NF-kappaB Protein Research Articles

Intravenous Injection of PEI-Decorated Iron Oxide Nanoparticles Impacts NF-kappaB Protein Expression in Immunologically Stressed Mice.

Nanoparticle-based formulations are considered valuable tools for diagnostic and treatment purposes. The surface decoration of nanoparticles with polyethyleneimine (PEI) is often used to enhance their targeting and functional properties. Here, we aimed at addressing the long-term fate in vivo and the potential "off-target" effects of PEI decorated iron oxide nanoparticles (PEI-MNPs) in individuals with low-grade and persistent systemic inflammation. For this purpose, we synthesized PEI-MNPs (core-shell method, PEI coating under high pressure homogenization). Further on, we induced a low-grade and persistent inflammation in mice through regular subcutaneous injection of pathogen-associated molecular patterns (PAMPs, from zymosan). PEI-MNPs were injected intravenously. Up to 7 weeks thereafter, the blood parameters were determined via automated fluorescence flow cytometry, animals were euthanized, and the organs analyzed for iron contents (atomic absorption spectrometry) and for expression of NF-κB associated proteins (p65, IκBα, p105/50, p100/52, COX-2, Bcl-2, SDS-PAGE and Western blotting). We observed that the PEI-MNPs had a diameter of 136 nm and a zeta-potential 56.9 mV. After injection in mice, the blood parameters were modified and the iron levels were increased in different organs. Moreover, the liver of animals showed an increased protein expression of canonical NF-κB signaling pathway members early after PEI-MNP application, whereas at the later post-observation time, members of the non-canonical signaling pathway were prominent. We conclude that the synergistic effect between PEI-MNPs and the low-grade and persistent inflammatory state is mainly due to the hepatocytes sensing infection (PAMPs), to immune responses resulting from the intracellular metabolism of the uptaken PEI-MNPs, or to hepatocyte and immune cell communications. Therefore, we suggest a careful assessment of the safety and toxicity of PEI-MNP-based carriers for gene therapy, chemotherapy, and other medical applications not only in healthy individuals but also in those suffering from chronic inflammation.

Combined Therapy with Simvastatin- and Coenzyme-Q10-Loaded Nanoparticles Upregulates the Akt-eNOS Pathway in Experimental Metabolic Syndrome.

In addition to their LDL-cholesterol-lowering effect, statins have pleiotropic beneficial effects on the cardiovascular system. However, long-term treatment with statins may be associated with serious side effects. With the aim to make statin therapy more effective, we studied the effects of simvastatin- and coenzyme-Q10-loaded polymeric nanoparticles on the lipid profile and nitric oxide (NO)/reactive oxygen species (ROS) balance in the heart and aorta of adult male obese Zucker rats. The rats were divided into an untreated group, a group treated with empty nanoparticles, and groups treated with simvastatin-, coenzyme Q10 (CoQ10)-, or a combination of simvastatin- and CoQ10-loaded nanoparticles (SIMV+CoQ10). After 6 weeks, the lipid profile in the plasma and the concentration of conjugated dienes in the liver were determined. Nitric oxide synthase (NOS) activity, Akt, endothelial NOS (eNOS), phosphorylated eNOS (p-eNOS), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, and nuclear factor kappaB (NF-kappaB) protein expressions were measured in the heart and aorta. All simvastatin, CoQ10, and SIMV+CoQ10 treatments decreased plasma LDL levels, but only the combined SIMV+CoQ10 treatment increased NOS activity and the expression of Akt, eNOS, and p-eNOS in both the heart and the aorta. Interestingly, NADPH oxidase in the heart and NF-kappaB protein expression in the aorta were decreased by all treatments, including nanoparticles alone. In conclusion, only combined therapy with SIMV- and CoQ10-loaded nanoparticles increased NOS activity and upregulated the Akt-eNOS pathway in obese Zucker rats, which may represent a promising tool for the treatment of cardiometabolic diseases.

