Upregulation Of NF-κB Activity Research Articles

Octreotide inhibits secretion of IGF-1 from orbital fibroblasts in patients with thyroid-associated ophthalmopathy via inhibition of the NF-κB pathway.

PurposeWe investigated the effect of octreotide, a long-acting somatostatin (SST) analogue, on IGF-1 secretion and its possible mechanism of action in orbital fibroblasts (OFs) from patients with thyroid-associated ophthalmopathy (TAO).Materials and methodsOFs were isolated from the orbital fat of patients with TAO or healthy individuals. The expression level of insulin-like growth factor (IGF)-1, at the protein and mRNA level, was determined with ELISA and quantitative RT-PCR, respectively. The expression pattern of somatostatin receptor (SSTR) 2, which has the highest affinity for octreotide, was examined by flow cytometry. The activity of NF-κB pathway was determined by examining the levels of phosphorylation of IKKα/β and p65, and degradation of IκB via western blot analysis, and by measuring the activity of NF-kB-dependent luciferase via transfection with plasmids containing luciferase and NF-κB binding site.ResultsOFs from patients with TAO showed significantly higher levels of IGF-1 secretion and NF-κB activity even in the absence of stimulation, compared to those from controls. Treatment with octreotide reduced the level of IGF-1 secretion in OFs from patients with TAO, but not in OFs from controls. OFs from patients with TAO expressed higher levels of SSTR2 on the cell surface, compared to controls. In addition, the expression of IGF-1 at the protein and mRNA level was dependent on the activity of NF-κB pathway in OFs from patients with TAO. Furthermore, treatment with octreotide reduced on the activity of NF-κB pathway in OFs from patients with TAO.ConclusionOFs from patients with TAO showed significantly higher levels of IGF-1 secretion via up-regulation of NF-κB activity. Treatment with octreotide inhibited the secretion of IGF-1 by reducing the NF-κB pathway in OFs, which expressed higher levels of SSRT2 on the cell surface, from patients with TAO.

Mucosa-Associated Lymphoid Tissue 1 Is an Oncogene Inducing Cell Proliferation, Invasion, and Tumor Growth via the Upregulation of NF-κB Activity in Human Prostate Carcinoma Cells.

Prostate cancer is one of the most common seen malignancies and the leading cause of cancer-related death among men. Given the importance of early diagnosis and treatment, it is worth to identify a potential novel therapeutic target for prostate cancer. Mucosa-associated lymphoid tissue 1 (MALT1) is a novel gene involved in nuclear factor κB (NF-κB) signal transduction by acting as an adaptor protein and paracaspase, with an essential role in inflammation and tumorigenesis in many cancers. This study investigated the functions and the potential regulatory mechanisms of MALT1 in the human prostate cancer cells. We found that MALT1 is abundant in prostate cancer tissues. MALT1 facilitated NF-κB subunits (p50 and p65) nuclear translocation to induce gene expression of interleukin 6 (IL-6) and C-X-C motif chemokine 5 (CXCL5) in prostate carcinoma cells. MALT1 promoted cell proliferation, invasion, and tumor growth in vitro and in vivo. MALT1 enhanced NF-κB activity in prostate carcinoma cells; moreover, NF-κB induced MALT1 expression determined by reporter and immunoblot assays, implying there is a positive feedback loop between MALT1 and NF-κB. In conclusion, MALT1 is a NF-κB-induced oncogene in the human prostate carcinoma cells.

