Melatonin treatment decreases c-fos expression in a headache model induced by capsaicin

The aim of the present work was to analyze c-fos response within the trigeminal nucleus caudalis (TNC) of pinealectomized rats and animals that received intraperitoneal melatonin, after intracisternal infusion of capsaicin, used to induce intracranial trigeminovascular stimulation. Experimental groups consisted of animals that received vehicle solution (saline-ethanol-Tween 80, 8:1:1, diluted 1:50) only (VEI, n = 5); animals that received capsaicin solution (200 nM) only (CAP, n = 6); animals submitted to pinealectomy (PX, n = 5); sham-operated animals (SH, n = 5); animals submitted to pinealectomy followed by capsaicin stimulation (200 nM) after 15 days (PX + CAP, n = 7); and animals that received capsaicin solution (200 nM) and intraperitoneal melatonin (10 mg/kg) (CAP + MEL, n = 5). Control rats, receiving vehicle in the cisterna magna, showed a small number of c-fos-positive cells in the TNC (layer I/II) as well as the sham-operated and pinealectomized rats, when compared to animals stimulated by capsaicin. On the other hand, pinealectomized rats, which received capsaicin, presented the highest number of c-fos-positive cells. Animals receiving capsaicin and melatonin treatment had similar expression of the vehicle group. Our data provide experimental evidence to support the role of melatonin and pineal gland in the pathophysiology of neurovascular headaches.

Cell death and immunohistochemistry of p53, c-Fos and c-Jun after spermine infection into the rat striatum

Administration of polyamines into the central nervous system results in tissue damage, possibly through the excitotoxic actions of the NMDA receptor. Direct injection of 100 nmol of spermine into the rat striatum produced a lesion equivalent to approximately 50% of the striatum. Analysis of the DNA in this region revealed the distinct ladder-like pattern of degradation often associated with apoptosis. This DNA fragmentation was confirmed in vivo using terminal deoxynucleotidyl-transferase-mediated biotinylated deoxyuridine triphosphate nick end labelling (TUNEL). The morphology of the TUNEL-positive cells showed marked differences at the needle tract when compared with cells in damaged areas away from the needle tract, suggesting a differential mechanism of cell death in these two regions. The patterns of p53, c-Fos and c-Jun protein expression were determined using immunohistochemistry. The number of p53-immunoreactive cells increased up to 14 h and returned to basal levels by 24 h. c-Fos protein expression transiently increased, peaking at 8 h after injection, c-Jun exhibited a protracted pattern of expression, remaining elevated up to 24 h. p53 protein expression was colocalised with TUNEL staining in areas away from the needle tract, but not in cells at the needle tract, suggesting once again a differential mechanism of cell death. At 14 h, c-Fos and c-Jun were not colocalised with TUNEL staining, suggesting that they are either not involved with the cell death process or that the time course of protein expression and the onset of DNA fragmentation do not overlap. This work represents the first characterisation of processes associated with cell death induced by spermine in vivo.

Cyhalothrin increased c-fos immunoreactivity at the paraventricular nucleus of the hypothalamus in rats, and suppressed macrophage activity in an adrenal-dependent fashion

Synthetic type II pyrethroid insecticides, such as cyhalothrin at certain dosage levels, simultaneously induce stress-like symptoms and innate immunosuppressive effects in laboratory animals. The present study was designed to further analyze the stress-like effects induced by cyhalotrin and also investigate the role of Hypothalamus-Hypophysis-Adrenal (HHA) axis and Sympathetic Nervous Systems (SNS) and their effects on macrophage activity of rats. Results showed that cyhalothrin treatment (3.0 mg/kg/day. for 7 days) increased corticosterone serum levels and c-fos immunoreactivity at the paraventricular nucleus of the hypothalamus (PVN) but induced no changes in c-fos expression at the basolateral amygdala (BLA). Both areas were related to HHA axis and SNS activations by stress. Further analysis showed that adrenalectomy partially abrogated the suppression effects of cyhalothrin on macrophage activity and that 6-OHDA-induced peripheral symphatectyomy had no effects on this innate immune cell activity. The present observed data support and reinforce the notion that cyhalotrin at this treatment schedule induces stress-like symptoms and suggest that other factors, beyond indirect neuroadaptative responses, are necessary for the suppression effects of insecticide on innate immune response. (C) 2008 Elsevier B.V. All rights reserved.