1668-P: Novel Mechanism of Negative Regulation of FOXP3 Expression in Autoimmune Diabetes

The NF-kappaB protein c-Rel is essential for the expression of forkhead box P3 (FOXP3), the master transcription factor that governs development and function of T regulatory cells. We found that O-GlcNAcylation of c-Rel at serine residue 350 suppresses the expression of FOXP3. Hyperglycemia-induced c-Rel O-GlcNAcylation-dependent suppression of FOXP3 expression was found in vivo in two mouse models of autoimmune diabetes; the streptozotocin (STZ)-induced diabetic mouse model and in the non-obese diabetic (NOD) mouse model. Mechanistically, we show that both hyperglycemia-induced and chemically enhanced cellular O-GlcNAcylation decreases the DNA binding ability of c-Rel at the FOXP3 promoter. Mutation of the O-GlcNAcylation site in c-Rel, (serine 350 to alanine), augments T cell receptor-induced FOXP3 expression and resists the O-GlcNAcylation-dependent repression of FOXP3 expression. This study reveals c-Rel S350 O-GlcNAcylation as a novel molecular mechanism regulating immunosuppressive FOXP3 expression in autoimmune diabetes with potential therapeutic implications. Disclosure P. Ramakrishnan: None. T.J. de Jesus: None. J. Centore: None. Funding National Institutes of Health (R01AI116730, R21AI144264)

Targeting c-Rel O-GlcNAcylation to control hyperglycemia-dependent transcription in T lymphocytes

Abstract The NF-kappaB protein c-Rel plays a critical role in controlling regulating T cell function and T regulatory cell development. We found that hyperglycemia increases O-GlcNAcylation of c-Rel at serine residue 350 and enhances the transcription of c-Rel-induced CD28RE-dependent pro-autoimmune cytokines, interleukin-2 (IL-2), interferon gamma (IFNG) and granulocyte macrophage colony stimulating factor (GM-CSF). Our recent results show that the regulatory effect of c-Rel O-GlcNAcylation is gene dependent and it suppresses the transcription of forkhead box P3 (FOXP3) expression in T cells. Mechanistically, we found that O-GlcNAcylation enhances the DNA binding of c-Rel at CD28RE and decreases it’s binding at the FOXP3 promoter. To specifically block O-GlcNAcylated c-Rel function, we developed a peptoid, called peptoid3 that specifically binds to O-GlcNAcylated c-Rel by molecular modeling and de novo synthesis. We found that peptoid3 treatment significantly decreased T cell receptor-induced, O-GlcNAcylation-dependent expression of proautoimmune cytokines and enhanced FOXP3 expression in T cells. This study reveals c-Rel S350 O-GlcNAcylation as a novel molecular mechanism inversely regulating proautoimmune gene expression and immunosuppressive FOXP3 expression in T cells with potential therapeutic implications in conditions such as type 1 diabetes.

Regulation of FOXP3 Expression by c-Rel O-GlcNAcylation

Abstract The NF-kappaB protein c-Rel plays a critical role in controlling autoimmunity through regulating T cell function and T regulatory cell development. Type 1 diabetes is a T cell-mediated autoimmune disease associated with hyperglycemia and chronic inflammatory complications. Previously, we discovered that hyperglycemia induces hyper O-GlcNAcylation of c-Rel at serine residue 350 and enhances the transcription of c-Rel-dependent pro-autoimmune cytokines interleukin-2 (IL-2), interferon gamma (IFNG) and granulocyte macrophage colony-stimulating factor (GM-CSF). In contrast, our recent results show that c-Rel O-GlcNAcylation decreases forkhead boxP3 (FOXP3) expression in T cells. Chemically enhancing cellular O-GlcNAcylation decreases the DNA binding ability of c-Rel at the FOXP3 promoter as demonstrated both by oligonucleotide pulldown and chromatin immunoprecipitation assays. Moreover, mutation of serine 350 in c-Rel to alanine augments T cell receptor-induced FOXP3 expression. O-GlcNAcylation-dependent suppression of FOXP3 was also found in vivo in streptozotocin-induced diabetic mouse model. This study reveals a novel molecular mechanism that regulates hyperglycemia-dependent c-Rel function in autoimmune diabetes, with potential to develop novel therapeutics targeting c-Rel O-GlcNAcylation.

TLR9 signaling activation at different stages in colorectal cancer and NF-kappaB expression

BackgroundThe relationship of inflammation and tumor is becoming more and more important in the study on the pathogenesis of colorectal cancer. The role of TLR9-mediated immune inflammation reaction in the process is not currently clear. The purpose of the study was to discuss the correlation of TLR9 signal activation with tumor progression by detecting the expression of TLR9 and its downstream molecule NF-kappaB in colorectal cancer tissues at different stages.MethodsTLR9 expression in colorectal cancer tissues was detected by immunohistochemical streptavidin-perosidase method and Western blot.ResultsThe result showed that the high expression of TLR9 was correlated with tumor poorly differentiation, invasion and liver metastasis, the abnomal increasing levels of CEA in blood. With the signal activation, the levels of TLR9 protein raised more in advanced colorectal cancer than in early colorectal cancer. Afterward, we found that the activation of specific expression of TLR9 signal was related to histologic origin. TLR9-C expression displayed in both advanced cancer and para-carcinoma tissues, and TLR9-R protein was predominat in partial sigmoid and rectal cancer tissues. With the differential expression of TLR9, the levels of its downstream molecule NF-kappaB protein increased in colon cancer tissues and decreased in rectal cancer tissues.ConclusionThe results confirmed that TLR9 signaling activation participated in the clinical process of colorectal cancer and influenced NF-kappaB expression.