Interaction between SDF1 and CXCR4 Promotes Photoreceptor Differentiation via Upregulation of NFκB Pathway Signaling Activity in Pax6 Gene-Transfected Photoreceptor Precursors

Background: CCL2 (also known as monocyte chemoattractant protein 1) and CX3CR1 (also known as Fractalkine receptor)-deficient mice have damaged photoreceptors. Objectives: We examined the interaction of SDF1 and CXCR4 on the differentiation of retinal progenitors into rhodopsin-positive photoreceptors. Methods: Cloned retinal progenitors were obtained by Pax6 gene transfection of mouse iPS cells followed by serial dilution. Clones were selected by expression of nestin, Musashi1, Six3, and Chx10 mRNA. Cell surface protein expression was analyzed by flow cytometry. The levels of mRNA and intracellular protein were examined by real-time PCR and immunochemistry, respectively. Transient transfection experiments of retinal progenitors were conducted using a human rhodopsin promoter luciferase plasmid. Results: We selected 10 clones that expressed Six3, Chx10, Crx, Rx1, Nrl, CD73, and rhodopsin mRNA, which, except for rhodopsin, are photoreceptor precursor markers. Clones expressed both CD73 and CXCR4 on the cell surface and differentiated into rhodopsin-positive photoreceptors, which was reinforced by the addition of exogenous SDF1. A CXCR4 inhibitor AMD3100 blocked SDF1-mediated differentiation of progenitors into photoreceptors. SDF1 enhanced human rhodopsin promoter transcription activity, possibly via the NFκB pathway. Addition of SDF1 to the cell culture induced nuclear translocation of NFκB on retinal progenitor cell clones. Neonatal and newborn mouse retinas expressed SDF1 and CXCR4. Cells in the outer nuclear layer where photoreceptors are located expressed CXCR4 at P14 and P56. Cells in the inner nuclear layer expressed SDF1. Conclusions: These findings suggest that retinal progenitor cell differentiation was at least partly regulated by SDF1 and CXCR4 via upregulation of NFκB activity.

94 - MALT1 is a downstream gene of WNT/β-catenin inducing cell proliferation and invasion potential via the upregulation of NFκB activity in human prostate carcinoma cells

94 - MALT1 is a downstream gene of WNT/β-catenin inducing cell proliferation and invasion potential via the upregulation of NFκB activity in human prostate carcinoma cells

Integrating GRK2 and NFkappaB in the Pathophysiology of Cardiac Hypertrophy.

G protein coupled receptor kinase type 2 (GRK2) plays an important role in the development and maintenance of cardiac hypertrophy and heart failure even if its exact role is still unknown. In this study, we assessed the effect of GRK2 on the regulation of cardiac hypertrophy. In H9C2 cells, GRK2 overexpression increased atrial natriuretic factor (ANF) activity and enhanced phenylephrine-induced ANF response, and this is associated with an increase of NFκB transcriptional activity. The kinase dead mutant and a synthetic inhibitor of GRK2 activity exerted the opposite effect, suggesting that GRK2 regulates hypertrophy through upregulation of NFκB activity in a phosphorylation-dependent manner. In two different in vivo models of left ventricle hypertrophy (LVH), the selective inhibition of GRK2 activity prevented hypertrophy and reduced NFκB transcription activity. Our results suggest a previously undisclosed role for GRK2 in the regulation of hypertrophic responses and propose GRK2 as potential therapeutic target for limiting LVH.

Sa1279 “Non Celiac Gluten Sensitivity” (NCGS) Is Uncommon in Patients Spontaneously Adhering to Gluten Free Diet (GFD) Without Medical Necessity

