Molecular Aspects Involved in the Mechanisms of Bothrops jararaca Venom-Induced Hyperalgesia: Participation of NK1 Receptor and Glial Cells.

Accidents caused by Bothrops jararaca (Bj) snakes result in several local and systemic manifestations, with pain being a fundamental characteristic. The inflammatory process responsible for hyperalgesia induced by Bj venom (Bjv) has been studied; however, the specific roles played by the peripheral and central nervous systems in this phenomenon remain unclear. To clarify this, we induced hyperalgesia in rats using Bjv and collected tissues from dorsal root ganglia (DRGs) and spinal cord (SC) at 2 and 4 h post-induction. Samples were labeled for Iba-1 (macrophage and microglia), GFAP (satellite cells and astrocytes), EGR1 (neurons), and NK1 receptors. Additionally, we investigated the impact of minocycline, an inhibitor of microglia, and GR82334 antagonist on Bjv-induced hyperalgesia. Our findings reveal an increase in Iba1 in DRG at 2 h and EGR1 at 4 h. In the SC, markers for microglia, astrocytes, neurons, and NK1 receptors exhibited increased expression after 2 h, with EGR1 continuing to rise at 4 h. Minocycline and GR82334 inhibited venom-induced hyperalgesia, highlighting the crucial roles of microglia and NK1 receptors in this phenomenon. Our results suggest that the hyperalgesic effects of Bjv involve the participation of microglial and astrocytic cells, in addition to the activation of NK1 receptors.

Taking advantage of fear generalization-associated destabilization to attenuate the underlying memory via reconsolidation intervention.

Upon retrieval, an aversive memory can undergo destabilization and reconsolidation. A traumatic-like memory, however, may be resistant to this process. The present study sought to contribute with a strategy to overcome this potential issue by investigating whether generalized fear retrieval is susceptible to destabilization-reconsolidation that can be pharmacologically modified. We hypothesized that exposure to a context that elicits moderate generalization levels would allow a malleable memory state. We developed a fear conditioning protocol in context A (cxt-A) paired with yohimbine administration to promote significant fear to a non-conditioned context B (cxt-B) in rats, mimicking the enhanced noradrenergic activity reported after traumatic events in humans. Next, we attempted to impair the reconsolidation phase by administering clonidine (CLO) immediately after exposure to cxt-A, cxt-B, or a third context C (cxt-C) neither conditioned nor generalized. CLO administered post-cxt-B exposure for two consecutive days subsequently resulted in decreased freezing levels in cxt-A. CLO after cxt-B only once, after cxt-A or cxt-C in two consecutive days, or independently of cxt-B exposures did not affect fear in a later test. A 6-h-delay in CLO treatment post-cxt-B exposures produced no effects, and nimodipine administered pre-cxt-B exposures precluded the CLO action. We then quantified the Egr1/Zif268 protein expression following cxt-B exposures and CLO treatments. We found that these factors interact to modulate this memory destabilization-reconsolidation mechanism in the basolateral amygdala but not the dorsal CA1 hippocampus. Altogether, memory destabilization can accompany generalized fear expression; thus, we may exploit it to potentiate reconsolidation blockers' action.

EGR1 upregulation following Venezuelan equine encephalitis virus infection is regulated by ERK and PERK pathways contributing to cell death.

Venezuelan equine encephalitis virus (VEEV) is a neurotropic virus that causes significant disease in both humans and equines. Here we characterized the impact of VEEV on signaling pathways regulating cell death in human primary astrocytes. VEEV productively infected primary astrocytes and caused an upregulation of early growth response 1 (EGR1) gene expression at 9 and 18 h post infection. EGR1 induction was dependent on extracellular signal-regulated kinase1/2 (ERK1/2) and protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), but not on p38 mitogen activated protein kinase (MAPK) or phosphoinositide 3-kinase (PI3K) signaling. Knockdown of EGR1 significantly reduced VEEV-induced apoptosis and impacted viral replication. Knockdown of ERK1/2 or PERK significantly reduced EGR1 gene expression, dramatically reduced viral replication, and increased cell survival as well as rescued cells from VEEV-induced apoptosis. These data indicate that EGR1 activation and subsequent cell death are regulated through ERK and PERK pathways in VEEV infected primary astrocytes.