Abstract 167: Targeting the NF-kappaB pathway with bardoxolone methyl to inhibit ovarian cancer spheroid formation

Abstract Over 70% of patients with advanced stage ovarian cancer relapse within 2 years, with long term overall survival being only 25%. Tumor-initiating cells (TICs) are thought to contribute to this high rate of recurrence as they can resist killing by platinum-based chemotherapy. In this study we sought to identify pathways unique to TICs and to disrupt these pathways in order to more effectively target this population of cells. We conducted an siRNA screen which identified NF-kappaB signaling as an important pathway in the maintenance of ovarian cancer cells grown in non-adherent spheroid cultures that mimic TICs. Downregulation of several known NF-kappaB target genes, including NXF1, RAC2, CDC42, BCL2L1 and SERPINB3, significantly decreased growth of TIC cultures but not the corresponding adherent cultures, thereby indicating that expression of these genes is necessary for growth in TIC cultures. These findings are consistent with previous studies which showed that higher NF-kappaB activity is linked with drug resistance in patients. In parallel, we conducted a drug screen to identify compounds that target TICs. One clear candidate was bardoxolone methyl, which has been shown to inhibit IKK, an upstream NF-kappaB signaling protein. We confirmed the results of the drug screen in a panel of ovarian cancer cell lines, showing that bardoxolone methyl decreased known TIC markers CD133 and ALDH by flow cytometry, and decreased NF-kappaB proteins by western blot. To further elucidate the mechanism of bardoxolone methyl on TICs we conducted an RNAi screen +/- bardoxolone methyl. Interestingly, the knockdown of genes including BRAF, MAP3K8, LMNA and SMAD6 acted in a synthetically lethal manner with bardoxolone methyl in the TICs. To begin translating to a clinically relevant model, we proceeded to test whether bardoxolone methyl could eliminate TICs that remained after treatment with platinum-based chemotherapy. We confirmed in vitro that bardoxolone methyl in combination with chemotherapy reduced TIC populations. Our ongoing studies will address whether this drug is effective in preventing relapse in vivo after chemotherapy treatment of ovarian cancer in mouse models. Citation Format: Michelle K. Ozaki, Carrie D. House, Krystyna Mazan-Mamczarz, Madhu Lal-Nag, Craig Thomas, Christina M. Annunziata. Targeting the NF-kappaB pathway with bardoxolone methyl to inhibit ovarian cancer spheroid formation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 167.

Abstract 1910: NF-kappaB classical versus alternative pathways support distinct populations of ovarian cancer tumor-initiating cells

Abstract Ovarian cancer is the most lethal gynecological malignancy in the United States with high morbidity and mortality due to recurrence and chemoresistance. Our data suggest tumor-initiating cells (TICs) play an important role in disease biology. We previously showed that a subset of ovarian cancer cells depends on NF-kappaB signaling, and that expression of NF-kappaB proteins is associated with poor survival. Given that NF-kappaB expression correlates with a poor outcome in ovarian cancer, and NF-kappaB activity supports drug resistance and tumorigenicity, we hypothesize that NF-kappaB supports a TIC program responsible for ovarian cancer relapse. To investigate this pathway in TICs, we designed a novel method to enrich for TICs from cell lines and patient samples by culturing non-adherent, floating cells in stem cell conditions defined by low attachment flasks and serum free media. Preliminary data confirm that these cells have higher stem cell marker expression, are chemoresistant, and are more tumorigenic in nude mice compared to their adherent counterparts. These TIC-enriched culture conditions enhance NF-kappaB expression and activity. More specifically, alternative NF-kappaB signaling through the RelB transcription factor supports TIC populations by regulating aldehyde dehydrogenase (ALDH), an enzyme with high activity in TICs. Using an inducible shRNA targeting RelB we show that spheroid formation, ALDH expression and activity, chemoresistance, and tumorigenesis in both subcutaneous and intrabursal xenograft models, significantly decreased in the absence of RelB. Interestingly, loss of classical NF-kappaB signaling, through an shRNA targeting the RelA transcription factor, was less effective in targeting TICs, but more effective at targeting a proliferative subpopulation with high Ki67 staining. We conclude that classical and alternative NF-kappaB signaling support ovarian tumor cells with distinct phenotypes and the collaboration of these pathways may be critical for supporting tumor repopulation following chemotherapy. Current studies will measure system level changes induced by these shRNAs, and identify a gene signature specific to each NF-kappaB pathway in ovarian TICs. Clarifying the nuances of NF-kappaB signaling in TICs will increase our understanding of ovarian cancer recurrence and further focus therapeutic strategies to prevent relapse. Citation Format: Carrie D. House, Christina M. Annunziata. NF-kappaB classical versus alternative pathways support distinct populations of ovarian cancer tumor-initiating cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1910. doi:10.1158/1538-7445.AM2017-1910

