Opposing Action Research Articles

Opposing effects of the purinergic P2X7 receptor on seizures in neurons and microglia in male mice

BackgroundThe purinergic ATP-gated P2X7 receptor (P2X7R) is increasingly recognized to contribute to pathological neuroinflammation and brain hyperexcitability. P2X7R expression has been shown to be increased in the brain, including both microglia and neurons, in experimental models of epilepsy and patients. To date, the cell type-specific downstream effects of P2X7Rs during seizures remain, however, incompletely understood. MethodsEffects of P2X7R signaling on seizures and epilepsy were analyzed in induced seizure models using male mice including the kainic acid model of status epilepticus and pentylenetetrazole model and in male and female mice in a genetic model of Dravet syndrome. RNA sequencing was used to analyze P2X7R downstream signaling during seizures. To investigate the cell type-specific role of the P2X7R during seizures and epilepsy, we generated mice lacking exon 2 of the P2rx7 gene in either microglia (P2rx7:Cx3cr1-Cre) or neurons (P2rx7:Thy-1-Cre). To investigate the protective potential of overexpressing P2X7R in GABAergic interneurons, P2X7Rs were overexpressed using adeno-associated virus transduction under the mDlx promoter. ResultsRNA sequencing of hippocampal tissue from wild-type and P2X7R knock-out mice identified both glial and neuronal genes, in particular genes involved in GABAergic signaling, under the control of the P2X7R following seizures. Mice with deleted P2rx7 in microglia displayed less severe acute seizures and developed a milder form of epilepsy, and microglia displayed an anti-inflammatory molecular profile. In contrast, mice lacking P2rx7 in neurons showed a more severe seizure phenotype when compared to epileptic wild-type mice. Analysis of single-cell expression data revealed that human P2RX7 expression is elevated in the hippocampus of patients with temporal lobe epilepsy in excitatory and inhibitory neurons. Functional studies determined that GABAergic interneurons display increased responses to P2X7R activation in experimental epilepsy. Finally, we show that viral transduction of P2X7R in GABAergic interneurons protects against evoked and spontaneous seizures in experimental temporal lobe epilepsy and in mice lacking Scn1a, a model of Dravet syndrome. ConclusionsOur results suggest a dual and opposing action of P2X7R in epilepsy and suggest P2X7R overexpression in GABAergic interneurons as a novel therapeutic strategy for acquired and, possibly, genetic forms of epilepsy.

A Versatile Assembly Approach toward Multifunctional Supramolecular Poly(Ionic Liquid) Nanoporous Membranes in Water.

Hydrogen (H)-bonding-integration of multiple ingredients into supramolecular polyelectrolyte nanoporous membranes in water, thereby achieving tailor-made porous architectures, properties, and functionalities, remains one of the foremost challenges in materials chemistry due to the significantly opposing action of water molecules against H-bonding. Herein, a strategy is described that allows direct fusing of the functional attributes of small additives into water-involved hydrogen bonding assembled supramolecular poly(ionic liquid) (PIL) nanoporous membranes (SPILMs) under ambient conditions. It discloses that the pore size distributions and mechanical properties of SPILMs are rationally controlled by tuning the H-bonding interactions between small additives and homo-PIL. It demonstrates that, benefiting from the synergy of multiple noncovalent interactions, small dye additives/homo-PIL solutions can be utilized as versatile inks for yielding colorful light emitting films with robust underwater adhesion strength, excellent stretchability, and flexibility on diverse substrates, including both hydrophilic and hydrophobic surfaces. This system provides a general platform for integrating the functional attributes of a diverse variety of additives into SPILMs to create multifunctional and programmable materials in water.