Anlotinib inhibits synovial sarcoma by targeting GINS1: a novel downstream target oncogene in progression of synovial sarcoma.

BACKGROUND: Synovial sarcoma (SS) is an aggressive soft-tissue sarcoma with a poor prognosis owing to its resistance to radiation and chemotherapy. Thus, novel therapeutic strategies for SS are urgently required. Anlotinib, a new oral tyrosine kinase inhibitor, is designed to primarily inhibit multi-targets in vasculogenesis and angiogenesis. This study was designed to characterize its antitumor efficacy and possible mechanism in patients with advanced refractory synovial sarcoma. METHODS: Anlotinib's antitumor effect was evaluated in vivo and vitro. Downstream targets of anlotinib in treating synovial sarcoma were analyzed through microarray assay. Cell proliferation and apoptosis analyses were performed to evaluate the impact of candidate downstream gene depletion in synovial sarcoma cells. Microarray assay were carried out to investigate potential signal network related with candidate downstream gene. RESULTS: Anlotinib significantly suppresses synovial sarcoma proliferation in PDTX model and cell lines. Additionally, GINS1 (also named as PSF1, Partner of SLD Five 1), rather than other conventional gene target, was demonstrated to be a vital target of anlotinib's antitumor effect in synovial sarcoma through microarray assay. Expression of GINS1 was remarkably higher in synovial sarcoma tumor samples and related with poor outcome. Knockdown of GINS1 expression could remarkably inhibit proliferation and promote apoptosis in vitro. Meanwhile, through microarray assay, CITED2, EGR1, SGK1 and SPP1 were identified and further validated by qPCR/WB as downstream targets of GINS1. CONCLUSION: Anlotinib might suppress proliferation of SS through a novel downstream GINS1-regulated network which plays a vital function in SS proliferation and also demonstrated that targeting the GINS1-regulated signal pathway could be a potential strategy for management of SS.

Memory corticalization triggered by REM sleep: mechanisms of cellular and systems consolidation.

Once viewed as a passive physiological state, sleep is a heterogeneous and complex sequence of brain states with essential effects on synaptic plasticity and neuronal functioning. Rapid-eye-movement (REM) sleep has been shown to promote calcium-dependent plasticity in principal neurons of the cerebral cortex, both during memory consolidation in adults and during post-natal development. This article reviews the plasticity mechanisms triggered by REM sleep, with a focus on the emerging role of kinases and immediate-early genes for the progressive corticalization of hippocampus-dependent memories. The body of evidence suggests that memory corticalization triggered by REM sleep is a systemic phenomenon with cellular and molecular causes.

The Host Factor Early Growth Response Gene (EGR-1) Regulates Vaccinia virus Infectivity during Infection of Starved Mouse Cells.

Evolution has equipped poxvirus genomes with the coding capacity for several virus-host interaction products which interfere with host cell gene expression and protein function, creating an adequate intracellular environment for a productive infection. We show here that Vaccinia virus (VACV) induces the expression of the cellular transcription factor EGR-1 (early growth response-1) in Mouse Embryonic Fibroblasts (MEFs) through the MEK (mitogen-activated protein kinase (MAPK)/ERK)/ERK (extracellular signal-regulated kinases) pathway, from 3 to 12 h post infection (h.p.i.). By using starved egr-1 knockout (egr-1-/-) MEFs, we demonstrate that VACV replication is reduced by ~1 log in this cell line. Although western blotting and electron microscopy analyses revealed no difference in VACV gene expression or morphogenesis, the specific infectivity of VACV propagated in egr-1-/- MEFs was lower than virus propagated in wild type (WT) cells. This lower infectivity was due to decreased VACV DNA replication during the next cycle of infection. Taken together, these results revealed that EGR-1 appears to facilitate VACV replication in starved fibroblasts by affecting viral particles infectivity.

c-Jun integrates signals from both MEK/ERK and MKK/JNK pathways upon vaccinia virus infection.