Abstract AP13: NF-KAPPAB CLASSICAL VERSUS ALTERNATIVE PATHWAYS SUPPORT DISTINCT POPULATIONS OF OVARIAN CANCER TUMOR-INITIATING CELLS

Abstract Ovarian cancer is the most lethal gynecological malignancy in the United States with high morbidity and mortality due to recurrence and chemoresistance. Our data suggest tumor-initiating cells (TICs) play an important role in disease biology. We previously showed that a subset of ovarian cancer cells depends on NF-kappaB signaling, and that expression of NF-kappaB proteins is associated with poor survival. Given that NF-kappaB expression correlates with a poor outcome in ovarian cancer, and NF-kappaB activity supports drug resistance and tumorigenicity, we hypothesize that NF-kappaB supports a TIC program responsible for ovarian cancer relapse. To investigate this pathway in TICs, we designed a novel method to enrich for TICs from cell lines and patient samples by culturing non-adherent, floating cells in stem cell conditions defined by low attachment flasks and serum free media. Preliminary data confirm that these cells have higher stem cell marker expression, are chemoresistant, and are more tumorigenic in nude mice compared to their adherent counterparts. These TIC-enriched culture conditions enhance NF-kappaB expression and activity. More specifically, alternative NF-kappaB signaling through the RelB transcription factor supports TIC populations by regulating aldehyde dehydrogenase (ALDH), an enzyme with high activity in TICs. Using an inducible shRNA targeting RelB we show that spheroid formation, ALDH expression and activity, chemoresistance, and tumorigenesis in both subcutaneous and intrabursal xenograft models, significantly decreased in the absence of RelB. Interestingly, loss of classical NF-kappaB signaling, through an shRNA targeting the RelA transcription factor, was less effective in targeting TICs, but more effective at targeting a proliferative subpopulation with high Ki67 staining. We conclude that classical and alternative NF-kappaB signaling support ovarian tumor cells with distinct phenotypes and the collaboration of these pathways may be critical for supporting tumor repopulation following chemotherapy. Current studies will measure system level changes induced by these shRNAs, and identify a gene signature specific to each NF-kappaB pathway in ovarian TICs. Clarifying the nuances of NF-kappaB signaling in TICs will increase our understanding of ovarian cancer recurrence and further focus therapeutic strategies to prevent relapse. Citation Format: Carrie D. House, Elizabeth A. Jordan, Christina M. Annunziata. NF-KAPPAB CLASSICAL VERSUS ALTERNATIVE PATHWAYS SUPPORT DISTINCT POPULATIONS OF OVARIAN CANCER TUMOR-INITIATING CELLS [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr AP13.

Donor Preconditioning After the Onset of Brain Death With Dopamine Derivate n-Octanoyl Dopamine Improves Early Posttransplant Graft Function in the Rat.

Heart transplantation is the therapy of choice for end-stage heart failure. However, hemodynamic instability, which has been demonstrated in brain-dead donors (BDD), could also affect the posttransplant graft function. We tested the hypothesis that treatment of the BDD with the dopamine derivate n-octanoyl-dopamine (NOD) improves donor cardiac and graft function after transplantation. Donor rats were given a continuous intravenous infusion of either NOD (0.882 mg/kg/h, BDD+NOD, n = 6) or a physiological saline vehicle (BDD, n = 9) for 5 h after the induction of brain death by inflation of a subdural balloon catheter. Controls were sham-operated (n = 9). In BDD, decreased left-ventricular contractility (ejection fraction; maximum rate of rise of left-ventricular pressure; preload recruitable stroke work), relaxation (maximum rate of fall of left-ventricular pressure; Tau), and increased end-diastolic stiffness were significantly improved after the NOD treatment. Following the transplantation, the NOD-treatment of BDD improved impaired systolic function and ventricular relaxation. Additionally, after transplantation increased interleukin-6, tumor necrosis factor TNF-α, NF-kappaB-p65, and nuclear factor (NF)-kappaB-p105 gene expression, and increased caspase-3, TNF-α and NF-kappaB protein expression could be significantly downregulated by the NOD treatment compared to BDD. BDD postconditioning with NOD through downregulation of the pro-apoptotic factor caspase-3, pro-inflammatory cytokines, and NF-kappaB may protect the heart against the myocardial injuries associated with brain death and ischemia/reperfusion.