Analyse der Informationsverarbeitung in CHL1-defizienten Mäusen mittels metabolischer Markierung, Expressionsstudien der Immediate-Early-Gene c-fos und arg3.1/arc sowie verhaltensbiologischer Tests

Close homolog of L1, neural cell recognition molecules, c-fos, arg3.1, arc, immediat early genes, novelty, information processing, behavioral tests

Fas-associated death domain protein (FADD) and caspase-8 mediate up-regulation of c-Fos by Fas ligand and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) via a FLICE inhibitory protein (FLIP)-regulated pathway.

Fas, a death domain-containing member of the tumor necrosis factor receptor family and its ligand FasL have been predominantly studied with respect to their capability to induce cell death. However, a few studies indicate a proliferation-inducing signaling activity of these molecules too. We describe here a novel signaling pathway of FasL and the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) that triggers transcriptional activation of the proto-oncogene c-fos, a typical target gene of mitogenic pathways. FasL- and TRAIL-mediated up-regulation of c-Fos was completely dependent on the presence of Fas-associated death domain protein (FADD) and caspase-8, but caspase activity seemed to be dispensable as a pan inhibitor of caspases had no inhibitory effect. Upon overexpression of the long splice form of cellular FADD-like interleukin-1-converting enzyme (FLICE) inhibitory protein (cFLIP) in Jurkat cells, FasL- and TRAIL-induced up-regulation of c-Fos was almost completely blocked. The short splice form of FLIP, however, showed a rather stimulatory effect on c-Fos induction. Together these data demonstrate the existence of a death receptor-induced, FADD- and caspase-8-dependent pathway leading to c-Fos induction that is inhibited by the long splice form FLIP-L.

Expression of the proto-oncogene c-fos in three-dimensional fetal brain cell cultures and the lack of correlation with maturation-inducing stimuli.

Previous work has shown that aggregating fetal brain cell cultures are able to attain a highly differentiated state, and that their development is greatly enhanced by growth and/or differentiation factors such as epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and the protein kinase C-activating tumor promoter mezerein. The present study shows that in these 3-dimensional cultures the peptide growth factors EGF and bFGF as well as mezerein are able to induce the expression of the proto-oncogene c-fos. This induction was rapid and transient, in good agreement with observations reported from a wide variety of cell types in vitro. The maximal levels of c-fos mRNA found after stimulation were low in immature cultures and increased greatly as maturation progressed. Of the three factors tested, mezerein was the most potent inducer of c-fos. In contrast to the peptide growth factors EGF and bFGF which were found to induce c-fos only in glial cells, mezerein was stimulatory in glial cells as well as in neurons. A similar cell type specificity has been observed previously for the maturation-enhancing response in immature aggregate cultures. However, in the present study no correlation was found between the degree of c-fos induction and the extent of the maturation-enhancing stimulation. Immature cultures known to be most sensitive and responsive to these maturation-enhancing agents required relatively high doses of peptide growth factors for the induction of c-fos, and the maximal levels of c-fos mRNA elicited were much lower than those in differentiated cultures which did not show any long-term response to these stimuli.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of c-fos expression by RNA polymerase elongation competence.

The molecular mechanisms underlying transcription elongation and their role in gene regulation are poorly characterized in eukaryotes. A number of genes, however, have been proposed to be regulated at the level of transcription elongation, including c-myc, c-fos and c-myb. Here, we analyze the control of transcription elongation at the mouse c-fos gene at the nucleotide level in intact cells. We find that RNA polymerases are engaged in the promoter-proximal part of the gene in the absence of gene activation signals and mRNA synthesis. Importantly, we determine that the engaged RNA polymerases originate from a continuous initiation of transcription which, in the absence of gene activation signals, terminate close to the promoter. We also observe that the c-fos gene presents an active chromatin conformation, with the promoter and upstream regulatory sequences constitutively occupied by proteins, accounting for the continuous initiation of RNA polymerase complexes. We propose that activation of c-fos gene expression results primarily from the assembly of elongation-competent RNA polymerases that can transcribe the complete gene. Our results suggest that the engaged RNA polymerases found downstream of a number of other eukaryotic promoters may be associated with transcription termination of elongation-incompetent polymerases in the absence of activating signals.

Silencing of c-Fos expression by microRNA-155 is critical for dendritic cell maturation and function.