Usurpation of the host's signalling pathways is a common strategy employed by viruses to promote their successful replication. Here we show that infection with the orthopoxvirus vaccinia virus (VACV) leads to sustained stimulation of c-Jun activity during the entire infective cycle. This stimulation is temporally regulated through MEK/ERK or MKK/JNK pathways, i.e. during the early/mid phase (1 to 6 hpi) and in the late phase (9 to 24 hpi) of the infective cycle, respectively. As a transcriptional regulator, upon infection with VACV, c-Jun is translocated from the cytoplasm to the nucleus, where it binds to the AP-1 DNA sequence found at the promoter region of its target genes. To investigate the role played by c-Jun during VACV replication cycle, we generated cell lines that stably express a c-Jun-dominant negative (DNc-Jun) mutation. Our data revealed that c-Jun is required during early infection to assist with viral DNA replication, as demonstrated by the decreased amount of viral DNA found in the DNc-Jun cells. We also demonstrated that c-Jun regulates the expression of the early growth response gene (egr-1), a gene previously shown to affect VACV replication mediated by MEK/ERK signalling. VACV-induced stimulation of the MKK/JNK/JUN pathway impacts viral dissemination, as we observed a significant reduction in both viral yield, during late stages of infection, and virus plaque size. Collectively, our data suggest that, by modulating the host's signalling pathways through a common target such as c-Jun, VACV temporally regulates its infective cycle in order to successfully replicate and subsequently spread.

Environmental enrichment protects against stress-induced anxiety: Role of glucocorticoid receptor, ERK, and CREB signaling in the basolateral amygdala.

Environmental enrichment (EE) is an experimental animal model that enhances an animal's opportunity to interact with sensory, motor, and social stimuli, compared to standard laboratory conditions. A prominent benefit of EE is the reduction of stress-induced anxiety. The relationship between stress and the onset of anxiety-like behavior has been widely investigated in experimental research, showing a clear correlation with structural changes in the hippocampus and basolateral amygdala (BLA). However, the mechanisms by which EE exerts its protective roles in stress and anxiety remain unclear, and it is not known whether EE reduces the effects of acute stress on animal behavior shortly following the cessation of stress. We found that EE can prevent the emergence of anxiety-like symptoms in rats measured immediately after acute restraint stress (1 h) and this effect is not due to changes in systemic release of corticosterone. Rather, we found that stress promotes a rapid increase in the nuclear translocation of glucocorticoid receptor (GR) in the BLA, an effect prevented by previous EE exposure. Furthermore, we observed a reduction of ERK (a MAPK protein) and CREB activity in the BLA promoted by both EE and acute stress. Finally, we found that EE decreases the expression of the immediate-early gene EGR-1 in the BLA, indicating a possible reduction of neuronal activity in this region. Hyperactivity of BLA neurons has been reported to accompany anxiety-like behavior and changes in this process may be one of the mechanism by which EE exerts its protective effects against stress-induced anxiety.

Venezuelan Equine Encephalitis Virus Induces Apoptosis through the Unfolded Protein Response Activation of EGR1.