Dual regulatory role of c-Rel O-GlcNAcylation in T cell function and Type 1 Diabetes

Abstract O-GlcNAcylation is a post-translational protein modification involving the addition of N-acetylglucosamine (GlcNAc) to serine and threonine residues on target proteins. Global O-GlcNAcylation levels rise under hyperglycemia such as that caused by Type 1 Diabetes (T1D). We found that hyperglycemia induces O-GlcNAcylation of the NF-kappaB protein c-Rel at a single serine residue, S350. O-GlcNAcylation of c-Rel enhances its DNA binding and the transcription of c-Rel-dependent pro-autoimmune Th1 cytokines in T cells. This direct link between hyperglycemia and augmented Th1 function imply a key regulatory role for c-Rel O-GlcNAcylation in the pathology of T1D. Interestingly, our recent results show that O-GlcNAcylation inhibits binding of c-Rel to the FOXP3 promoter, leading to reduced FOXP3 expression. Together, these data suggest that c-Rel O-GlcNAcylation has a novel, dual regulatory role in controlling T cell function by: 1) Enhancing pro-autoimmune T cell function and 2) Suppressing Treg function. This creates an immune environment prone to heightened inflammation and autoimmunity that may exacerbate the frequency of autoimmunity in Western countries. Our current work focuses on defining the molecular mechanisms by which c-Rel O-GlcNAcylation regulates transcriptional activation in T cells and Treg cells. We are studying the non-obese diabetic (NOD) mouse model of spontaneous diabetes to examine the pathophysiological role of c-Rel O-GlcNAcylation. Knowledge on the regulation of autoimmunity by c-Rel O-GlcNAcylation will enhance our understanding of the etiology immune-mediated pathologies in diabetes and reveal a new therapeutic target to treat autoimmune diabetes.

Abstract A72: Dissecting the role of NF-kappaB signaling in ovarian cancer tumor-initiating cells.

Abstract Ovarian cancer is the most lethal gynecological malignancy in the United States with high morbidity and mortality due to recurrence and chemoresistance. Our data suggest tumor-initiating cells (TICs) play an important role in disease biology. We previously showed that a subset of ovarian cancer cells depends on NF-kappaB signaling, and that expression of NF-kappaB proteins is associated with poor survival. To investigate this pathway in TICs, we designed a novel method to enrich for TICs from cell lines and patient samples by culturing non-adherent, floating cells in stem cell conditions. Preliminary data show these cells have higher stem cell marker expression, are chemoresistant, and are more tumorigenic in nude mice compared to their adherent counterparts. Given that NF-kappaB expression correlates with a poor outcome in ovarian cancer, and NF-kappaB activity supports drug resistance and tumorigenicity, we hypothesize that NF-kappaB supports a TIC program responsible for ovarian cancer relapse. Preliminary data show TIC-enriched culture conditions increased NF-kappaB expression and activity in TICs and stimulation of NF-kappaB signaling enhanced chemoresistance and stem cell marker expression. Current studies focus on dissecting the role of individual NF-kappaB proteins in TICs using inducible shRNAs to measure changes in spheroid formation, chemoresistance, tumorigenesis, and disease relapse following chemotherapy in a xenograft model. Future studies will measure system level changes induced by these shRNAs, and identify a gene signature specific to NF-kappaB in ovarian TICs. Clarifying the nuances of NF-kappaB signaling in TICs will increase our understanding of ovarian cancer recurrence and may lead to improved therapeutic strategies. Citation Format: Carrie D. House, Christina M. Annunziata. Dissecting the role of NF-kappaB signaling in ovarian cancer tumor-initiating cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A72.

Abstract A16: Regulation of TRAIL and NF-kappaB pathways in ovarian cancer.

