Suppression of NF-κB and NF-κB regulated oxidative stress and neuroinflammation by BAY 11-7082 (IκB phosphorylation inhibitor) in experimental diabetic neuropathy

Inflammation is an emerging patho-mechanism of diabetes and its complications. NF-κB pathway is one of the central machinery initiating and propagating inflammatory responses. The present study envisaged the involvement of NF-κB inflammatory cascade in the pathophysiology of diabetic neuropathy using BAY 11-7082, an IκB phosphorylation inhibitor. Streptozotocin was used to induce diabetes in Sprauge Dawley rats. BAY 11-7082 (1 &; 3 mg/kg) was administered to diabetic rats for 14 days starting from the end of six weeks post diabetic induction. Diabetic rats developed deficits in nerve functions and altered nociceptive parameters and also showed elevated expression of NF-κB (p65), IκB and p-IκB along with increased levels of IL-6 &; TNF-α and inducible enzymes (COX-2 and iNOS). Furthermore, there was an increase in oxidative stress and decrease in Nrf2/HO-1 expression. We observed that BAY 11-7082 alleviated abnormal sensory responses and deficits in nerve functions. BAY 11-7082 also ameliorated the increase in expression of NF-κB, IκB and p-IκB. BAY 11-7082 curbed down the levels of IL-6, TNF-α, COX-2 and iNOS in the sciatic nerve. Lowering of lipid peroxidation and improvement in GSH levels was also seen along with increased expression of Nrf2/HO-1. Thus it can be concluded that NF-κB expression and downstream expression of proinflammatory mediators are prominent features of nerve damage leading to inflammation and oxidative stress and BAY 11-7082 was able to ameliorate experimental diabetic neuropathy by modulating neuroinflammation and improving antioxidant defence.     

Stress-induced phosphoprotein 1 restrains spinal cord ischaemia-reperfusion injury by modulating NF-κB signalling

Spinal cord injury (SCI), a major cause of disability, causes high global disease and economic burdens. Stress-induced phosphoprotein 1 (STIP1) has been identified to be involved in spinal cord ischaemia-reperfusion injury (SCII); however, the effect of STIP1 on SCII remains unclear until now. This study aimed to examine the role of STIP1 in SCII and unravel the possible mechanisms. Western blotting and immunohistochemical staining showed that STIP1 expression rapidly increased and then decreased in rat spinal cord following SCII treatment. Neurological function scoring, HE staining, immunohistochemical staining and Western blotting revealed that STIP1 overexpression alleviated SCII-induced motor dysfunction of hind limbs, neuronal loss and inflammation in spinal cord, and inhibited activity of nuclear factor kappa B (NF-κB) signalling in rats. Immunoprecipitation identified that STIP1 was co-located with Iba-1. In addition, STIP1 was found to ameliorate oxygen and glucose deprivation (OGD)-induced inflammation and activation of NF-κB signalling in mouse microglia BV2 cells, and STIP1 resulted in decrease of heat shock protein family A member 8 (HSPA8), increase of IκBβ expression and reduced binding of IκBβ to HSPA8 in BV2 cells. The results of the present study demonstrate that STIP1 alleviates ischaemia/reperfusion-induced neuronal injury and inflammation in rat spinal cord and mouse microglial cells by deactivating NF-κB signalling. These findings may provide novel insights for the clinical diagnosis and treatment of SCI.     

