94ATTG variants was found to be irrelevant for CD. For the remaining markers, a susceptibility role was attributed also confirming that markers on CARD15 gene, in particular G908R and L1007fsinsC, are involved with CD to the same extent as FcGIIIA G559T and TNF-alpha-G308A. Moreover, an odds ratio of 3.99(p<1.0 x 10(-4)) was assigned to this combination which is greater than the best odds ratio found in the single SNP analysis. Our methodology allowed to statistically measure the association of a panel of SNPs with a specific phenotype. Therefore this approach could be suitable for a population screening program with simultaneous evaluation of a large set of gene polymorphism.

Myocardial Function After Gut Ischemia/Reperfusion: Does NFκB Play a Role?

Mesenteric ischemia/reperfusion (I/R) is a common problem in critically ill patients and is frequently associated with myocardial dysfunction. Several potential mechanisms have been proposed to be involved in the myocardial dysfunction associated with mesenteric I/R, including nuclear factor kappa B (NF kappaB)-mediated tumor necrosis factor alpha (TNF-alpha) release leading to cardiodepression. Thus, we sought to investigate the effect of NF kappaB inhibition on mesenteric I/R-associated myocardial dysfunction in a large animal model (dog). A total of 21 mongrel dogs were anesthetized and mechanically ventilated. Animals were instrumented with a Swan-Ganz Catheter, left ventricle (LV) pressure manometer, and ultrasonic crystals. Mesenteric I/R consisted of 60 min of ischemia followed by 180 min of reperfusion. Seven animals received pyrrolidine dithiocarbamate (PDTC, 100 mg/kg) prior to mesenteric I/R (I/R PDTC). Another group of animals (n = 7) without mesenteric ischemia received PDTC following baseline measurements and served as control for the effect of PDTC alone (PDTC). Preload recruitable stroke work, +/-dp/dt(max), isovolumic relaxation (tau), and cardiac output were measured. Myocardial tissue was analyzed for NF kappaB activity, TNF-alpha production, and myocardial apoptosis. Mesenteric I/R impaired both LV systolic and diastolic function. Administration of PDTC worsened LV function impairment following I/R. In addition, PDTC resulted in decreased LV function even without mesenteric I/R. NF kappaB, TNF-alpha, and myocardial apoptosis were not different among the groups. Mesenteric I/R affects LV function independent of NF kappaB and TNF-alpha pathways. PDTC acts as a cardiac depressant through a thus far unknown mechanism. Therefore, evaluation of cardiac and hemodynamic function in experimental setups using PDTC has to be carefully interpreted.

Marked regression of liver metastasis by combined therapy of ultrasound‐mediated NFkB‐decoy transfer and transportal injection of paclitaxel, in mouse

Nuclear factor-kappaB (NF kappaB) plays a pivotal role in cancer progression. In this study, we developed a decoy cis-element oligo-deoxyribonucleic acid against NF kappaB-binding site (NF kappaB-decoy), which effectively inhibits NF kappaB activity, and tested the effect of combined therapy comprising local transfection of NF kappaB-decoy into the liver and transportal injection of paclitaxel on cancer growth and metastasis using an orthotopic murine model of colon cancer liver metastasis. For NF kappaB-decoy transfection, we employed a novel approach using ultrasound exposure with an echocardiographic contrast agent, Optison. We examined the influence of NF kappaB-decoy transfer on susceptibility to paclitaxel in cancer cells and the mechanism involved using several in vitro analysis systems. We then studied the in vivo effect of combined NF kappaB-decoy transfer and paclitaxel in preventing cancer progression using a murine model of liver metastasis created by splenic injection of a human colon cancer cell line, HT29. In vitro experiments, including MTT-assay, fluorescence-activated cell sorter and cDNA array analysis, revealed that NF kappaB-decoy transfer significantly increased the susceptibility of cancer cells to paclitaxel, and that decreased expression of anti-apoptotic genes along with increased expression of genes relevant to the apoptosis-promotor may be involved. In vivo experiments showed that local transfection of NF kappaB-decoy into the liver followed by portal injection of paclitaxel effectively induced cancer cell apoptosis in the liver metastasis, and significantly prolonged animal survival compared to controls, without notable side effects. In conclusion, a combination of local NF kappaB-decoy transfer into the liver and transportal injection of paclitaxel may be a safe and effective new therapy for liver metastasis.