Managing shame and guilt in addiction: A pathway to recovery

A dominant view of guilt and shame is that they have opposing action tendencies: guilt- prone people are more likely to avoid or overcome dysfunctional patterns of behaviour, making amends for past misdoings, whereas shame-prone people are more likely to persist in dysfunctional patterns of behaviour, avoiding responsibility for past misdoings and/or lashing out in defensive aggression. Some have suggested that addiction treatment should make use of these insights, tailoring therapy according to people’s degree of guilt-proneness versus shame-proneness. In this paper, we challenge this dominant view, reviewing empirical findings from others as well as our own to question (1) whether shame and guilt can be so easily disentangled in the experience of people with addiction, and (2) whether shame and guilt have the opposing action tendencies standardly attributed to them. We recommend a shift in theoretical perspective that explains our main finding that both emotions can be either destructive or constructive for recovery, depending on how these emotions are managed. We argue such management depends in turn on a person’s quality of self-blame (retributive or ‘scaffolding’), impacting upon their attitude towards their own agency as someone with fixed and unchanging dispositions (shame and guilt destructive for recovery) or as someone capable of changing themselves (shame and guilt productive for recovery). With an eye to therapeutic intervention, we then explore how this shift in attitude towards the self can be accomplished. Specifically, we discuss empathy-driven affective and narratively-driven cognitive components of a process that allow individuals to move away from the register of retributive self-blame into a register of scaffolding ‘reproach’, thereby enabling them to manage their experiences of both shame and guilt in a more generative way.

Valeriana officinalis Counteracts Rotenone Effects on Spreading Depression in the Rat Brain in vivo and Protects Against Rotenone Cytotoxicity Toward Rat Glioma C6 Cells in vitro.

Astrocytes can protect neurons against oxidative stress and excitability-dependent disorders, such as epilepsy. Valeriana officinalis has been used as anticonvulsant and can exert an antioxidant effect, which may underlie its opposing action against the toxic effects of the pesticide rotenone. We investigated the V. officinalis/rotenone interaction in the cortical spreading depression (CSD), a phenomenon that depends upon brain excitability (in vivo model). In addition, we analyzed the protective action of V. officinalis against the cytotoxic effects of rotenone in cultures of rat C6 glioma cells (in vitro model). For the CSD study, Wistar rats received either V. officinalis (250 mg/kg/day via gavage for 15 days; n = 8) or 10 mg/kg/day rotenone via subcutaneous injections for 7 days (n = 7), or they received both substances (n = 5). Two control groups received either saline (vehicle for V. officinalis; n = 8) or 1% Tween-80 aqueous solution (vehicle for rotenone; n = 9). After treatment, CSD was recorded for 4 h. The rotenone- and V. officinalis-treated groups presented, respectively, with lower (2.96 ± 0.14 mm/min), and higher CSD propagation velocity (3.81 ± 0.10 mm/min) when compared with the controls (Tween-80, 3.37 ± 0.06 mm/min and saline, 3.35 ± 0.08 mm/min; p < 0.05). The rotenone plus V. officinalis-treated group displayed a CSD velocity (3.38 ± 0.07 mm/min) that was similar to controls. In line with these results, in vitro experiments on rat glioma C6 cells revealed a protective effect (MTT assay) of V. officinalis against rotenone-induced cytotoxicity. These results suggest the therapeutic potential of V. officinalis for treating neurological diseases involving redox imbalance and astrocyte dysfunction.

Probabilistic reversal learning under acute tryptophan depletion in healthy humans: a conventional analysis.

The involvement of serotonin in responses to negative feedback is well established. Acute serotonin reuptake inhibition has enhanced sensitivity to negative feedback (SNF), modelled by behaviour in probabilistic reversal learning (PRL) paradigms. Whilst experiments employing acute tryptophan depletion (ATD) in humans, to reduce serotonin synthesis, have shown no clear effect on SNF, sample sizes have been small. We studied a large sample of healthy volunteers, male and female, and found ATD had no effect on core behavioural measures in PRL. These results indicate that ATD effects can differ from other manipulations of serotonin expected to have a parallel or opposing action.