Endothelial ICAM-1 protein induction is regulated by cytosolic phospholipase A2α via both NF-κB and CREB transcription factors

The regulated expression of ICAM-1 plays an important role in inflammatory processes and immune responses. The present study aimed to determine the in vivo involvement of cytosolic phospholipase A(2)α (cPLA(2)α) in ICAM-1 overexpression during inflammation and to elucidate the cPLA(2)α-specific role in signal events leading to ICAM-1 upregulation in endothelial cells. cPLA(2)α and ICAM-1 upregulation were detected in inflamed paws of mice with collagen-induced arthritis and in periepididymal adipose tissue of mice fed a high-fat diet. Intravenous injection of 2 mg/kg oligonucleotide antisense against cPLA(2)α (AS) that reduced cPLA(2)α upregulation also decreased ICAM-1 overexpression, suggesting a key role of cPLA(2)α in ICAM-1 upregulation during inflammation. Preincubation of endothelial ECV-304 cells that express ICAM-1 and of HUVEC that express ICAM-1 and VCAM-1 with 1 μM AS prevented cPLA(2)α and the adhesion molecule upregulation induced by TNF-α and inhibited their adherence to phagocyte like-PLB cells. Whereas AS did not inhibit NADPH oxidase 4-NADPH oxidase activity, inhibition of oxidase activity attenuated cPLA(2)α activation, suggesting that NADPH oxidase acts upstream to cPLA(2)α. Attenuating cPLA(2)α activation by AS or diphenylene iodonium prevented the induction of cyclooxygenase-2 and the production of PGE(2) that were essential for ICAM-1 upregulation. Inhibition of cPLA(2)α activity by AS inhibited the phosphorylation of both p65 NF-κB on Ser(536) and protein kinase A-dependent CREB. To our knowledge, our results are the first to show that CREB activation is involved in ICAM-1 upregulation and suggest that cPLA(2)α activated by NADPH oxidase is required for sequential phosphorylation of NF-κB by an undefined kinase and CREB activation by PGE(2)-mediated protein kinase A.     

Regulation of NF-κB signaling by caspases and MALT1 paracaspase

Caspases are intracellular proteases that are best known for their function in apoptosis signaling. It has become evident that many caspases also function in other signaling pathways that propagate cell proliferation and inflammation, but studies on the inflammatory function of caspases have mainly been limited to caspase-1-mediated cytokine processing. Emerging evidence, however, indicates an important contribution of caspases as mediators or regulators of nuclear factor-κB (NF-κB) signaling, which plays a key role in inflammation and immunity. Much still needs to be learned about the mechanisms that govern the activation and regulation of NF-κB by caspases, and this review provides an update of this area. Whereas apoptosis signaling is dependent on the catalytic activity of caspases, they mainly act as scaffolding platforms for other signaling proteins in the case of NF-κB signaling. Caspase proteolytic activity, however, counteracts the pro-survival function of NF-κB by cleaving specific signaling molecules. A striking exception is the paracaspase mucosa-associated lymphoid tissue 1 (MALT1), whose adaptor and proteolytic activity are both needed to initiate a full blown NF-κB response in antigen-stimulated lymphocytes. Understanding the role of caspases and MALT1 in the regulation of NF-κB signaling is of high interest for therapeutic immunomodulation.     

Coordinate Regulation of TPL-2 and NF-κB Signaling in Macrophages by NF-κB1 p105

The role of IκB kinase (IKK)-induced proteolysis of NF-κB1 p105 in innate immune signaling was investigated using macrophages from Nfkb1(SSAA/SSAA) mice, in which the IKK target serines on p105 are mutated to alanines. We found that the IKK/p105 signaling pathway was essential for TPL-2 kinase activation of extracellular signal-regulated kinase (ERK) mitogen-activate protein (MAP) kinase and modulated the activation of NF-κB. The Nfkb1(SSAA) mutation prevented the agonist-induced release of TPL-2 from its inhibitor p105, which blocked activation of ERK by lipopolysaccharide (LPS), tumor necrosis factor (TNF), CpG, tripalmitoyl-Cys-Ser-Lys (Pam(3)CSK), poly(I · C), flagellin, and R848. The Nfkb1(SSAA) mutation also prevented LPS-induced processing of p105 to p50 and reduced p50 levels, in addition to decreasing the nuclear translocation of RelA and cRel. Reduced p50 in Nfkb1(SSAA/SSAA) macrophages significantly decreased LPS induction of the IκBζ-regulated Il6 and Csf2 genes. LPS upregulation of Il12a and Il12b mRNAs was also impaired although specific blockade of TPL-2 signaling increased expression of these genes at late time points. Activation of TPL-2/ERK signaling by IKK-induced p105 proteolysis, therefore, induced a negative feedback loop to downregulate NF-κB-dependent expression of the proinflammatory cytokine interleukin-12 (IL-12). Unexpectedly, TPL-2 promoted soluble TNF production independently of IKK-induced p105 phosphorylation and its ability to activate ERK, which has important implications for the development of anti-inflammatory drugs targeting TPL-2.     