Abstract Objective: The equilibrium between apoptotic and survival pathways determines the fate of cells. Dysregulation in any one mechanism often leads to cancer. The present study was designed to determine the crosstalk between TRAIL and NF-kappaB pathways through study of the much controversial role of DcR2 in the induction of NF-kappaB. Methodology: A total of 50 epithelial ovarian tissue samples were used for immunohistochemical staining of DcR2 and NF-kappaB (p65) proteins along with the other players of TRAIL pathway including DR4, DR5, DcR1, FLIP and TRAIL. Correlations were drawn and survival analysis was performed using Kaplan Meier's method. Results: A total of 72% ovarian carcinomas showed positive DR4 expression, 68% showed positive DR5 expression, 42% showed positive DcR1 expression, 48% showed positive DcR2 expression, 44% had positive FLIP expression, 46% had positive TRAIL expression in epithelium while 28% had positive TRAIL expression in stroma. NF-kappaB was positive in 46% of the tissue samples. A strong but non-significant correlation was seen between DcR2 and NF-kappaB expression. Interestingly a similar correlation was also found between DR4 and NF-kappaB expression. Survival analysis showed better survival for patients with positive NF-kappaB expression as compared to negative expression. Conclusion: Coexpression of DcR2 and NF-kappaB suggests a potential therapeutic approach of targeting NF-kappaB through TRAIL pathway by using anti-DcR2 antibodies. However, the study may be elaborated with increased sample size to get significant results. Note: This abstract was not presented at the conference. Citation Format: Mariam Anees, Umaima Batool, Sidra Bukhari, Sohail Yousaf. Regulation of TRAIL and NF-kappaB pathways in ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A16.

PD98059 Influences Immune Factors and Enhances Opioid Analgesia in Model of Neuropathy.

Neuropathic pain treatment remains challenging due to ineffective therapy and resistance to opioid analgesia. Mitogen-activated protein kinase kinase (MAPKK) have been identified as the crucial regulators of pro- and antinociceptive factors. We used PD98059, an inhibitor of the MAPKK family members MEK1/2. The aim of study was to examine the influence of single and/or repeated PD98059 on nociception and opioid effectiveness in neuropathy. Moreover, we examined how PD98059 influences selected members of cellular pathways and cytokines. The PD98059 (2.5 mcg) was intrathecally preemptively administered before chronic constriction injury (CCI), and then once daily for 7 days. Additionally, at day 7 after CCI the PD98059-treated rats received a single injection of opioids. Using Western blot and qRT-PCR techniques in PD98059-treated rats we analyzed the mRNA and/or protein level of p38, ERK1/2, JNK, NF-kappaB, IL-1beta, IL-6, iNOS and IL-10 in the lumbar spinal cord. Our results indicate that PD98059 has an analgesic effects and potentiates morphine and/or buprenorphine analgesia. Parallel we observed that PD98059 inhibit upregulation of the CCI-elevated p38, ERK1/2, JNK and NF-kappaB protein levels. Moreover, PD98059 also prevented increase of pro- (IL-1beta, IL-6, and iNOS) but enhances anti-nociceptive (IL-10) factors. Summing up, PD98059 diminished pain and increased the effectiveness of opioids in neuropathy. The inhibition of MEKs might inactivate a variety of cell signaling pathways that are implicated in nociception.

Molecular regulation of autoimmunity by O-GlcNAcylation of NF-kappaB c-Rel in T lymphocytes (IRM11P.633)

Abstract The NF-kappaB protein c-Rel plays a critical role in controlling autoimmunity through regulating T cell function and T regulatory cell development. Type 1 diabetes is an autoimmune disease associated with hyperglycemia and chronic inflammatory complications. We found that hyperglycemia induces O-GlcNAcylation of c-Rel at serine residue 350 and activates the transcription of c-Rel-dependent pro-autoimmune cytokines interleukin-2 (IL-2), interferon gamma (IFNG) and granulocyte macrophage colony-stimulating factor (GM-CSF). Our recent results show that c-Rel O-GlcNAcylation decreases FOXP3 expression in T cells. These results suggest that the glucose metabolism-dependent c-Rel O-GlcNAcylation serves as a key regulatory switch with dual roles in controlling T cell and T regulatory cell function in autoimmune diabetes. Despite decades of research, drugs based on molecular targets to treat type 1 diabetes have remained elusive. This study reveals a novel molecular mechanism that regulates NF-kappaB c-Rel and autoimmune diabetes, with potential to develop novel therapeutics targeting c-Rel O-GlcNAcylation. Moreover, this study serves as the basis to explore the role of c-Rel O-GlcNAcylation in other autoimmune diseases such as celiac disease, lupus and arthritis, where c-Rel function have been implicated.