Enteropathy-associated T-cell lymphoma (EATL) is a rare intestinal lymphoma that arises from intraepithelial lymphocytes. Clinical outcome of patients with EATL is very poor, due to chemotherapy-resistance and high relapse rates. Therefore, new therapeutic options for EATL are urgently needed. Many studies in other types of lymphoma have shown that inhibition of apoptosis may cause chemotherapy-resistance and that restoration of defective apoptosis can induce cell death in these lymphomas. Preliminary data in EATL samples have demonstrated an increased expression of a fraction of NFκB target genes, suggesting upregulation of NF-κB activity in EATL tumor cells. The proteasome inhibitor bortezomib inhibits NF-κB activity and can induce apoptosis via upregulation of the pro-apoptotic BH3only protein Noxa. In the present study, we evaluated if the intrinsic apoptosis pathway is disrupted in EATL and if bortezomib can restore apoptosis in cultured EATL cells. Lasercapture microdissection was applied to 19 frozen EATL samples to obtain purified tumor cells for RNA isolation. Intraepithelial lymphocytes (IEL) of healthy controls were obtained from fresh duodenal biopsies and isolated by FACS cell sorting. RT-MLPA analysis revealed that expression of the pro-apoptotic BH3-only gene Noxa was significantly downregulated in EATL cells compared to healthy donor IEL. Treatment with bortezomib resulted in induction of apoptosis in all EATL samples tested. The lethal dose (LD50) varied between 5 nM and 15 nM after 36 and 48 hours of incubation. Bortezomib-induced cell death in EATL cells was caspase-9 mediated. mRNA and protein expression analyses showed upregulation of Noxa after incubation with bortezomib. Downregulation of Noxa using siRNA analysis decreased bortezomib-induced apoptosis in EATL cells. In conclusion, our study showed that bortezomib induces apoptosis by upregulation of Noxa in EATL cells. Therefore, bortezomib should be considered as a potential drug in the treatment of patients with EATL to improve their prognosis.

Stromal Adipocyte Enhancer-binding Protein (AEBP1) Promotes Mammary Epithelial Cell Hyperplasia via Proinflammatory and Hedgehog Signaling

Disruption of mammary stromal-epithelial communication leads to aberrant mammary gland development and induces mammary tumorigenesis. Macrophages have been implicated in carcinogenesis primarily by creating an inflammatory microenvironment, which promotes growth of the adjacent epithelial cells. Adipocyte enhancer-binding protein 1 (AEBP1), a novel proinflammatory mediator, promotes macrophage inflammatory responsiveness by inducing NF-κB activity, which has been implicated in tumor cell growth and survival by aberrant sonic hedgehog (Shh) expression. Here, we show that stromal macrophage AEBP1 overexpression results in precocious alveologenesis in the virgin AEBP1 transgenic (AEBP1(TG)) mice, and the onset of ductal hyperplasia was accelerated in AEBP1(TG) mice fed a high fat diet, which induces endogenous AEBP1 expression. Transplantation of AEBP1(TG) bone marrow cells into non-transgenic (AEBP1(NT)) mice resulted in alveolar hyperplasia with up-regulation of NF-κB activity and TNFα expression as displayed in the AEBP1(TG) mammary macrophages and epithelium. Shh expression was induced in AEBP1(TG) macrophages and RAW264.7 macrophages overexpressing AEBP1. The Shh target genes Gli1 and Bmi1 expression was induced in the AEBP1(TG) mammary epithelium and HC11 mammary epithelial cells co-cultured with AEBP1(TG) peritoneal macrophages. The conditioned AEBP1(TG) macrophage culture media promoted NF-κB activity and survival signal, Akt activation, in HC11 cells, whereas such effects were abolished by TNFα neutralizing antibody treatment. Furthermore, HC11 cells displayed enhanced proliferation in response to AEBP1(TG) macrophages and their conditioned media. Our findings highlight the role of AEBP1 in the signaling pathways regulating the cross-talk between mammary epithelium and stroma that could predispose the mammary tissue to tumorigenesis.

Down-regulation of PKHD1 induces cell apoptosis through PI3K and NF-κB pathways

Mutations in PKHD1 (polycystic kidney and hepatic disease gene 1) gene cause the autosomal recessive polycystic kidney disease (ARPKD). Fibrocystin/polyductin (FPC), encoded by PKHD1, is a membrane-associated receptor-like protein. Although it is widely accepted that cystogenesis is mostly due to aberrant cell proliferation and apoptosis, it is still unclear how apoptosis is regulated. The aim of this study is to analyze the relationship among apoptosis, phosphatidylinositol 3-kinase (PI3K)/Akt and nuclear factor κB (NF-κB) in FPC knockdown kidney cells. We show that PKHD1-silenced HEK293 cells demonstrate a higher PI3K/Akt activity. Selective inhibition of PI3K/Akt using LY294002 or wortmannin in these cells increases serum starvation-induced HEK293 cell apoptosis with a concomitant decrease in cell proliferation and higher caspase-3 activity. PI3K/Akt inhibition also leads to increased NF-κB activity in these cells. We conclude that the PI3K/Akt pathway is involved in apoptotic function in PKHD1-silenced cells, and PI3K/Akt inhibition correlates with upregulation of NF-κB activity. These observations provide a potential platform for determining FPC function and therapeutic investigation of ARPKD.

