Frequent downregulation of the transcription factor Foxa2 in lung cancer through epigenetic silencing

Purpose: We sought to determine the mechanisms of downregulation of the airway transcription factor Foxa2 in lung cancer and the expression status of Foxa2 in non-small-cell lung cancer (NSCLC). Methods: A series of 25 lung cancer cell lines were evaluated for Foxa2 protein expression, FOXA2 mRNA levels, FOXA2 mutations, FOXA2 copy number changes and for evidence of FOXA2 promoter hypermethylation. In addition, 32 NSCLCs were sequenced for FOXA2 mutations and 173 primary NSCLC tumors evaluated for Foxa2 expression using an immunohistochemical assay. Results: Out of the 25 cell lines, 13 (52%) had undetectable FOXA2 mRNA. The expression of FOXA2 mRNA and Foxa2 protein were congruent in 19/22 cells (p = 0.001). FOXA2 mutations were not identified in primary NSCLCs and were infrequent in cell lines. Focal or broad chromosomal deletions involving FOXA2 were not present. The promoter region of FOXA2 had evidence of hypermethylation, with an inverse correlation between FOXA2 mRNA expression and presence of CpG dinucleotide methylation (p < 0.0001). In primary NSCLC tumor specimens, there was a high frequency of either absence (42/173, 24.2%) or no/low expression (96/173,55.4%) of Foxa2. In 130 patients with stage I NSCLC there was a trend towards decreased survival in tumors with no/low expression of Foxa2 (HR of 1.6, 95%CI 0.9-3.1; p = 0.122). Conclusions: Loss of expression of Foxa2 is frequent in lung cancer cell lines and NSCLCs. The main mechanism of downregulation of Foxa2 is epigenetic silencing through promoter hypermethylation. Further elucidation of the involvement of Foxa2 and other airway transcription factors in the pathogenesis of lung cancer may identify novel therapeutic targets. (C) 2012 Elsevier Ireland Ltd. All rights reserved.

Molecular Characterization of the Putative Transcription Factor SebA Involved in Virulence in Aspergillus fumigatus

Aspergillus fumigatus is a major opportunistic pathogen and allergen of mammals. Nutrient sensing and acquisition mechanisms, as well as the capability to cope with different stressing conditions, are essential for A. fumigatus virulence and survival in the mammalian host. This study characterized the A. fumigatus SebA transcription factor, which is the putative homologue of the factor encoded by Trichoderma atroviride seb1. The Delta sebA mutant demonstrated reduced growth in the presence of paraquat, hydrogen peroxide, CaCl2, and poor nutritional conditions, while viability associated with sebA was also affected by heat shock exposure. Accordingly, SebA:GFP (SebA:green fluorescent protein) was shown to accumulate in the nucleus upon exposure to oxidative stress and heat shock conditions. In addition, genes involved in either the oxidative stress or heat shock response had reduced transcription in the Delta sebA mutant. The A. fumigatus Delta sebA strain was attenuated in virulence in a murine model of invasive pulmonary aspergillosis. Furthermore, killing of the Delta sebA mutant by murine alveolar macrophages was increased compared to killing of the wild-type strain. A. fumigatus SebA plays a complex role, contributing to several stress tolerance pathways and growth under poor nutritional conditions, and seems to be integrated into different stress responses.