STRATEGIC ACCORD AND TENSION FOR BUSINESS MODEL INNOVATION: EXAMINING DIFFERENT TACIT KNOWLEDGE TYPES AND OPEN ACTION STRATEGIES

Both academics and practitioners are underlining the relevance of business model innovation in unlocking lasting, competitive advantages. To understand how business model innovation is achieved in the team level and which contingency factors drive or inhibit the process, this study scrutinizes the strategic accord and tension of analogous and opposing action strategies, as potential contingency factors controlling and balancing the effects of external and internal tacit knowledge on business model innovation. Using a research sample of 162 team projects, our empirical study supported the research hypotheses regarding how tacit knowledge fuels business model innovation, highlighting differed moderating roles of three open action strategies (team autonomy, team creativity, and team diversity) on the relationship between two transferred knowledge types and business model innovation of technology-intensive companies. This study shed light on how team management strategies, namely manifestations of open action strategies, moderate the effectiveness of tacit knowledge on business model innovation.

Foxo1 Corepressor (FCoR) and Foxo1 Regulate a-Cell and ß-Cell Identity

We previously identified a Foxo1 binding protein; Foxo1 CoRepressor (FCoR), in adipocytes. FCoR inhibits Foxo1 activity by enhancing its acetylation. FCoR is expressed in pancreatic α- and β-cells from the embryonic stage. Fcor knockout mice (FcorKO) show impaired glucose tolerance, decreased insulin secretion, increased glucagon secretion and increased α-cell mass. We have shown that FCoR suppresses the expression of master α-cell regulatory transcription factor Aristaless-related homeobox (Arx) by increasing the methylation of the CpG-rich promoter region, while Foxo1 induces Arx expression by hypomethylation of this area. These results suggest that FCoR and Foxo1 have an opposing action in the regulation of Arx expression and α-cell mass. We studied how Foxo1 regulates Arx expression. Arx promoter region contains a conserved forkhead responsive element (FRE). Foxo1 binds to the FRE and induces Arx expression. Binding of Foxo1 to the Arx promoter dissociates binding of DNA methyltransferase 3a (Dnmt3a), and this may result in hypomethylation of the CpG rich region. Our results suggest that Arx is the target gene of Foxo1. Immunohistochemical staining of FcorKO islets showed Foxo1 nuclear localization and decreased Foxo1 in the cytosol. In isolated islets from FcorKO, we observed significant deacetylation of Foxo1, and decreased endogenous Foxo1 protein compared to control. These results are consistent with the previous finding that deacetylated Foxo1 is the active form, and is quickly ubiquitinated and degraded. To study the relationship between FCoR and Foxo1, we generated β-cell-specific Foxo1 knockout mice in the background of FcorKO (DKO). DKO showed further aggravation of glucose tolerance and insulin secretion compared to FcorKO. Foxo1 single knockout has normal glucose metabolism, thus, our results suggest that Foxo1 is activated to maintain β-cell function in FcorKO. We provide evidence that FCoR and Foxo1 function in harmonization to regulate pancreatic α-cell and β-cell identity. Disclosure N. Kodani: None. J. Nakae: None. H. Itoh: None.

Relativistic magnetised perturbations: magnetic pressure versus magnetic tension

We study the linear evolution of magnetised cosmological perturbations in the post-recombination epoch. Using full general relativity and adopting the ideal magnetohydrodynamic approximation, we refine and extend the previous treatments. More specifically, this is the first relativistic study that accounts for the effects of the magnetic tension, in addition to those of the field’s pressure. Our solutions show that on sufficiently large scales, larger than the (purely magnetic) Jeans length, the perturbations evolve essentially unaffected by the magnetic presence. The magnetic pressure dominates on small scales, where it forces the perturbations to oscillate and decay. Close to the Jeans length, however, the field’s tension takes over and leads to a weak growth of the inhomogeneities. These solutions clearly demonstrate the opposing action of the aforementioned two magnetic agents, namely of the field’s pressure and tension, on the linear evolution of cosmological density perturbations.

Investigation of mechanisms involved in regulation of progesterone catabolism using an overfed versus underfed ewe-lamb model.