Sustained NF‐kappaB activity in chronic lymphocytic leukemia is independent of genetic and epigenetic alterations in the TNFAIP3 (A20) locus

Inappropriate nuclear factor (NF) κB activity is one major hallmark of B-cell malignancies and chronic lymphocytic leukemia (CLL). NFκB-dependent genes are involved in antiapoptosis, cell proliferation and metastasis and are responsible for survival and proliferation of tumors. However, the mechanisms of NFκB activity in CLL still need to be elucidated. Previously, we identified translocations in a region on chromosome 6q that encodes tumor necrosis factor alpha-induced protein 3, which is a key player in negative feedback loop regulation of NFκB. Inactivation of this ubiquitin-editing enzyme is involved in immunopathologies and in tumorigenesis. Frequent mutations in the A20 locus--leading to sustained NFκB activity--could be shown to play a dominant role in development of different B-cell malignancies. To check if A20 is involved in upregulation of NFκB activity in CLL, we sequenced Exons 2-9 of the A20 gene in 55 CLL DNA samples. Furthermore, we determined the methylation status of the promoter region in 63 CLL DNA samples and compared to 10 control DNAs of B cells from healthy donors. Contrary to reports from other B-cell malignancies, the A20 region showed neither mutations nor aberrant DNA methylation. Moreover, its expression could be confirmed by immunoblotting and showing comparable results to healthy B cells. These results indicate that malignant development in CLL differs from most of other B-cell malignancies, which show frequent inactivation of A20.

Tumor Necrosis Factor-α Contributes to Microenvironmental up- Regulation of NF-κB Activity in Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia: Implication for a Novel Therapeutic Target.

Constitutively activated NF-κB has been demonstrated in primary blast cells and cell lines derived from Philadelphia chromosome (Ph- positive acute lymphoblastic leukemia(Ph- ALL). In our previous study, we have shown the essential role for NF-κB in growth and survival of Ph- ALL cells. To gain insight into the microenvironmental (cytokines and/or stromal cell) regulation of NF-κB activity in Ph- ALL, we lentivirally transduced Ph- ALL cells, IMS-PhL1 and Sup-B15 cells, with NF-κB/luciferase (kB/Luc) reporter construct and established a bioluminescence imaging model of Ph- ALL for in vitro and in vivo analysis. In in vitro study, we checked NF-κB/Luc activity by luminoter. It showed that NF-κB activity of Ph- ALL cells was significantly up-regulated by TNFa stimulation and synergistically augmented by seeding them onto a layer of murine HESS5 stromal cells, which singly didn't change NF-κB activity in Ph- ALL cells. DHMEQ, a specific inhibitor of nuclear translocation of p65, eradicated constitutive and TNFa inducible NF-κB activity of Ph- ALL cells and induced their substantial apoptosis dose-dependently. However, the inhibitory effect of DHMEQ on TNFa induced NF-κB activity as well as viability of Ph- ALL was alleviated in the presence of HESS5. This alleviatory effect of DHMEQ induced NF-κB suppression by HESS5 was overcame by addition of TNFa inhibitor, Etanercept, in a dose of less than 1ug/ml. (Fig.1) Taken together, TNFa plays an essential role in up-regulation of NF-κB activity in the absence or presence of HESS5 cells. When Ph- ALL cells were treated with TPCA-1, an IKK-2 inhibitor, the TNFa induced NF-κB activity in Ph- ALL cells was suppressed even in the presence of HESS5 cells. Cell proliferation assay also showed inhibitory effect on proliferation of Ph- ALL cells by TPCA-1. In in vivo study, we transplanted Ph- ALL cells into NOD-Scid mice and periodically monitored the NF-κB activity of Ph- ALL cells by a CCD camera. Intriguingly, strong signal was detected in liver, stomach and ovary in addition to bone marrow, which was the predominant site of leukemic cell infiltration. QR-PCR analysis and immunohistochemistry staining for mouse tissue verified tumor infiltration up-regulate murine TNFa production in these tissues. It suggests the essential role of TNFa in the up-regulation of NF-κB signaling in mouse model. However high dose Etanercept, 1mg, subcutaneous injection into Ph- ALL cells transplanted mouse didn't show significant reduction of NF-κB activity and partial response of NF-κB suppression was seen in the mouse injected with 5mg of Etanercept intraperitoneally. (Fig.2) This result suggests that factors other than TNFa may contribute to in vivo maintenance and/or up-regulation of NF-κB activity in Ph- ALL cells. In conclusion, TNFa-dependent and independent pathways are involved in microenvironmental up-regulation of NF-κB activity, which contribute to survival, expansion and presumably drug-resistance of Ph- ALL cells. The present bioimaging model helps us to dissect the regulatory mechanism of NF-κB signal in Ph- ALL and the results suggest us microenvironment as a novel therapeutic target in the treatment of Ph- ALL. [Display omitted] [Display omitted]