Norepinephrine activates NF-κB transcription factor in cultured rat pineal gland

AIMS: The circadian rhythm in mammalian pineal melatonin secretion is modulated by norepinephrine (NE) released at night. NE interaction with β1-adrenoceptors activates PKA that phosphorylates the transcription factor CREB, leading to the transcription and translation of the arylalkylamine-N-acetyltransferase (AANAT) enzyme. Several studies have reported the interplay between CREB and the nuclear factor-κB (NF-κB) and a circadian rhythm for this transcription factor was recently described in the rat pineal gland. In this work we studied a direct effect of NE on NF-κB activation and the role played by this factor on melatonin synthesis and Aanat transcription and activity. MAIN METHODS: Cultured rat pineal glands were incubated in the presence of two different NF-κB inhibitors, pyrrolidine-dithiocarbamate or sodium salicylate, and stimulated with NE. Melatonin content was quantified by HPLC with electrochemical detection. AANAT activity was measured by a radiometric assay and the expression of Aanat mRNA was analyzed by real-time PCR. Gel shift assay was performed to study the NF-κB activation in cultured rat pineal glands stimulated by NE. KEY FINDINGS: Our results showed that the p50/p50 homodimer of NF-κB is activated by NE and that it has a role in melatonin synthesis, acting on Aanat transcription and activity. SIGNIFICANCE: Here we present evidence that NF-κB is an important transcription factor that acts, directly or indirectly, on Aanat transcription and activity leading to a modulation of melatonin synthesis. NE plays a role in the translocation of NF-κB p50/p50 homodimer to the nucleus of pinealocytes, thus probably influencing the nocturnal pineal melatonin synthesis

Influence of the dopaminergic system, CREB, and transcription factor-κB on cocaine neurotoxicity

Cocaine is a widely used drug and its abuse is associated with physical, psychiatric and social problems. Abnormalities in newborns have been demonstrated to be due to the toxic effects of cocaine during fetal development. The mechanism by which cocaine causes neurological damage is complex and involves interactions of the drug with several neurotransmitter systems, such as the increase of extracellular levels of dopamine and free radicals, and modulation of transcription factors. The aim of this review was to evaluate the importance of the dopaminergic system and the participation of inflammatory signaling in cocaine neurotoxicity. Our study showed that cocaine activates the transcription factors NF-κB and CREB, which regulate genes involved in cellular death. GBR 12909 (an inhibitor of dopamine reuptake), lidocaine (a local anesthetic), and dopamine did not activate NF-κB in the same way as cocaine. However, the attenuation of NF-κB activity after the pretreatment of the cells with SCH 23390, a D1 receptor antagonist, suggests that the activation of NF-κB by cocaine is, at least partially, due to activation of D1 receptors. NF-κB seems to have a protective role in these cells because its inhibition increased cellular death caused by cocaine. The increase in BDNF (brain-derived neurotrophic factor) mRNA can also be related to the protective role of both CREB and NF-κB transcription factors. An understanding of the mechanisms by which cocaine induces cell death in the brain will contribute to the development of new therapies for drug abusers, which can help to slow down the progress of degenerative processes.

Eye enucleation activates the transcription nuclear factor kappa-B in the rat superior colliculus

Ocular enucleation produces significant morphological and physiological changes in central visual areas. However, our knowledge of the molecular events resulting from eye enucleation in visual brain areas remains elusive. We characterized here the transcription nuclear factor kappa-B (NF-kappa B) activation induced by ocular enucleation in the rat superior colliculus (SC). We also tested the effectiveness of the synthetic glucocorticoid dexamethasone in inhibiting its activation. Electrophoretic mobility shift assays to detect NF-kappa B indicated that this transcription factor is activated in the SC from 1 h to day 15 postlesion. The expression of p65 and p50 proteins in the nuclear extracts was also increased. Dexamethasone treatment was able to significantly inhibit NF-kappa B activation. These findings suggest that this transcriptional factor is importantly involved in the visual system short-term processes that ensue after retinal lesions in the adult brain. (C) 2012 Elsevier Ireland Ltd. All rights reserved.

Cocaine induces cell death and activates the transcription nuclear factor kappa-b in pc12 cells