UNLABELLED: Venezuelan equine encephalitis virus (VEEV) is a previously weaponized arthropod-borne virus responsible for causing acute and fatal encephalitis in animal and human hosts. The increased circulation and spread in the Americas of VEEV and other encephalitic arboviruses, such as eastern equine encephalitis virus and West Nile virus, underscore the need for research aimed at characterizing the pathogenesis of viral encephalomyelitis for the development of novel medical countermeasures. The host-pathogen dynamics of VEEV Trinidad donkey-infected human astrocytoma U87MG cells were determined by carrying out RNA sequencing (RNA-Seq) of poly(A) and mRNAs. To identify the critical alterations that take place in the host transcriptome following VEEV infection, samples were collected at 4, 8, and 16 h postinfection and RNA-Seq data were acquired using an Ion Torrent PGM platform. Differential expression of interferon response, stress response factors, and components of the unfolded protein response (UPR) was observed. The protein kinase RNA-like endoplasmic reticulum kinase (PERK) arm of the UPR was activated, as the expression of both activating transcription factor 4 (ATF4) and CHOP (DDIT3), critical regulators of the pathway, was altered after infection. Expression of the transcription factor early growth response 1 (EGR1) was induced in a PERK-dependent manner. EGR1(-/-) mouse embryonic fibroblasts (MEFs) demonstrated lower susceptibility to VEEV-induced cell death than isogenic wild-type MEFs, indicating that EGR1 modulates proapoptotic pathways following VEEV infection. The influence of EGR1 is of great importance, as neuronal damage can lead to long-term sequelae in individuals who have survived VEEV infection. IMPORTANCE: Alphaviruses represent a group of clinically relevant viruses transmitted by mosquitoes to humans. In severe cases, viral spread targets neuronal tissue, resulting in significant and life-threatening inflammation dependent on a combination of virus-host interactions. Currently there are no therapeutics for infections cause by encephalitic alphaviruses due to an incomplete understanding of their molecular pathogenesis. Venezuelan equine encephalitis virus (VEEV) is an alphavirus that is prevalent in the Americas and that is capable of infecting horses and humans. Here we utilized next-generation RNA sequencing to identify differential alterations in VEEV-infected astrocytes. Our results indicated that the abundance of transcripts associated with the interferon and the unfolded protein response pathways was altered following infection and demonstrated that early growth response 1 (EGR1) contributed to VEEV-induced cell death.

Activation of Tax protein by c-Jun-N-terminal kinase is not dependent on the presence or absence of the early growth response-1 gene product.

The Tax protein of human T cell leukemia virus type 1 plays a major role in the pathogenesis of adult T cell leukemia (ATL), an aggressive neoplasia of CD4+ T cells. In the present study, we investigated whether the EGR-1 pathway is involved in the regulation of Tax-induced JNK expression in human Jurkat T cells transfected to express the Tax protein in the presence or absence of PMA or ionomycin. Overexpression of EGR-1 in Jurkat cells transfected to express Tax, promoted the activation of several genes, with the most potent being those that contained AP-1 (Jun/c-Fos), whereas knockdown of endogenous EGR-1 by small interfering RNA (siRNA) somewhat reduced Tax-mediated JNK-1 transcription. Additionally, luciferase-based AP-1 and NF-κB reporter gene assays demonstrated that inhibition of EGR-1 expression by an siRNA did not affect the transcriptional activity of a consensus sequence of either AP-1 or NF-κB. On the other hand, the apoptosis assay, using all-trans retinoic acid (ATRA) as an inducer of apoptosis, confirmed that siRNA against EGR-1 failed to suppress ATRA-induced apoptosis in Jurkat and Jurkat-Tax cells, as noted by the low levels of both DEVDase activity and DNA fragmentation, indicating that the induction of apoptosis by ATRA was Egr-1-independent. Finally, our data showed that activation of Tax by JNK-1 was not dependent on the EGR-1 cascade of events, suggesting that EGR-1 is important but not a determinant for the activity for Tax-induced proliferation of Jurkat cells.

Interdependent adrenergic receptor regulation of Arc and Zif268 mRNA in cerebral cortex.