Thioredoxin-like 6 protects retinal cell line from photooxidative damage by upregulating NF-κB activity

Apoptosis is the common pathway to photoreceptor cell death in many eye diseases including age-related macular degeneration which affects more than 8 million individuals in the United States alone. RdCVF, a truncated mouse thioredoxin is specifically expressed by rod photoreceptor cells and prevents the apoptosis of cone cells. However the protective mechanism of RdCVF and the implications of its human homologue, thioredoxin-like 6 (TXNL6), on the apoptosis of retinal cells remain unknown. In this study, we examined the function of TXNL6 and investigated its mechanism of protection using a cone photoreceptor cell line, 661W. We found that the photooxidative stress-induced degradation of NF-κB proteins is rescued by overexpression of TXNL6, which enabled the NF-κB transactivation activity. Furthermore, the overexpression of TXNL6 rescued the photooxidative stress-induced apoptosis of 661W cells. Interestingly, this protective effect was significantly blocked by NF-κB specific inhibitors demonstrating that TXNL6 exerts its protective effect against apoptosis via NF-κB. Taken together, our study shows that the TXNL6 probably protects retinal cells from photooxidative damage-induced apoptosis via upregulation of NF-κB activity. The identification of TXNL6 and the demonstration of its protective mechanism offer new insights into treatment possibilities for photoreceptor cell degradation.

Nuclear factor-κB activation in response to active site-inhibited factor VIIa pretreatment during acute pancreatitis in the rat

Objective. Active site-inhibited factor VII (fVIIai) has previously been reported to reduce remote organ inflammation during acute pancreatitis. The aim of this study was to investigate whether the anti-inflammatory properties of fVIIai in remote organs are mediated through nuclear factor (NF)-κB. Material and methods. Acute pancreatitis in the rat was induced using retrograde intraductal infusion of sodium taurodeoxycholate. Upregulation of NF-κB activity was evaluated using an enzyme-linked immunosorbent assay. Results. An increase in NF-κB activity could be seen in the lungs, which was reduced to normal levels at both 1 and 6 h following pretreatment with fVIIai. In the liver, the greatest treatment effects were seen at 1 h, but were still notable at 6 h. In the ileum, treatment effects were seen only at 6 h. Simultaneous fVIIai and N-acetylcysteine treatment ameliorated many of the treatment effects of fVIIai. In the pancreas, no differences could be seen. Conclusion. fVII inhibition reduces NF-κB act...