Cocaine is a worldwide used drug and its abuse is associated with physical, psychiatric and social problems. The mechanism by which cocaine causes neurological damage is very complex and involves several neurotransmitter systems. For example, cocaine increases extracellular levels of dopamine and free radicals, and modulates several transcription factors. NF-κB is a transcription factor that regulates gene expression involved in cellular death. Our aim was to investigate the toxicity and modulation of NF-κB activity by cocaine in PC 12 cells. Treatment with cocaine (1 mM) for 24 hours induced DNA fragmentation, cellular membrane rupture and reduction of mitochondrial activity. A decrease in Bcl-2 protein and mRNA levels, and an increase in caspase 3 activity and cleavage were also observed. In addition, cocaine (after 6 hours treatment) activated the p50/p65 subunit of NF-κB complex and the pretreatment of the cells with SCH 23390, a D1 receptor antagonist, attenuated the NF-κB activation. Inhibition of NF-κB activity by using PDTC and Sodium Salicilate increased cell death caused by cocaine. These results suggest that cocaine induces cell death (apoptosis and necrosis) and activates NF-κB in PC12 cells. This activation occurs, at least partially, due to activation of D1 receptors and seems to have an anti-apoptotic effect on these cells.

Plasma corticosterone elevation inhibits the activation of nuclear factor kappa B (NFKB) in the Syrian hamster pineal gland

We evaluated how the mild stress-induced increase in endogenous corticosterone affected the pineal gland in Syrian hamsters (Mesocricetus auratus). The animals were maintained under constant light for 1 day, instead of a cycle of 14:10-h, to increase the circulating corticosterone levels during the daytime. The nuclear translocation of nuclear factor kappa B (NFKB), which is the pivotal transcription factor for stress and injury, presented a daily rhythm in normal animals. NFKB nuclear content increased linearly from the onset of light [Zeitgeber Time 0 (ZT0)] until ZT11 and decreased after ZT12 when the plasma corticosterone peak was detected in normal animals. However, the 24-h profiles of the two curves were different, and they did not clearly support an exclusive relationship between corticosterone levels and NFKB content. Therefore, we tested the effect of increased endogenous corticosterone through inducing mild stress by maintaining daytime illumination for one night. This stressful condition, which increased daytime corticosterone levels, resulted in a daytime decrease in NFKB nuclear content, and this was inhibited by mifepristone. Overall, this study shows that NFKB has a daily rhythm in Syrian hamster pineal glands and, by increasing endogenous corticosterone with a stressful condition, NFKB activity is regulated. Therefore, this study suggests that the pineal gland in the Syrian hamster is a sensor of stressful conditions.

Nuclear Factor (NF) κB polymorphism is associated with heart function in patients with heart failure

Abstract Background Cardiac remodeling is generally an adverse sign and is associated with heart failure (HF) progression. NFkB, an important transcription factor involved in many cell survival pathways, has been implicated in the remodeling process, but its role in the heart is still controversial. Recently, a promoter polymorphism associated with a lesser activation of the NFKB1 gene was also associated with Dilated Cardiomyopathy. The purpose of this study was to evaluate the association of this polymorphism with clinical and functional characteristics of heart failure patients of different etiologies. Methods A total of 493 patients with HF and 916 individuals from a cohort of individuals from the general population were investigated. The NFKB1 -94 insertion/deletion ATTG polymorphism was genotyped by High Resolution Melt discrimination. Allele and genotype frequencies were compared between groups. In addition, frequencies or mean values of different phenotypes associated with cardiovascular disease were compared between genotype groups. Finally, patients were prospectively followed-up for death incidence and genotypes for the polymorphism were compared regarding disease onset and mortality incidence in HF patients. Results We did not find differences in genotype and allelic frequencies between cases and controls. Interestingly, we found an association between the ATTG1/ATTG1 genotype with right ventricle diameter (P = 0.001), left ventricle diastolic diameter (P = 0.04), and ejection fraction (EF) (P = 0.016), being the genotype ATTG1/ATTG1 more frequent in patients with EF lower than 50% (P = 0.01). Finally, we observed a significantly earlier disease onset in ATTG1/ATTG1 carriers. Conclusion There is no genotype or allelic association between the studied polymorphism and the occurrence of HF in the tested population. However, our data suggest that a diminished activation of NFKB1, previously associated with the ATTG1/ATTG1 genotype, may act modulating on the onset of disease and, once the individual has HF, the genotype may modulate disease severity by increasing cardiac remodeling and function deterioration.