Brachyury‐downstream gene sets in a chordate, Ciona intestinalis: integrating notochord specification, morphogenesis and chordate evolution

In vertebrates, Brachyury, a T-box transcription factor gene, seems to have a dual role in the differentiation of axial midline mesoderm cells into notochord and gastrulation cell movements regulated by non-canonical Wnt/planar cell polarity (Wnt/PCP) signaling. To understand the function of Brachyury-downstream genes in chordate embryos, from a series of our survey on differential expression, including subtractive hybridization, dot-blot assays, EST sequences and the expression patterns in whole-mount in situ hybridization at embryonic stages, we developed a knowledge database called "CINOBI: CionaNotochord and Brachyury-downstream gene Index" to create comprehensive catalogues of Brachyury-downstream gene sets in Ciona intestinalis. Combining genome and large-scale cDNA data, we were able to characterize 450 non-redundant Brachyury-downstream genes: Twenty-four genes were newly annotated as notochord-expressed genes. Several genes are components of signaling pathways such as Wnt/PCP, Nf kappaB and TGF-beta signaling. We propose that Brachyury is linked to these pathways regulating the expression of each component, and such a regulatory mechanism might be conserved among chordates.

Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) Augments Acute Lung Injury via Its Neutrophil Priming Effects

Granulocyte macrophage-colony stimulating factor (GM-CSF) has immuno-stimulatory effects. We hypothesized that GM-CSF plays an important role both in lipopolysaccharide (LPS)- and hemorrhage-induced acute lung injury (ALI). We also postulated that GM-CSF augments LPS-induced inflammation by priming neutrophils. ALI was induced in GM-CSF-/- or control C57BL mice either by LPS injection or by hemorrhage. Lung inflammation (by lung expression for tumor necrosis factor-α (TNF-α), macrophage inflammatory protein-2 (MIP-2), interleukin-1β (IL-1β), interleukin-6 (IL-6), and keratinocyte-derived chemokine) and lung injury (by myeloperoxidase and evans blue dye assay) were evaluated after ALI. Incremental doses of LPS (0, 1, 10, and 100 ng/mL) and GM-CSF (0, 1, 10, and 100 ng/mL) were added to bone marrow neutrophils. The expression of TNF-α, MIP-2, and IL-1β was evaluated with enzyme linked immunosorbent assay. The mRNA expression of three cytokines, and the nuclear translocation of nuclear factor kappa B (NFκ-B) were evaluated by reverse transcriptase-polymerase chain reaction and electropnoretic mobility shift assay, respectively. GM-CSF -/- mice showed decreased neutrophil infiltration, less leakage, and lower expression of cytokines in the lung after LPS or hemorrhage. GM-CSF augmented LPS-induced protein and mRNA expression of TNF-α, MIP-2 and IL-1β, which was mediated by increased intra-nuclear translocation of NF-κB. GM-CSF plays an important role in high-dose LPS and hemorrhage-induced ALI, which appears to be mediated by its priming effect on neutrophils.

NF-kappa B Plays a Key Role in the Hematopoietic Niche Function of Osteoblast after Ionizing Radiation.