MicroRNAs (miRNAs) are small, noncoding RNAs that regulate target mRNAs by binding to their 3' untranslated regions. There is growing evidence that microRNA-155 (miR155) modulates gene expression in various cell types of the immune system and is a prominent player in the regulation of innate and adaptive immune responses. To define the role of miR155 in dendritic cells (DCs) we performed a detailed analysis of its expression and function in human and mouse DCs. A strong increase in miR155 expression was found to be a general and evolutionarily conserved feature associated with the activation of DCs by diverse maturation stimuli in all DC subtypes tested. Analysis of miR155-deficient DCs demonstrated that miR155 induction is required for efficient DC maturation and is critical for the ability of DCs to promote antigen-specific T-cell activation. Expression-profiling studies performed with miR155(-/-) DCs and DCs overexpressing miR155, combined with functional assays, revealed that the mRNA encoding the transcription factor c-Fos is a direct target of miR155. Finally, all of the phenotypic and functional defects exhibited by miR155(-/-) DCs could be reproduced by deregulated c-Fos expression. These results indicate that silencing of c-Fos expression by miR155 is a conserved process that is required for DC maturation and function.

Expressão dos protooncogenes c-fos, c-myc e c-jun em miométrio normal e mioma humanos

OBJETIVO: Comparar a expressão gênica (mRNA) e protéica dos protooncogenes c-fos, c-myc e c-jun em miométrio normal e mioma humanos. MÉTODOS: Foi realizado um estudo do tipo caso-controle. O material foi coletado de 12 pacientes submetidas a histerectomia no Hospital de Clínicas de Porto Alegre. A expressão do mRNA específico para c-myc, c-fos, c-jun e beta-microglobulina foi avaliada pela técnica de RT-PCR, utilizando primers específicos para cada gene. A expressão protéica destes protooncogenes foi avaliada através de Western blot com anticorpos específicos. RESULTADOS: Não houve diferença significativa para expressão gênica desses protooncogenes entre miométrio normal e mioma (c-myc: 0,87 ± 0,08 vs 0,87 ± 0,08, p = 0,952; c-fos: 1,10 ± 0,17 vs 1,01 ± 0,11, p = 0,21; c-jun: 1,03 ± 0,12 vs 0,96 ± 0,09, p = 0,168, respectivamente). Não houve diferença significativa para expressão protéica desses protooncogenes entre miométrio normal e mioma (c-myc: 1,36 ± 0,48 vs 1,53 ± 0,29, p = 0,569; c-fos: 8,85 ± 5,5 vs 6,56 ± 4,22, p = 0,434; e c-jun: 6,47 ± 3,04 vs 5,42 ± 2,03, p = 0,266, respectivamente). CONCLUSÃO: A expressão gênica (transcrição) e a expressão protéica (tradução) dos protooncogenes c-myc, c-fos e c-jun em mioma e miométrio normal são semelhantes.

c-Fos expression induced by electroacupuncture at the Zusanli point in rats submitted to repeated immobilization

In laboratory animals, acupuncture needs to be performed on either anesthetized or, if unanesthetized, restrained subjects. Both procedures up-regulate c-Fos expression in several areas of the central nervous system, representing therefore a major pitfall for the assessment of c-Fos expression induced by electroacupuncture. Thus, in order to reduce the effect of acute restraint we used a protocol of repeated restraint for the assessment of the brain areas activated by electroacupuncture in adult male Wistar rats weighing 180-230 g. Repeated immobilization protocols (6 days, 1 h/day and 13 days, 2 h/day) were used to reduce the effect of acute immobilization stress on the c-Fos expression induced by electroacupuncture at the Zusanli point (EA36S). Animals submitted to immobilization alone or to electroacupuncture (100 Hz, 2-4 V, faradic wave) in a non-point region were compared to animals submitted to electroacupuncture at EA36S (4 animals/subgroup). c-Fos expression was measured in 41 brain areas by simple counting of cells and the results are reported as number of c-Fos-immunoreactive cells/10,000 µm². The protocols of repeated immobilization significantly reduced the immobilization-induced c-Fos expression in most of the brain areas analyzed (P < 0.05). Animals of the EA36S groups had significantly higher levels of c-Fos expression in the dorsal raphe nucleus, locus coeruleus, posterior hypothalamus and central medial nucleus of the thalamus. Furthermore, the repeated immobilization protocols intensified the differences between the effects of 36S and non-point stimulation in the dorsal raphe nucleus (P < 0.05). These data suggest that high levels of stress can interact with and mask the evaluation of specific effects of acupuncture in unanesthetized animals.