Norepinephrine is a neurotransmitter that signals by stimulating the α1, α2 and ß adrenergic receptor (AR). We determined the role of these receptors in regulating the immediate early genes, Activity Regulated Cytoskeleton Associated Protein (Arc) and Zif268 in the rat cerebral cortex. RX821002, an α2-AR antagonist, produced Arc and Zif268 elevations across cortical layers. Next we examined the effects of delivering RX821002 with an α1-AR antagonist, prazosin, and a ß-AR antagonist, propranolol. RX821002 given with a prazosin and propranolol cocktail, or with each of these antagonists individually, decreased Arc and Zif268 to saline-treated control levels in most cortical layers. Arc and Zif268 levels were also similar to saline-treated control levels when rats were given a prazosin and propranolol cocktail alone, or when each of these antagonists were delivered individually. Taken together, these data reveal that α2-AR uniquely exert a tonic inibitory regulation of both Arc and Zif268 compared to α1 and ß-AR. However, the ability of RX821002 to increase Arc and Zif268 is interdependent with α1 and ß-AR signaling.

Prevalência de dor crônica e sua associação com a situação sociodemográfica e atividade física no lazer em idosos de Florianópolis, Santa Catarina: estudo de base populacional / Prevalence of chronic pain and its association with the sociodemographic situation and physical activity in leisure of elderly in Florianópolis, Santa Catarina: population-based study

OBJETIVO: Estimar a prevalência de dor crônica e sua associação com a situação socioeconômica, demográfica e atividade física no lazer em idosos. MÉTODOS: Este estudo é parte do inquérito epidemiológico e transversal de base populacional e domiciliar EpiFloripa Idoso 2009-2010 realizado com 1.705 idosos (≥ 60 anos), residentes em Florianópolis, Santa Catarina. A partir da resposta afirmativa de dor crônica, foram investigadas as associações com as variáveis obtidas por meio de entrevista estruturada. Realizou-se a estatística descritiva, incluindo cálculos de proporções e intervalos de confiança 95% (IC95%). Na análise bruta e ajustada, empregou-se regressão de Poisson, estimando-se as razões de prevalência, com intervalos de confiança de 95% e valores p ≤ 0,05. RESULTADOS: Dentre os idosos investigados, 29,3% (IC95% 26,5 - 32,2) relataram dor crônica. Na análise ajustada, observou-se que as variáveis sexo feminino, menor escolaridade e pior situação econômica ficaram associadas significativamente com maior prevalência de dor crônica; ser fisicamente ativo no lazer ficou associado significativamente com menor prevalência do desfecho. CONCLUSÕES: Percebe-se que a dor crônica é um agravo que acomete considerável parcela de idosos, havendo desigualdades sociais na sua frequência e sendo beneficamente afetada pela atividade física no lazer. É necessário que políticas públicas de saúde subsidiem programas multidisciplinares de controle da dor incluindo a prática regular de atividade física, voltada especificamente à promoção da saúde do idoso, evitando assim que a dor crônica comprometa a qualidade de vida desta população. .

OBJECTIVE: To estimate the prevalence of chronic pain and its association with socioeconomic and demographic status, and leisure physical activity in the elderly population. METHODS: This study is part of an epidemiological cross-sectional population-based household survey called EpiFloripa Elderly 2009-2010, which was conducted with 1,705 elderly individuals (≥ 60 years) residents of Florianópolis, Santa Catarina. From the positive response to chronic pain, the associations with the variables were investigated through a structured interview. Descriptive statistics were conducted, including ratio calculation and 95% confidence intervals. In crude and adjusted analysis, Poisson regression was utilized, estimating prevalence ratios, with 95% confidence intervals and ≤ 0.05 p-values. RESULTS: Among the subjects, 29.3% (IC95% 26.5 - 32.2) reported chronic pain. Adjusted analysis showed that being female, having less years of schooling, and being in worse economic situation were significantly associated with a higher prevalence of chronic pain. Being physically active during leisure time was significantly associated with lower prevalence of the outcome. CONCLUSIONS: Therefore, it is clear that chronic pain affects a considerable amount of elderly individuals. Social inequalities are a harmful influence in these individuals' quality of life, inasmuch as those inequalities increase the frequency with which chronic pain afflicts them. At the same time, physical activity during leisure time decreases chronic pain frequency. It is fundamental that public health policies subsidize multidisciplinary pain management programs, which should include health targeted physical activity for the elderly, thus preventing the decrease in quality of life that chronic pain brings to this population. .