Targeted Immunomodulation of the NF-κB Pathway in Airway Epithelium Impacts Host Defense against Pseudomonas aeruginosa

We investigated the impact of inflammatory signaling in airway epithelial cells on host defense against Pseudomonas aeruginosa, a major cause of nosocomial pneumonia. In mice, airway instillation of P. aeruginosa resulted in NF-kappaB activation in the lungs that was primarily localized to the bronchial epithelium at 4 h, but was present in a variety of cell types by 24 h. We modulated NF-kappaB activity in airway epithelium by intratracheal delivery of adenoviral vectors expressing RelA (AdRelA) or a dominant inhibitor of NF-kappaB before P. aeruginosa infection. Bacterial clearance was enhanced by up-regulation of NF-kappaB activity following AdRelA administration and was impaired by treatment with a dominant inhibitor of NF-kappaB. The TNF-alpha concentration in lung lavage was increased by AdRelA treatment and beneficial effects of NF-kappaB up-regulation were abrogated in TNF-alpha-deficient mice. In contrast, NF-kappaB inhibition reduced MIP-2 expression and neutrophil influx following P. aeruginosa infection. Therefore, inflammatory signaling through the NF-kappaB pathway in airway epithelial cells critically regulates the innate immune response to P. aeruginosa.

Loss of New Chemokine CXCL14 in Tumor Tissue Is Associated with Low Infiltration by Dendritic Cells (DC), while Restoration of Human CXCL14 Expression in Tumor Cells Causes Attraction of DC Both In Vitro and In Vivo

Breast and kidney-expressed chemokine (BRAK) CXCL14 is a new CXC chemokine with unknown function and receptor selectivity. The majority of head and neck squamous cell carcinoma (HNSCC) and some cervical squamous cell carcinoma do not express CXCL14 mRNA, as opposed to constitutive expression by normal oral squamous epithelium. In this study, we demonstrate that the loss of CXCL14 in HNSCC cells and at HNSCC primary tumor sites was correlated with low or no attraction of dendritic cell (DC) in vitro, and decreased infiltration of HNSCC mass by DC at the tumor site in vivo. Next, we found that recombinant human CXCL14 and CXCL14-positive HNSCC cell lines induced DC attraction in vitro, whereas CXCL14-negative HNSCC cells did not chemoattract DC. Transduction of CXCL14-negative HNSCC cell lines with the human CXCL14 gene resulted in stimulation of DC attraction in vitro and increased tumor infiltration by DC in vivo in chimeric animal models. Furthermore, evaluating the biologic effect of CXCL14 on DC, we demonstrated that the addition of recombinant human CXCL14 to DC cultures resulted in up-regulation of the expression of DC maturation markers, as well as enhanced proliferation of allogeneic T cells in MLR. Activation of DC with recombinant human CXCL14 was accompanied by up-regulation of NF-kappaB activity. These data suggest that CXCL14 is a potent chemoattractant and activator of DC and might be involved in DC homing in vivo.

Expression of NF-kappaB in prostate cancer lymph node metastases.

Nuclear factor-kappaB (NF-kappaB) is a transcription factor that transactivates genes involved in the regulation of cell growth, apoptosis, angiogenesis, and metastasis. Our aim was to assess NF-kappaB expression in lymph node (LN) metastases of prostate cancer. Immunohistochemical staining was performed using the p65 anti-NF-kappaB antibody. Seventy-seven paraffin-embedded LN specimens obtained from 54 prostate cancer patients were analyzed. Of the 54 patients, 32 had positive LN metastases, while 22 showed no evidence of metastasis and were considered as controls. The overall percentage of NF-kappaB-nuclear localization was assessed, as well as the intensity of staining. Nuclear localization of NF-kappaB was significantly greater in the metastatic LN group compared to controls. In patients with positive-LN metastases, 84.4% showed >10% nuclear staining in tumor cells. Moreover, 64.4% of the malignant LN specimens had >10% nuclear staining in lymphocytes compared to 0% in controls. Intensity of cytoplasmic and nuclear staining was higher in the metastatic LN group than in controls (P < 0.01). Nuclear localization/activation of NF-kappaB is up-regulated in prostate cancer LN metastasis. Such up-regulation of NF-kappaB activity is observed in the tumor cells as well as in the surrounding lymphocytes.