Recent studies demonstrated that osteoblasts in the endosteal region of bone are a key cellular component of the hematopoietic niche that directly regulates hematopoietic stem cell (HSC) survival, proliferation and differentiation. In experiments with the human fetal osteoblast cell line hFOB1.19, we found survival of gamma-irradiated primary human hematopoietic CD34+ cells was significantly enhanced by co-culture with hFOB cells or by conditioned medium (CM) from hFOB cells, as shown by increased colony efficiency and colony size. In clonogenic assays of CD34+ cells starting with 5 × 103 cells/dish, co-culture with hFOB cells increased colony number from 428±53 (control) to 988±65 (co-culture) after 2 Gy (p<0.01), 301±44 to 739±56 after 4 Gy (p<0.01), and 45±15 to 72±10 after 6 Gy (p<0.05). Surprisingly, although radiation induces osteoblast cell damage, CM from 6 Gy-irradiated hFOB cells had a greater effect than CM from unirradiated hFOB cells in supporting CD34+ clonogenicity and resulted in colony number increase from 430±40 (CM from unirradiated hFOB cells) to 773±48 (CM from 6 Gy-irradiated hFOB cells) after 2 Gy IR (p<0.01). Using a cytokine array for 120 cytokines and chemokines, we detected 11 factors released from hFOB cells, and 4 of them (IL-6, G-CSF, GRO, and IL-8) were enhanced by ionizing radiation (IR). IR (2–8 Gy) caused hFOB cell cycle arrest in G2 phase after 24 h, and irreversible cell cycle block and apoptotic cell death were observed 72 h after IR in a radiation dose-dependent manner. IR induced NF-kappaB (NFkB) p65 phosphorylation and NFkB activity in hFOB cells. Inhibition of NFkB expression with siRNA blocked the ability of hFOB CM to support clonogenic survival of CD34+ cells, and abrogated the effect of IR on release of IL-6, G-CSF, GRO, and IL-8. Radiation upregulated p53 (ser15) phosphorylation within 4 h, and p21 expression after 24 h in hFOB cells. However, NFkB gene silencing accelerated p21 expression and induced a massive apoptosis of hFOB cells. We propose that IR induces NFkB and p53 activation simultaneously in hFOB cells. The fate of osteoblasts after irradiation depends on the amount and severity of injury, and on repair processes regulated by NFkB and p53. Silencing the NFkB gene disrupted this balance and resulted in hFOB cell apoptosis.

Flt3 Receptor Inhibition Abolishes Constitutive NFκB Activation in High-Risk Myelodysplastic Syndrome and Acute Myeloid Leukemia.

High-risk myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) are characterized by the constitutive activation of the anti-apoptotic transcription factor NF-kappaB, via the activation of the IKK complex. We show that constitutive activation of the receptor tyrosine kinase Flt3 is responsible for IKK activation and this activation of the NF-kappaB pathway was found to involve a not yet described phosphorylation of the IKK and IkBa complex involving tyrosine residues compared to serine residues in the classical NF-kappaB pathway. Chemical inhibition or knockdown of Flt3 with small interfering RNAs abolished NF-kappaB activation in MDS and AML cell lines, as well as in primary CD34+ bone marrow cells from patients, causing mitochondrial apoptosis. Epistatic analysis involving the simultaneous inhibition of Flt3 and IKK indicated that both kinases act via the same anti-apoptotic pathway. An IKK2 mutant with a constitutive kinase activity and a plasma membrane-tethered mutant of NEMO that activates IKK1/2 prevented the cytocidal action of Flt3 inhibition. IKK2 and Flt3 physically associated in MDS and AML cells and Flt3 inhibition caused the release of IKK2 from a preferential association with the plasma membrane. Flt3 inhibition only killed CD34+ bone marrow cells from high-risk MDS and AML patients, in correlation with the blast numbers and the NF-kappaB activity, yet had no lethal effect on healthy CD34+ cells or cells from low-risk MDS. These results suggest that Flt3 inhibitors might exert an anti-neoplastic effect in high-risk MDS and AML through inhibition of constitutive NF kappaB activation.