Alterations in progesterone (P4) catabolism due to high feed intake underlie some effects of nutrition on reproduction. Based on previous research, we hypothesized that high feed intake could potentially increase P4 catabolism, likely due to increased liver blood flow. However, there could also be an opposing action due to increased circulating insulin, which has been shown to inhibit hepatic expression of key enzymes involved in P4 catabolism. To test which effect would have the greatest impact on circulating P4 during a 1- and 2 -mo time frame, we used a noncyclic ewe model. The plane of nutrition was controlled, and effects on circulating insulin, P4 catabolism in response to exogenous P4, and steady state mRNA for key hepatic enzymes were evaluated. Twenty-four F Dorper × Santa Inês ewe lambs (5 mo old and approximately 25 kg BW) were used. After 14 d of adaptation, ewes were randomized into 2 groups: ad libitum fed (Ad), with intake of 3.8% DM/kg BW, or restricted feed intake (R), with 2% DM/kg BW, for 8 wk. At wk 4 and 8, ewes received an intravaginal P4 implant to evaluate P4 catabolism. As designed, Ad ewes had greater daily feed intake than R ewes (means of 1.8 [SE 0.03] and 0.6 kg/ewe [SE 0.01]; < 0.001) and greater weekly gain in BW (means of 1.7 [SE 0.12] vs. -0.1 kg/ewe [SE 0.03]; < 0.001). Mean circulating insulin of samples collected from -0.5 to 7 h after the start of feeding was over 5-fold greater in Ad ewes than in R ewes (least squares means of 8.2 [SE 0.93] vs. 1.5 μIU/mL [SE 0.16], respectively, at wk 4 and 12.0 [SE 1.02] vs. 2.2 μIU/mL [SE 0.18], respectively, at wk 8; < 0.001). Although both groups received the same P4 treatment, mean circulating P4 of samples collected from -0.5 to 7 h after feeding was much lower in Ad ewes than in R ewes (least squares means of 3.2 [SE 0.32] vs. 5.5 ng/mL [SE 0.32], respectively, at wk 4 and 2.8 [SE 0.28] vs. 5.2 ng/mL [SE 0.28], respectively, at wk 8; < 0.001) indicating much greater P4 catabolism in ewes with high feed intake. Unexpectedly, there was no effect of diet on hepatic mRNA concentrations for , , , or at wk 4 or 8 in spite of dramatically elevated insulin. Therefore, high energy/feed intake primarily increased P4 catabolism with no evidence for offsetting effects due to insulin-induced changes in hepatic P4 metabolizing enzymes.

Metformin augments doxorubicin cytotoxicity in mammary carcinoma through activation of adenosine monophosphate protein kinase pathway.

Since the incidence of breast cancer increases dramatically all over the world, the search for effective treatment is an urgent need. Metformin has demonstrated anti-tumorigenic effect both in vivo and in vitro in different cancer types. This work was designed to examine on molecular level the mode of action of metformin in mice bearing solid Ehrlich carcinoma and to evaluate the use of metformin in conjunction with doxorubicin as a combined therapy against solid Ehrlich carcinoma. Ehrlich ascites carcinoma cells were inoculated in 60 female mice as a model of breast cancer. The mice were divided into four equal groups: Control tumor, metformin, doxorubicin, and co-treatment. Metformin (15 mg/kg) and doxorubicin (4 mg/kg) were given intraperitoneally (i.p.) for four cycles every 5 days starting on day 12 of inoculation. The anti-tumorigenic effect of metformin was mediated by enhancement of adenosine monophosphate protein kinase activity and elevation of P53 protein as well as the suppression of nuclear factor-kappa B, DNA contents, and cyclin D1 gene expression. Metformin and doxorubicin mono-treatments exhibited opposing action regarding cyclin D1 gene expression, phosphorylated adenosine monophosphate protein kinase, and nuclear factor-kappa B levels. Co-treatment markedly decreased tumor volume, increased survival rate, and improved other parameters compared to doxorubicin group. In parallel, the histopathological findings demonstrated enhanced apoptosis and absence of necrosis in tumor tissue of co-treatment group. Metformin proved chemotherapeutic effect which could be mediated by the activation of adenosine monophosphate protein kinase and related pathways. Combining metformin and doxorubicin, which exhibited different mechanisms of action, produced greater efficacy as anticancer therapeutic regimen.