Protection by DHA of Early Hippocampal Changes in Diabetes: Possible Role of CREB and NF-κB

The mechanisms underlying diabetic encephalopathy, are only partially understood. In this study, we try to address the mechanisms of diabetes induced damage and whether docosahexaenoic acid (DHA) could attenuate the degenerative changes in diabetic hippocampus in a rodent model of diabetes. Diabetes was induced in rats by an intraperitoneal injection of streptozotocin. Animals were divided into the following experimental groups: control rats; control animals treated with DHA; untreated diabetic rats; diabetic rats treated with insulin; diabetic rats treated with DHA; diabetic rats treated with insulin and DHA. At the end of week 12, rats were killed and one of the hemispheres was cryosectioned and the other was dissected and hippocampi homogenized. The number of bromodeoxyuridine positive cells in the hippocampus of diabetic rats was decreased, and the latency time to find the platform in the Morris Water maze was significantly increased in the diabetic rats when compared to controls. No changes where observed in the expression of p21 in the hippocampus of control and diabetic rats. Biochemical markers of oxidative stress were altered in hippocampus of diabetic rats, and NFκB-positive cells were increased in the hippocampus of diabetic rats when compared to controls. Treatment with DHA, or the combination of DHA with insulin, significantly restored to control levels all the values mentioned above. Our findings confirm a pivotal role for oxidative stress as well as NF-κB, but not p21, in diabetes-induced hippocampal impairments. Administration of DHA as well as insulin prevented the changes induced by diabetes in hippocampus.     

Identification of NF-κB1 and NF-κBIΑ Polymorphisms Using PCR–RFLP Assay in a Turkish Population

A polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) assay was used in a Turkish population to determine the frequency of polymorphisms of the nuclear factor-kappa (NF-κB1) and NF-κBIA genes, which have been shown to be related to several inflammatory diseases and cancer pathogenesis. Total genomic DNA was isolated from peripheral blood samples taken from 565 healthy volunteers living in Ayd?n Province. The genomic regions in question were amplified by PCR, and the polymorphisms in these regions were detected by a PCR–RFLP assay. The frequencies were 10.3% for the NF-κB1 ?94ins/delATTG del/del genotype, 49.1% for del/ins, and 40.6% for ins/ins. The genotype frequencies of the NF-κBIA 3′UTR A → G genotypes were A/A 19.2%, A/G 42.3%, and G/G 38.5%. Distribution of genotype frequencies was tested by Hardy–Weinberg; the NF-κB1 gene was in Hardy–Weinberg equilibrium (χ² = 3.402, P > 0.05), the NF-κBIA gene was not (χ² = 8.293, P < 0.05).     

Activation of NF-κB by alloferon through down-regulation of antioxidant proteins and IκBα