Involvement of nuclear transcription factor-κB in low-dose doxorubicin-induced drug resistance of cervical carcinoma cells

Administration of suboptimal doses of anticancer drugs not only fails to control tumor but often results in increased drug resistance of tumor cells. However, little is known about the effects of transient exposure to a minimally cytotoxic dose of chemotherapy on the development of drug resistance of the tumors. Previous studies have shown that upregulation of drug-exporter proteins (ATP-binding-cassette proteins) may be one of the key mechanisms involved in inducible drug resistance. In this study, we demonstrated that upregulation of NF-κB is another possible mechanism. SiHa cells were exposed to low-dose doxorubicin (100 nM; ic 30) for 3 hr, and then were continuously cultured in drug-free culture media (designated as SiHa/DR cells). SiHa/DR cells at up to 9 passages showed increased resistance to doxorubicin and cross-resistance to cisplatin. Results of quantitative real-time PCR and flow cytometry assay indicated that the increased drug-resistance in SiHa/DR cells was not due to upregulation of drug-exporter proteins or to the decrease of intracellular concentration of anticancer drugs. Both the basal and drug-induced NF-κB activity were shown to be increased in SiHa/DR cells by EMSA and NF-κB-driven luciferase reporter gene assay. Suppression of NF-κB activation by transfection of a dominant negative IκBα prevented the development of drug resistance, indicating that the upregulated NF-κB activity was positively correlated with the low-dose doxorubicin-induced drug resistance. These results suggest that even a transient exposure to a small dose of anticancer drugs may induce drug resistance in some cancer cells via upregulation of NF-κB activity.

Ceramide-induced and Age-associated Increase in Macrophage COX-2 Expression Is Mediated through Up-regulation of NF-κB Activity

We have shown that the age-associated increase in lipopolysaccharide (LPS)-stimulated macrophages (M phi) prostaglandin E(2) (PGE(2)) production is because of ceramide-induced up-regulation of cyclooxygenase (COX)-2 transcription that leads to increased COX-2 expression and enzyme activity. To determine the mechanism of the age-related and ceramide-dependent increase in COX-2 transcription, we investigated the role of various transcription factors involved in COX-2 gene expression. The results showed that LPS-initiated activations of both consensus and COX-2-specific NF-kappa B, but not AP-1 and CREB, were significantly higher in M phi from old mice than those from young mice. We further showed that the higher NF-kappa B activation in old M phi was because of greater I kappa B degradation in the cytoplasm and p65 translocation to the nucleus. An I kappa B phosphorylation inhibitor, Bay 11-7082, inhibited NF-kappa B activation, as well as PGE(2) production, COX activity, COX-2 protein, and mRNA expression in both young and old M phi. Similar results were obtained by blocking NF-kappa B binding activity using a NF-kappa B decoy. Furthermore, NF-kappa B inhibition resulted in significantly greater reduction in PGE(2) production and COX activity in old compared with young M phi. Addition of ceramide to the young M phi, in the presence or absence of LPS, increased NF-kappa B activation in parallel with PGE(2) production. Bay 11-7082 or NF-kappa B decoy prevented this ceramide-induced increase in NF-kappa B binding activity and PGE(2) production. These findings strongly suggest that the age-associated and ceramide-induced increase in COX-2 transcription is mediated through higher NF-kappa B activation, which is, in turn, because of a greater I kappa B degradation in old M phi.

The Transcription Factors NF-κB and AP-1 Are Differentially Regulated in Skeletal Muscle during Sepsis