Methylosome Protein 50 and PKCδ/p38δ Protein Signaling Control Keratinocyte Proliferation via Opposing Effects on p21Cip1 Gene Expression

Protein arginine methyltransferase 5 (PRMT5) is a key epigenetic regulator that symmetrically dimethylates arginine residues on histones H3 and H4 to silence gene expression. PRMT5 is frequently observed in a complex with the cofactor methylosome protein 50 (MEP50), which is required for PRMT5 activity. PKCδ/p38δ signaling, a key controller of keratinocyte proliferation and differentiation, increases p21(Cip1) expression to suppress keratinocyte proliferation. We now show that MEP50 enhances keratinocyte proliferation and survival via mechanisms that include silencing of p21(Cip1) expression. This is associated with enhanced PRMT5-MEP50 interaction at the p21(Cip1) promoter and enhanced arginine dimethylation of the promoter-associated histones H3 and H4. It is also associated with a MEP50-dependent reduction in the level of p53, a key controller of p21(Cip1) gene expression. We confirm an important biological role for MEP50 and PRMT5 in regulating keratinocyte proliferation using a stratified epidermal equivalent model that mimics in vivo epidermal keratinocyte differentiation. In this model, PRMT5 or MEP50 knockdown results in reduced keratinocyte proliferation. We further show that PKCδ/p38δ signaling suppresses MEP50 expression, leading to reduced H3/H4 arginine dimethylation at the p21(Cip1) promoter, and that this is associated with enhanced p21(Cip1) expression and reduced cell proliferation. These findings describe an opposing action between PKCδ/p38δ MAPK signaling and PRMT5/MEP50 epigenetic silencing mechanisms in regulating cell proliferation.

Opposing Role of NMDA Receptor GluN2B and GluN2D in Somatosensory Development and Maturation

Development of correct topographical connections between peripheral receptors and central somatosensory stations requires activity-dependent synapse refinement, in which the NMDA type of glutamate receptors plays a key role. Here we compared functional roles of GluN2B (GluRε2 or NR2B) and GluN2D (GluRε4 or NR2D), two major regulatory subunits of neonatal NMDA receptors, in development of whisker-related patterning at trigeminal relay stations. Compared with control littermates, both the appearance of whisker-related patterning and the termination of the critical period, as assessed by unilateral infraorbital nerve transection, were delayed by nearly a day in the somatosensory cortex of GluN2B(+/-) mice but advanced by nearly a day in GluN2D(-/-) mice. Similar temporal shifts were found at subcortical relay stations in the thalamus and brainstem of GluN2B(+/-) and GluN2D(-/-) mice. In comparison, the magnitude of lesion-induced critical period plasticity in the somatosensory cortex, as assessed following row-C whisker removal, was normal in both mutants. Thus, GluN2B and GluN2D play counteractive roles in temporal development and maturation of somatosensory maps without affecting the magnitude of critical period plasticity. To understand the opposing action, we then examined neuronal and synaptic expressions of the two subunits along the trigeminal pathway. At each trigeminal station, GluN2B was predominant at asymmetrical synapses of non-GABAergic neurons, whereas GluN2D was selective to asymmetrical synapses of GABAergic neurons. Together, our findings suggest that GluN2B expressed at glutamatergic synapses on glutamatergic projection neurons facilitates refinement of ascending pathway synapses directly, whereas GluN2D expressed at glutamatergic synapses on GABAergic interneurons delays it indirectly.

Understanding the Cost of Conflict and an Approach to Conflict-Management Design

INTRODUCTION Conflict is inevitable in human interactions. Its Latin stem, conflictus, means “the act of striking together.” It is defined as “competitive or opposing action of incompatibles: antagonistic state or action (as of divergent ideas, interests, or persons)” [1]. More than simple disagreement, features of conflict include intense negative feelings, poor communication, and active efforts to hinder the other side. Although not always apparent, the cost of conflict within organizations can be massive. Hidden costs include lost productivity from job dissatisfaction and stress, employee or faculty turnover, and time spent on conflict management. One study found that in 2008 in the United States, workers spent 2.8 hours per week dealing with conflict-related issues, which translated to a $359 billion loss [2]. Additional hidden costs are the lost collaboration due to failed relationships, weakened organizational effectiveness due to inadequate conflict management, and an increase in patient-care errors [3]. Considering the high costs, it would