Inhibitory effects of intervention of the TNFa/NF-kappaB signaling pathway activation on hepatoma cell proliferation

To investigate the inhibitory effects of intervention of the tumor necrosis factor-alpha (TNFa)/nuclear factor-kappa B (NF-kappaB) signaling pathway activation on hepatoma cell proliferation and to explore its mechanism. A rodent hepatoma model was established by feeding N-2-fluorenylacetamide (2-N-FAA) to male Sprague-Dawley rats. Human subjects with various liver diseases were enrolled in the study, and serum and peripheral blood nuclear cells were collected for analysis. HepG2 cells were cultured in vitro and treated with anti-TNFa (monoclonal antibody, mAb) to down-regulate its expression or transfected with siRNA targeting the p65 subunit of NF-kappaB to inhibit its activation. The liver cell line L02 was used as a control. Changes in protein and gene expression levels of NF-kappaB and TNFa were analyzed by Western blotting or enzyme-linked immunosorbent assay and real-time PCR, respectively. Changes in the cell cycle or apoptosis were evaluated by flow cytometry or Annexin-V/PI double-labeling assay, respectively. TNFa and NF-kappaB expression showed increasing trends during the malignant transformation of rat hepatocytes, and the differential expression patterns showed association with histopathological alterations in the hepatocytes. Following treatment with the TNFa mAb, the HepG2 cells showed a higher percentage of apoptotic cells than the untreated control cells (21.45% +/- 4.07% vs. 5.63% +/- 0.93%, q =10.07, P less than 0.01).There was a significant difference in the rate of cells in the G0/G1 phase in the p65-siRNA transfected cells (66.23% +/- 1.29% vs. untreated control cells: 59.00% +/- 1.02%, q =10.98, P less than 0.01). The decreased expression of TNFa and NF-kappaB in cell culture supernatants was positively correlated with the dose of treatment (r =0.89, P less than 0.01), with the most robust decreases being achieved with the highest concentrations ( P less than 0.01). NF-kappaB expression was significantly higher in the HepG2 cells than in the L02 cells, and transfection of p65-siRNA reduced the mRNA (93%) and protein (62%) levels and increased the cell apoptosis index (to 85%). Proliferation of hepatoma cells may be significantly inhibited by intervening in the activation of the TNFa/NF-kappaB signaling pathway, which promotes cell apoptosis and blocks cell cycling.

HSV-1 activates NF-kappaB in mouse astrocytes and increases TNF-alpha and IL-6 expression via Toll-like receptor 3

Objective: To investigate the effect of HSV-1 infection via TLR3 on the transcriptional activity of NF-kappaB and the expression of cytokines TNF-alpha and IL-6 in astrocytes.Methods: HSV-1-infected primary astrocytes were cultured until the third passage and the mRNA and protein levels of TLR3, NF-kappaB, TNF-alpha, and IL-6 were assessed by immunofluorescence, RT-PCR, and Western blot. The effects of the NF-kappaB inhibitor pyrrolidine dithiocarbamate (PDTC) and TLR3-neutralizing antibody on the expression of NF-kappaB, TNF-alpha, and IL-6 were investigated.Results: Uninfected astrocytes expressed TLR3 and NF-kappaB at the mRNA and protein levels. After infection with HSV-1, the TLR3 mRNA and protein levels were up-regulated and NF-kappaB protein was highly expressed. Also, the mRNA and protein levels of TNF-alpha and IL-6 were up-regulated. Pyrrolidine dithiocarbamate inhibited NF-kappaB activation, resulting in the down-regulation of nuclear NF-kappaB protein, which led to the down-regulation of the mRNA and protein levels of TNF-alpha and IL-6. After blocking astrocyte membrane TLR3, the nuclear NF-kappaB protein expression was down-regulated and the mRNA and protein levels of TNF-alpha and IL-6 were increased. The antiviral functions of astrocytes were weaker, as reflected by higher HSV-1 glycoprotein D (gD) mRNA expression and increased HSV-1 titers.Conclusion: Astrocytes infected with HSV-1 can activate NF-kappaB via TLR3 so as to up-regulate the expression of TNF-alpha and IL-6 that have antiviral functions.