Alloferon is a 13-amino acid peptide isolated from the bacteria-challenged larvae of the blow fly Calliphora vicina. The pharmaceutical value of the peptide has been well demonstrated by its capacity to stimulate NK cytotoxic activity and interferon (IFN) synthesis in animal and human models, as well as to enhance antiviral and antitumor activities in mice. Antiviral and the immunomodulatory effectiveness of alloferon have also been supported clinically proved in patients suffering with herpes simplex virus (HSV) and human papilloma virus (HPV) infections. To elucidate molecular response to alloferon treatment, we initially screened a model cell line in which alloferon enhanced IFN synthesis upon viral infection. Among the cell lines tested, Namalva was chosen for further proteomic analysis. Fluorescence difference gel electrophoresis (DIGE) revealed that the levels of a series of antioxidant proteins decreased after alloferon treatment, while at least three glycolytic enzymes and four heat-shock proteins were increased in their expression levels. Based on the result of our proteomic analysis, we speculated that alloferon may activate the NF-kappaB signaling pathway. IkappaB kinase (IKK) assay, Western blot analysis on IkappaBalpha and its phosphorylated form at Ser 32, and an NF-kappaB reporter assay verified our proteomics-driven hypothesis. Thus, our results suggest that alloferon potentiates immune cells by activating the NF-kappaB signaling pathway through regulation of redox potential. Since NF-kappaB activation is involved in IFN synthesis, our results provide further clues as to how the alloferon peptide may stimulate IFN synthesis.     

Alpha-Catulin Contributes to Drug-Resistance of Melanoma by Activating NF-κB and AP-1

Melanoma is the most dangerous type of skin cancer accounting for 48,000 deaths worldwide each year and an average survival rate of about 6-10 months with conventional treatment. Tumor metastasis and chemoresistance of melanoma cells are reported as the main reasons for the insufficiency of currently available treatments for late stage melanoma. The cytoskeletal linker protein α-catulin (CTNNAL1) has been shown to be important in inflammation, apoptosis and cytoskeletal reorganization. Recently, we found an elevated expression of α-catulin in melanoma cells. Ectopic expression of α-catulin promoted melanoma progression and occurred concomitantly with the downregulation of E-cadherin and the upregulation of mesenchymal genes such as N-cadherin, Snail/Slug and the matrix metalloproteinases 2 and 9. In the current study we showed that α-catulin knockdown reduced NF-κB and AP-1 activity in malignant melanoma cells. Further, downregulation of α-catulin diminished ERK phosphorylation in malignant melanoma cells and sensitized them to treatment with chemotherapeutic drugs. In particular, cisplatin treatment led to decreased ERK-, JNK- and c-Jun phosphorylation in α-catulin knockdown melanoma cells, which was accompanied by enhanced apoptosis compared to control cells. Altogether, these results suggest that targeted inhibition of α-catulin may be used as a viable therapeutic strategy to chemosensitize melanoma cells to cisplatin by down-regulation of NF-κB and MAPK pathways.     

Analysis of nuclear factor-κB (NF-κB) essential modulator (NEMO) binding to linear and lysine-linked ubiquitin chains and its role in the activation of NF-κB

Nuclear factor-κB (NF-κB) essential modulator (NEMO), a component of the inhibitor of κB kinase (IKK) complex, controls NF-κB signaling by binding to ubiquitin chains. Structural studies of NEMO provided a rationale for the specific binding between the UBAN (ubiquitin binding in ABIN and NEMO) domain of NEMO and linear (Met-1-linked) di-ubiquitin chains. Full-length NEMO can also interact with Lys-11-, Lys-48-, and Lys-63-linked ubiquitin chains of varying length in cells. Here, we show that purified full-length NEMO binds preferentially to linear ubiquitin chains in competition with lysine-linked ubiquitin chains of defined length, including long Lys-63-linked deca-ubiquitins. Linear di-ubiquitins were sufficient to activate both the IKK complex in vitro and to trigger maximal NF-κB activation in cells. In TNFα-stimulated cells, NEMO chimeras engineered to bind exclusively to Lys-63-linked ubiquitin chains mediated partial NF-κB activation compared with cells expressing NEMO that binds to linear ubiquitin chains. We propose that NEMO functions as a high affinity receptor for linear ubiquitin chains and a low affinity receptor for long lysine-linked ubiquitin chains. This phenomenon could explain quantitatively distinct NF-κB activation patterns in response to numerous cell stimuli.