Interleukin-1ß-induced memory reconsolidation impairment is mediated by a reduction in glutamate release and zif268 expression and α-melanocyte-stimulating hormone prevented these effects.

The immune system is an important modulator of learning, memory and neural plasticity. Interleukin 1ß (IL-1ß), a pro-inflammatory cytokine, significantly affects several cognitive processes. Previous studies by our group have demonstrated that intrahippocampal administration of IL-1ß impairs reconsolidation of contextual fear memory. This effect was reversed by the melanocortin alpha-melanocyte-stimulating hormone (α-MSH). The mechanisms underlying the effect of IL-1ß on memory reconsolidation have not yet been established. Therefore, we examined the effect of IL-1ß on glutamate release, ERK phosphorylation and the activation of the transcription factor zinc finger- 268 (zif268) during reconsolidation. Our results demonstrated that IL-1ß induced a significant decrease of glutamate release after reactivation of the fear memory and this effect was related to calcium concentration in hippocampal synaptosomes. IL-1ß also reduced ERK phosphorylation and zif268 expression in the hippocampus. Central administration of α-MSH prevented the decrease in glutamate release, ERK phosphorylation and zif268 expression induced by IL-1ß. Our results establish possible mechanisms involved in the detrimental effect of IL-1ß on memory reconsolidation and also indicate that α-MSH may exert a beneficial modulatory role in preventing IL-1ß effects.

Association of increased levels of TGF-ß1 and p14ARF in prostate carcinoma cell lines overexpressing Egr-1.

The present study examined the effect of the overexpression of early growth response gene (Egr-1) on transforming growth factor ß-1 (TGF-ß1) and p14ARF levels, in PC-3 and LNCaP prostate carcinoma cell lines. Amplification of EGR-1, TGF-ß1 and p14ARF were observed in the two cell lines treated with different stimuli and resulted in a corresponding mRNA and protein expression. The downregulation of TGF-ß1 and the attenuation of p14ARF expression by siRNA against Egr-1 predominantly suggested that TGF-ß1 and p14ARF may be regulated by the transcription factor EGR-1. A marginal attenuation of cell growth in PC-3 and LNCaP prostate carcinoma cell lines overexpressing p14ARF was observed. Cells transfected with Egr-1 wild-type were able to grow and avoid cell cycle arrest and apoptosis in the presence or absence of p14ARF. In addition, EGR-1 stimulated the expression of TGF ß-l as well as the accumulation of the p14ARF proteins. The results suggested that TGF-ß1 and p14ARF activities in the presence of EGR-1 overexpression can exist independently of the presence of cells carrying a mutant p53 (PC-3 cells) or cells carrying a wild­type p53 (LNCaP cells). Thus, the effect of EGR-1 on the growth of prostate carcinoma cells may occur through multiple mechanisms, but be independent of p53 expression control.

Decreased c-Abl activity in PC-3 and LNCaP prostate cancer cells overexpressing the early growth response-1 protein.

Early growth response-1 (Egr-1) and the non-receptor protein tyrosine kinase (c-Abl) are 2 response genes that can act as regulators of cell growth and apoptosis in response to stress. Both Egr-1 and c-Abl regulate cell proliferation and survival in different types of cancer cells. To study the effect of overexpression of EGR-1 on the activity of c-Abl in prostate cancer cells, human PC-3 and LNCaP cells were transfected with a control vector or a vector containing the murine Egr-1 cDNA and assessed for the expression of the c-Abl gene. Cells overexpressing Egr-1 were studied with respect to apoptosis (Annexin V)/DEVDase activity, Egr-1/c-Abl activation (western blotting) and cell proliferation (MTT assay). The cells were exposed to tumor necrosis factor α (TNF-α), a known inductor of Egr-1, to c-Abl inhibitor STI-571 and to small interfering RNA (siRNA)-Egr-1, respectively. The results from our studies strongly suggest that overexpression of Egr-1 decreased c-Abl activity independent of endogenous Egr-1 inhibition by siRNA-Egr-1.