Study on effect of total coumarins from Urtica dentata on dextran sulfate sodium-induced colitis in mice

To study the effect of total coumarins (TC) from Urtica dentata on dextran sulfate sodium (DSS)-induced colitis in mice. The colitis model was established by administering DSS. Having been treated with TC, their body weight was determined. Concentrations of IL-6, IL-10, TGF-beta1 and IFN-gamma were monitored by ELISA. Colon samples were collected for the histopathological examination. Western blot was used to detect TLR4 and NF-kappaB protein expression in colonic tissues. TCs from U. dentata effectively controlled the body weight loss of mice with colitis, down-regulated the concentration of IL-6 and IFN-gamma and increased the suppressive cytokines IL-10 and TGF-beta1 in the serum. Additionally, TC alleviated the mucosal damage and decreased the expressions of TLR4 and NF-kappaB in colonic tissues. TCs from U. dentata shows the anti-inflammatory effect on colitis in mice by reducing the expressions of TLR4 and NF-kappaB in colonic tissues and regulating pro-and anti-inflammatory cytokines.

Effect of sophoridine and TLR4/MD-2 blocking agent on pathway of LPS-induced RAW264.7 macrophage TLR4-NF-κB-TNF-α

To study the effect of sophoridine and TLR4/MD-2 blocking agent on pathway of LPS-induced RAW264. 7 macrophage TLR4-NF-kappaB-TNF-alpha in and its pharmacological mechanism of antiendotoxin. RAW264. 7 macrophages were cultured and divided into 5 groups, namely the blank control group, the LPS model group, the sophoridine + LPS group, the TLR4/MD-2 blocking agent + LPS group and the anti-TLR4/MD-2 + sophoridine + LPS group. Cells and cell culture fluids were collected at 120 min after the each group was processed. The expression of TLR4 protein was measured by western blot, the distribution and expression of NF-kappaB protein were measured by immunocytochemistry, and the expression of NF-kappaB and TNF-alpha mRNA were measured by western blot and reverse transcriptase polymerase chain reaction (RT-PCR). The content of TNF-alpha in the cell supernatant was detected by using radioimmunoassay. Compared with the LPS group, the expression of TLR4 protein, NF-kappaB mRNA, the rate of NF-kappaB entry the nucleus, TNF-alpha mRNA and TNF-alpha content in the cell supernatant were significantly decreased in the sophoridine + LPS group (P < 0.01). The rate of NF-kappaB entry the nucleus and TNF-alpha in the TLR4/MD-2 blocking agent + LPS group and the TLR4/MD-2 blocking agent + sophoridine + LPS group were notablly lower than that of the LPS model group (P < 0.01), close to that of the blank control group. However, there was no statistical significance between the two groups. TLR4/MD-2 may be one of sophoridine's targets. Sophordine's inhibitory effect on pathway activity of TLR4-NF-kappaB-TNF-alpha may be one of its antiendotoxin mechanisms.

Toll-like receptor 4 signaling is involved in PACAP-induced neuroprotection in BV2 microglial cells under OGD/reoxygenation

Object: The neuroprotective effects of pituitary adenylate cyclise-activating polypeptide (PACAP) have been well documented in vivo and in vitro. However, the mechanisms by which PACAP protected microglia from ischemic/hypoxic injury via inhibition of microglia activation remain unclear. Toll-like receptor 4 (TLR4) plays a considerable role in the induction of innate immune and inflammatory responses. The purpose of this study is to investigate the effect of PACAP on the oxygen and glucose deprivation (OGD)/reoxygenation BV2 microglia and to explore the role of TLR4/myeloid differentiation protein 88 (MyD88)/nuclear factor-kappa B (NF-kappaB) pathway in the neuroprotective effects of PACAP.Methods: We conducted OGD/reoxygenation by placing BV2 microglia into an airtight chamber and in glucose-free medium. BV2 microglia cell viability was determined by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide] assay. Western blot was utilized to detect TLR4, MyD88 expression, inhibitory protein of NF-kappaB (IkappaB) phosphorylation/degradation, NF-kappaB activation. Level of tumor necrosis factor-alpha (TNF-alpha) in culture medium was measured with enzyme-linked immunosorbent assay (ELISA). Apoptosis was determined by flow cytometry.Results: We found that pretreatment with PACAP to BV2 cells immediately before OGD/reoxygenation significantly alleviated microglia hypoxic injury. PACAP inhibited upregulation of TLR4, MyD88 and NF-kappaB in BV2 microglial cells exposed to OGD/reoxygenation. PACAP administration also significantly reduced the production of proinflammatory cytokines and apoptosis in BV2 microglia exposed to OGD/reoxygenation.Discussion: Pretreatment with PACAP inhibited activation of the TLR4/MyD88/NF-kappaB signaling pathway and decreased inflammatory cytokine levels, as well as apoptosis in microglia, thereby attenuating microglia hypoxic injury. Our results suggested that TLR4-mediated MyD88-dependent signaling pathway contributed to neuroprotection of PACAP to microglia against OGD/reoxygenation.