Pim-1 kinase inhibits the activation of reporter gene expression in Elk-1 and c-Fos reporting systems but not the endogenous gene expression: an artifact of the reporter gene assay by transient co-transfection

We have studied the molecular mechanism and signal transduction of pim-1, an oncogene encoding a serine-threonine kinase. This is a true oncogene which prolongs survival and inhibits apoptosis of hematopoietic cells. In order to determine whether the effects of Pim-1 occur by regulation of the mitogen-activated protein kinase pathway, we used a transcriptional reporter assay by transient co-transfection as a screening method. In this study, we found that Pim-1 inhibited the Elk-1 and NFkappaB transcriptional activities induced by activation of the mitogen-activated protein kinase cascade in reporter gene assays. However, Western blots showed that the induction of Elk-1-regulated expression of endogenous c-Fos was not affected by Pim-1. The phosphorylation and activation of neither Erk1/2 nor Elk-1 was influenced by Pim-1. Also, in the gel shift assay, the pattern of endogenous NFkappaB binding to its probe was not changed in any manner by Pim-1. These data indicate that Pim-1 does not regulate the activation of Erk1/2, Elk-1 or NFkappaB. These contrasting results suggest a pitfall of the transient co-transfection reporter assay in analyzing the regulation of transcription factors outside of the chromosome context. It ensures that results from reporter gene expression assay should be verified by study of endogenous gene expression.

Gentle handling temporarily increases c-Fos in the substantia nigra pars compacta

Dopaminergic neurotransmission is involved in the regulation of sleep. In particular, the nigrostriatal pathway is an important center of sleep regulation. We hypothesized that dopaminergic neurons located in substantia nigra pars compacta (SNpc) could be activated by gentle handling, a method to obtain sleep deprivation (SD). Adult male C57/BL6J mice (N = 5/group) were distributed into non-SD (NSD) or SD groups. SD animals were subjected to SD once for 1 or 3 h by gentle handling. Two experiments were performed. The first determined the activation of SNpc neurons after SD, and the second examined the same parameters after pharmacologically induced dopaminergic depletion using intraperitoneal reserpine (2 mg/kg). After 1 or 3 h, SD and NSD mice were subjected to motor evaluation using the open field test. Immediately after the behavioral test, the mice were perfused intracardially to fix the brain and for immunohistochemical analysis of c-Fos protein expression within the SNpc. The open field test indicated that SD for 1 or 3 h did not modify motor behavior. However, c-Fos protein expression was increased after 1 h of SD compared with the NSD and 3-h SD groups. These immunohistochemistry data indicate that these periods of SD are not able to produce dopaminergic supersensitivity. Nevertheless, the increased expression of c-Fos within the SNpc suggests that dopaminergic nigral activation was triggered by SD earlier than motor responsiveness. Dopamine-depleted mice (experiment 2) exhibited a similar increase of c-Fos expression compared to control animals indicating that dopamine neurons are still activated in the 1-h SD group despite the exhaustion of dopamine. This finding suggests that this range (2-5-fold) of neuronal activation may serve as a marker of SD.

Effects of sciatic nerve transection on ultrastructure, NADPH-diaphorase reaction and serotonin-, tyrosine hydroxylase-, c-Fos-, glucose transporter 1- and 3-like immunoreactivities in frog dorsal root ganglion

Frogs have been used as an alternative model to study pain mechanisms. Since we did not find any reports on the effects of sciatic nerve transection (SNT) on the ultrastructure and pattern of metabolic substances in frog dorsal root ganglion (DRG) cells, in the present study, 18 adult male frogs (Rana catesbeiana) were divided into three experimental groups: naive (frogs not subjected to surgical manipulation), sham (frogs in which all surgical procedures to expose the sciatic nerve were used except transection of the nerve), and SNT (frogs in which the sciatic nerve was exposed and transected). After 3 days, the bilateral DRG of the sciatic nerve was collected and used for transmission electron microscopy. Immunohistochemistry was used to detect reactivity for glucose transporter (Glut) types 1 and 3, tyrosine hydroxylase, serotonin and c-Fos, as well as nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-diaphorase). SNT induced more mitochondria with vacuolation in neurons, satellite glial cells (SGCs) with more cytoplasmic extensions emerging from cell bodies, as well as more ribosomes, rough endoplasmic reticulum, intermediate filaments and mitochondria. c-Fos immunoreactivity was found in neuronal nuclei. More neurons and SGCs surrounded by tyrosine hydroxylase-like immunoreactivity were found. No change occurred in serotonin- and Glut1- and Glut3-like immunoreactivity. NADPH-diaphorase occurred in more neurons and SGCs. No sign of SGC proliferation was observed. Since the changes of frog DRG in response to nerve injury are similar to those of mammals, frogs should be a valid experimental model for the study of the effects of SNT, a condition that still has many unanswered questions.