Calcitriol supplementation effects on Ki67 expression and transcriptional profile of breast cancer specimens from post-menopausal patients.

BACKGROUND & AIMS: High concentration of 1,25(OH)2D3 (50-100 nM), which cause hypercalcemia in vivo, induce the hormone transcriptional targets and exert antiproliferative effects in cultured breast cancer lineages, however, no studies investigated whether these effects might be reproduced in tumor specimens in vivo. Our aim was to evaluate the effects of calcitriol supplementation on the proliferative index (Ki67 expression) and gene expression profile of post-menopausal breast cancer samples. METHODS & RESULTS: Tumor samples were collected from 33 patients, most of whom (87.5%) presenting 25(OH)D3 insufficiency, before and after a short term calcitriol supplementation (0.50 µg/day PO, for 30 days). Tumor dimension remained stable in ultrasound evaluations. A slight reduction in Ki67 immunoexpression was detected, however in only 10/32 post-calcitriol samples an expressively low proliferative index [Ln (%Ki67+) < 1] was achieved. Gene expression from 15 matched pre/post-supplementation samples was analyzed by microarray (U133 Plus 2.0 GeneChip, Affymetrix) and 15 genes were over-expressed in post-supplementation tumors, including FOS and EGR1, which were previously shown to be regulated by vitamin D. However, these results were not confirmed in another four breast cancer samples. CONCLUSIONS: Calcitriol supplementation is neither sufficient to expressively elicit an antiproliferative response nor to induce the hormone transcriptional signaling pathway in breast cancer specimens.

Increased expression of p21Waf1/Cip1 and JNK with costimulation of prostate cancer cell activation by an siRNA Egr-1 inhibitor.

The p21Waf1/Cip1 protein (hereafter, p21) and the c­Jun N-terminal kinase (JNK) are two well-characterized cell modulators that play a crucial role in cell differentiation, senescence and apoptosis. Here, we report that transcription of the p21Waf1/Cip1 and JNK-1 genes is affected by inhibition of the early growth response-1 (Egr-1) in response to a small interfering RNA [siRNA)-Egr-1] in LNCaP and PC-3 prostate carcinoma cell lines. The expression levels of protein were determined by western blotting, and apoptosis was measured by propidium iodide staining and flow cytometric analysis. Inhibition of Egr-1, p21 and JNK-1 was carried out by siRNAs. LNCaP and PC-3 cells exhibited readily detectable Egr-1, JNK and p21, even in low serum medium without the addition of other exogenous agents. The expression of Egr-1, p21 and JNK was strongly increased after treatment of the cells with TPA, tumor necrosis factor-α (TNF-α) or arsenite. Suppression of Egr-1 expression by siRNA abrogated the ability of TPA to induce Egr-1 and JNK-1 activities, moderately increasing the p21 activity and abrogating the anti-apoptotic effect of Egr-1 observed in the prostate cancer cell lines. Moreover, blockade of p21 and JNK was unable to decrease the activity of Egr-1, while siRNA against p21 abrogated the pro­apoptotic effect of p21. The results demonstrated that Egr-1 acts as a key player in prostate tumor cell growth and survival, while p21 plays a key pro­apoptotic role in LNCaP and PC-3 prostate carcinoma cell lines.

Maintenance of long-term memory storage is dependent on late posttraining Egr-1 expression.