Sepsis is associated with increased muscle proteolysis and upregulated transcription of several genes in the ubiquitin-proteasome proteolytic pathway. Glucocorticoids are the most important mediator of sepsis-induced muscle cachexia. Here, we examined the influence of sepsis in rats on the transcription factors NF-κB and AP-1 in skeletal muscle and the potential role of glucocorticoids in the regulation of these transcription factors. Sepsis was induced by cecal ligation and puncture (CLP). Control rats were sham-operated. NF-κB and AP-1 DNA binding activity was determined by electrophoretic mobility shift assay (EMSA) in extensor digitorum longus muscles at different time points up to 16 h after sham-operation or CLP. Sepsis resulted in an early (4 h) upregulation of NF-κB activity followed by inhibited NF-κB activity at 16 h. AP-1 binding activity was increased at all time points studied during the septic course. When rats were treated with the glucocorticoid receptor antagonist RU38486, NF-κB activity increased, whereas AP-1 activity was not influenced by RU38486. The results suggest that NF-κB and AP-1 are differentially regulated in skeletal muscle during sepsis and that glucocorticoids may regulate some but not all transcription factors in septic muscle.

The NF-κB Family Member RelB Is Required for Innate and Adaptive Immunity toToxoplasma gondii

AbstractThe NF-κB family of transcription factors are associated with the regulation of innate and adaptive immunity to infection. Infection of C57BL/6 mice with Toxoplasma gondii resulted in up-regulation of NF-κB activity that included the NF-κB family member RelB. To assess the role of RelB in the regulation of the immune response to this infection, we challenged RelB-deficient mice (RelB−/−) and wild-type (WT) littermate controls with T. gondii. Although WT controls were resistant to T. gondii, RelB−/− mice succumbed 10–15 days after infection. Examination of accessory cell functions associated with resistance to T. gondii revealed that RelB−/− macrophages stimulated with IFN-γ plus LPS or TNF-α produced IL-12 as well as reactive nitrogen intermediates and inhibited parasite replication similar to WT macrophages. Analysis of the systemic responses of RelB−/− and WT mice revealed that infected mice had similar serum levels of IL-12. However, RelB−/− mice challenged with T. gondii produced negligible levels of IFN-γ and had reduced NK cell activity compared with WT mice. Similarly, splenocytes from uninfected RelB−/− mice stimulated with polyclonal stimuli were deficient in their ability to produce IFN-γ. Together, our results demonstrate that RelB is essential for the development of innate NK and adaptive T cell responses that lead to the production of IFN-γ and resistance to T. gondii.

Regulation of interleukin 1 signalling through integrin binding and actin reorganization: disparate effects on NF-kappaB and stress kinase pathways.

Interleukin 1 (IL-1)-mediated gene regulation is dependent on cell-matrix interactions. Both IL-1-activated pathways, nuclear factor kappaB (NF-kappaB) and the stress-activated protein kinase (SAPK), can be regulated by cell adhesion and changes in the cytoskeleton, suggesting that cell-matrix effects on IL-1 responses are initiated in part though effects on signal transduction. Here we show that IL-1-induced transient alterations in cell shape and in the cytoskeleton in fibronectin attached cells are correlated with effects on peak activity of NF-kappaB and SAPK. Cells on fibronectin showed a 1.5-2-fold enhancement in IL-1-induced NF-kappaB activity compared with levels in cells on poly(l-lysine) or bare tissue culture plates. The effect was increased with increasing concentrations of fibronectin and was most prominent at lower concentrations of IL-1. In contrast, fibronectin attachment caused an approx. 50% decrease in the IL-1 activation of SAPK, eliminating the peak activity after 15 min of stimulation with IL-1. IL-1-induced NF-kappaB activity showed a successive, substratum-independent increase during 4 h of attachment and spreading, whereas the inhibitory effect of fibronectin on the SAPK pathway was induced at the initial stages of attachment. Further, the addition of a peptide containing the motif RGD resulted in a 40% decrease in NF-kappaB activity in cells on fibronectin, largely accounted for by an effect on the p50/p65 heterodimer. Similarly, blocking of integrin aggregation by RGD-containing peptide resulted in a total abrogation of the fibronectin effect on IL-1-induced SAPK activity. The results demonstrate disparate effects of cell adhesion on the activation by IL-1 of the NF-kappaB and SAPK pathways. Thus fibronectin attachment causes an up-regulation of NF-kappaB activity in the presence of IL-1, whereas in contrast it results in a pronounced decrease in IL-1-induced SAPK activity.