Imunorreatividade à proteína C-FOS após estimulação periférica nociva e tratamento com morfina no sistema nervoso central do caracol terrestre Megalobulimus abbreviatus

Utilizando as técnicas de imunoistoquímica e densitometria óptica, foi investigada a localização e a expressão da proteína c-Fos no SNC do caracol Megalobulimus abbreviatus. Neurônios imunorreativos foram encontrados nos gânglios cerebrais, pedais, parietal direito e visceral de caracóis submetidos ao estímulo térmico aversivo (50oC), e sacrificados em diferentes tempos (3, 6, 12, 18 e 24 h) após a estimulação. A análise da imunorreatividade à c-Fos através do método de medida da densidade óptica (DO) revelou uma diferença significativa no sentido de apresentar uma maior expressão (p<0,05) na área do lobo pedal do pós-cérebro do gânglio cerebral em relação às outras regiões analisadas no mesmo gânglio (mesocérebro, pró-cérebro e lobo pleural do pós-cérebro). Além disso, também houve expressão significativamente maior (p<0,05) quando comparada a densitometria da região do mesocérebro em relação ao lobo pleural do pós-cérebro nos grupos controle, 3h e 18h. O lobo pleural do pós-cérebro apresentou uma expressão significativamente menor (p<0,05) na imunorreatividade da proteína c-Fos quando comparado ao pró-cérebro em animais sacrificados 12h e 24h após e estímulo aversivo. Em relação ao grupo controle, a DO da proteína c-Fos não variou nos diferentes tempos de sacrifício quando comparada a mesma região do gânglio (cerebral, pedal, parietal direito ou visceral) ao longo do tempo na maioria das regiões. A única diferença estatisticamente significativa (p<0,05) foi encontrada no mesocérebro do gânglio de animais sacrificados 12 h após o estímulo térmico aversivo, mostrando uma diminuição da imunorreatividade. Nos animais tratados com salina (1ml) ou morfina (20mg/kg) 15 min antes do estímulo térmico aversivo, os mesmos grupos neuronais nos gânglios do SNC de M. abbreviatus mostraram imunomarcação à proteína c-Fos. Em relação ao grupo controle, observou-se uma expressão significativamente menor (p<0,01) na DO da imunorreatividade da proteína c-Fos nos neurônios anteriores do gânglio pedal nos animais sacrificados 3 h e 6 h após o estímulo térmico aversivo. No momento em que a comparação foi feita entre os grupos salina e morfina de animais sacrificados ao mesmo tempo, na grande maioria dos grupos observou-se uma diminuição na imunorreatividade da proteína c-Fos. Esta diferença, porém, mostrou-se significativa (p<0,01) no mesocérebro de animais do grupo 3h, no lobo pedal do pós cérebro de animais dos grupos 3 h, 6 h e 18 h, nos neurônios anteriores do gânglio pedal nos grupos 6 h e 12 h, nos neurônios mediais do gânglio pedal do grupo 3 h, nos neurônios posteriores do gânglio pedal do grupo 6 h, nos neurônios da região anterior do gânglio parietal direito no grupo 12 h e nos neurônios do gânglio visceral no grupo experimental 12 h. A diferença na DO da proteína c-Fos apresentou uma diminuição extremamente significativa (p<0,001) nos neurônios mediais do gânglio pedal de animais sacrificados 12 h após o estímulo térmico aversivo, nos neurônios posteriores do gânglio pedal dos animais sacrificados 12 h após o estímulo e nos neurônios do gânglio visceral dos animais do grupo experimental 6 h. A partir destes dados e da correlação com estudos realizados em M. abbreviatus para detecção de mediadores químicos envolvidos na nocicepção, podemos concluir que as áreas imunorreativas que apresentaram estas variações na densidade óptica da imunorreatividade à proteína c-Fos em diferentes tempos de sacrifício e tratamento com morfina estão envolvidas no processo nociceptivo neste caracol.