Expression of a pathogen-response program in peripheral blood cells defines a subgroup of Rheumatoid Arthritis patients

Rheumatoid arthritis (RA) is a heterogeneous disease with unknown etiology. Here we aimed to distinguish RA subtypes based on peripheral blood (PB) gene expression profiles in comparison with a pathogen-response transcriptional program. PB was obtained from 35 RA patients and 15 healthy individuals. For expression profiling we used DNA microarrays. A combined cluster analysis of RA and control samples together with samples from a viral infection model revealed that the gene expression profile of a subgroup of RA patients (RA(A)) was reminiscent to that of poxvirus-infected macaques. Statistical analysis, followed by Gene Ontology analysis of the RA(A) patients confirmed that these patients form a distinct group, with activation of several host defense mechanisms that resemble a common host-pathogen response. Analysis of the promoter region of genes that were overexpressed in the RA(A) patients, revealed an enrichment of transcription factor binding sites for NF kappaB and interferon-activated transcription factors. Moreover, this subgroup of RA patients expressed significantly increased titers of anti-cyclic citrullinated peptide antibodies. We conclude that activation of a host-pathogen response defines a subgroup of RA patients characterized by increased autoreactivity against citrullinated proteins.

Sequence determination of variable regions within the genomes of gallid herpesvirus-2 pathotypes

Comparative genomic studies of attenuated and virulent strains of Gallid herpesvirus 2 (GaHV-2) have identified 6 regions of sequence variability. These regions include the open reading frames (ORFs) encoding UL36 and UL49 and regions devoid of large ORFs (132-bp repeats, a-like sequences and the junctions flanking the unique short region). Our data indicate that the carboxyl terminus of UL36 contains regions of heterogeneity that are unique to CVI988-derived attenuated strains. A deletion of the TKSERT domain and a glycine(245) polymorphism in the UL49 proteins were also identified in these derivatives. Phylogenetic analyses of both UL36 and UL49 sequences indicate that CVI988-derived strains partition differently from other attenuated strains (RM-1 and R2/23), indicating that additional mutations contribute to attenuation. In very virulent and very virulent plus strains a single nucleotide polymorphism (SNP) was identified within the 132-bp tandem repeats. Within the junctions flanking the unique short region, these strains also contain deletions in sequences that are predicted to bind the transcription factor NF kappaB. In some attenuated strains, deletions were also identified in the latency-associated transcript (LAT) promoters and adjacent regions encoding microRNAs. These results indicate that virulence is likely multi-factorial with contributions from both multiple genes and cis-acting sites.

Knocking Out Peroxisome Proliferator-Activated Receptor (PPAR) α Inhibits Radiation-Induced Apoptosis in the Mouse Kidney through Activation of NF-κB and Increased Expression of IAPs

Peroxisome proliferator-activated receptor (PPAR) alpha, a member of the ligand-activated nuclear receptor superfamily, plays an important role in lipid metabolism and glucose homeostasis and is highly expressed in the kidney. The present studies were aimed at testing the hypothesis that PPARalpha knockout mice would exhibit decreased radiation-induced apoptosis due to exacerbated activation of NF-kappaB (NFKB) and expression of pro-survival factors. Thirty wild-type mice (29S1/SvImJ) and 30 PPARalpha knockout mice were irradiated with a single total-body dose 10 Gy of (137)Cs gamma rays; controls were sham-irradiated. Tissue samples were collected at 3, 6, 12, 24 and 48 h postirradiation. Apoptosis was quantified using immunohistochemical staining for apoptotic bodies and cleaved caspase 3. Radiation-induced apoptosis was observed in both mouse strains in a time-dependent manner. However, the level of apoptosis was significantly suppressed in PPARalpha knockout mice compared with wild-type mice at 6 h postirradiation (P < 0.05). This inhibition of radiation-induced apoptosis was associated with time-dependent increases in NF-kappaB DNA-binding activity, IkappaBalpha phosphorylation, and expression of other antiapoptosis factors in the PPARalpha knockout mouse kidneys but not in wild-type animals. These data support the hypothesis that the loss of PPARalpha expression leads to the suppression of radiation-induced apoptosis in the mouse kidney, mediated through activation of NF-kappaB and up-regulation of anti-apoptosis factors.