Expression of immediate-early genes, like Egr-1, has been shown to be induced by activity-dependent synaptic plasticity or behavioral training and is widely thought to play an important role in long-term memory (LTM) formation. However, little is known about the role of Egr-1 in the maintenance of memory storage. Here we show that dorsal hippocampal Egr-1 protein expression is upregulated between 12 and 24 h after strong inhibitory avoidance (IA) training in rats. Local infusion of antisense oligodeoxynucleotide (ASO) to specifically knockdown Egr-1 in the dorsal hippocampus 8 h posttraining impairs LTM tested 7 days, but not 1 day after training, indicating that a delayed learning-associated expression of Egr-1 is necessary for the persistence of LTM storage. In addition, we show that consolidation of the IA memory is accompanied by an increase in Egr-1 protein levels 3 h, but not immediately or 1 h after training. Local infusion of egr-1 ASO 30 min before training in the dorsal hippocampus persistently hinders memory formation measured 1 and 7 days after IA training, indicating the crucial role of Egr-1 in memory formation. Our findings demonstrate that there are at least two waves of Egr-1 expression in the dorsal hippocampus after IA training, an early wave which is involved in IA LTM formation, and a lasting late wave that peaks around 12-24 h after a strong training protocol which is specifically involved in the maintenance of LTM storage.

Timed maternal melatonin treatment reverses circadian disruption of the fetal adrenal clock imposed by exposure to constant light.

Surprisingly, in our modern 24/7 society, there is scant information on the impact of developmental chronodisruption like the one experienced by shift worker pregnant women on fetal and postnatal physiology. There are important differences between the maternal and fetal circadian systems; for instance, the suprachiasmatic nucleus is the master clock in the mother but not in the fetus. Despite this, several tissues/organs display circadian oscillations in the fetus. Our hypothesis is that the maternal plasma melatonin rhythm drives the fetal circadian system, which in turn relies this information to other fetal tissues through corticosterone rhythmic signaling. The present data show that suppression of the maternal plasma melatonin circadian rhythm, secondary to exposure of pregnant rats to constant light along the second half of gestation, had several effects on fetal development. First, it induced intrauterine growth retardation. Second, in the fetal adrenal in vivo it markedly affected the mRNA expression level of clock genes and clock-controlled genes as well as it lowered the content and precluded the rhythm of corticosterone. Third, an altered in vitro fetal adrenal response to ACTH of both, corticosterone production and relative expression of clock genes and steroidogenic genes was observed. All these changes were reversed when the mother received a daily dose of melatonin during the subjective night; supporting a role of melatonin on overall fetal development and pointing to it as a 'time giver' for the fetal adrenal gland. Thus, the present results collectively support that the maternal circadian rhythm of melatonin is a key signal for the generation and/or synchronization of the circadian rhythms in the fetal adrenal gland. In turn, low levels and lack of a circadian rhythm of fetal corticosterone may be responsible of fetal growth restriction; potentially inducing long term effects in the offspring, possibility that warrants further research.

Inhibition of Egr-1 by siRNA in prostate carcinoma cell lines is associated with decreased expression of AP-1 and NF-κB.

Previous studies suggest that the effects of Egr-1 on tumorigenesis are critically dependent on the levels of Egr-1 and the cellular context. For this reason, we examined the effects of blocking the Egr-1 activity by a short interfering RNA (siRNA) against Egr-1 on the expression of nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) signaling in the PC-3 and LNCaP prostate carcinoma cell lines. We observed that the reduction in expression of Egr-1 in PC-3 and LNCaP cells by effects of the siRNA vector resulted in lower cell viability and increased apoptosis at 24 and 120 h after transfection. This reduced cell viability correlated well with a reduced activity of the NF-κB and AP-1 factors. We analyzed the expression and activity of these factors and found that p65 and IκB but not p50 were reduced in the siRNA-treated cells. Similarly, we found that c-Fos but not c-Jun was reduced in the siRNA treated cells. These effects were translated to reduced transcriptional activity of NF-κB over cellular inhibitor of apoptosis (cIAP) and p21 genes, as assayed by RT-PCR and of AP-1 over a luciferase reporter activated by AP-1 response elements. Egr-1 was also found to interact directly with p65 and IκB members of the NF-κB family, thereby was able to regulate directly their activity by post-transcriptional effects. These results suggest that Egr-1 may promote prostate cancer development by modulating the activity of factors NF-κB and AP-1, which are involved in cell proliferation and apoptosis.