Baroreceptor and Chemoreceptor Reflex Contribution in Cardiovascular Changes during Exercise

During exercise, both baroreceptors and chemoreceptors contribute in bringing about the cardiovascular changes, but less is documented about the contribution made by either of these mechanisms. The purpose of the present study was to demonstrate the differential effects of baroreceptors and chemoreceptors on cardiovascular parameters during exercise. Twelve healthy individuals were subjected to exercise maneuver on stationary cycling machine. Systolic blood pressure (SBP), diastolic blood pressure (DBP) and electrocardiogram (ECG) were recorded during rest and immediately after exercise. Heart rate (HR) and heart rate variability (HRV) was computed from ECG. SBP, DBP, mean arterial blood pressure (MABP) and HR significantly increased due to exercise. Increase in SBP, DBP, Pulse pressure (PP), MABP and HR were more pronounced when chemoreceptor influence was not considered as against when changes due to both baroreceptors and chemoreceptors were considered. The HRV analysis reflected increase in the low-frequency (LF) power % and decrease in high-frequency (HF) power % along with reduction in LF/HF ratio due to exercise, but these changes were statistically not significant. Baroreceptors through baroreflex mechanism overall increase the CVS parameters. The novel finding in this study was the opposing action of chemoreceptor mechanism to that of baroreceptor mechanism in cardiovascular homeostasis during exercise.

24 A novel anti-hypertrophic pathway in cardiomyocytes regulated by the tumour suppressor mammalian STE20-like kinase 1 (Mst1)

The mammalian STE20-like kinase 1 (Mst1) is a pro-apoptotic kinase implicated in organ size control and tumour suppression. We have recently discovered that in the heart tumour suppressors may act...

The life instinct

In psychoanalytic writing an oversimplified interpretation of Freud’s concept of the life and death instincts sometimes colours the presentation. Roughly, there is an implication that the life instinct is ‘good’ and the death instinct ‘bad’. Freud however is clear that: “Neither of these instincts is any less essential than the other; the phenomena of life arise from the concurrent or mutually opposing action of both“(1933b, p. 209). In this paper I look in detail at the characteristics of the life instinct as conceptualized by Freud, and draw on Bion’s work ‘on linking’ to elaborate Freud’s view that binding is the life instinct’s key characteristic. I suggest that there are pathological forms of both the life and death instinct if defused (separated off) from the other, and I explore a pathological variation of the life instinct in which binding is without the negation, rest, limit or end provided by the ‘opposing action’ of the death instinct. I consider an instance of the kind that any analyst might meet clinically, in which an inhibited patient experiences severe anxiety that life‐giving connections threaten to proliferate indiscriminately and to an overwhelming intensity and size.

Decreased phosphorylation of delta and epsilon subunits of the acetylcholine receptor coincides with delayed postsynaptic maturation in PKC theta deficient mouse

Protein kinase C (PKC) activity is involved in the nicotinic acetylcholine receptor (nAChR) redistribution at the neuromuscular junction in vivo during postnatal maturation. Here we studied, in PKC theta (PKCθ) deficient mice (KO), how the theta isoform of PKC is involved in the nAChR cluster maturation that is accompanied by the developmental activity-dependent neuromuscular synapse elimination process. We found that axonal elimination and dispersion of nAChR from the postsynaptic plaques and its redistribution to form the mature postsynaptic apparatus were delayed but not totally suppressed in PKCθ deficient mice. Moreover, the delay in the maturation of the morphology of the nAChR clusters during the early postnatal synapse elimination period in the PKCθ deficient mice coincides with a reduction in the PKCθ-mediated phosphorylation on the delta subunit of the nAChR. In addition, we show evidence for PKCθ regulation of PKA in normally phosphorylating the epsilon subunit of nAChR. We have also found that the θ isoform of PKC is located on the postsynaptic component of the neuromuscular junction but is also expressed by motoneurons in the spinal cord and in the motor nerve terminals. The results allow us to hypothesize that a spatially specific and opposing action of PKCθ and PKA may result in activity-dependent alterations to synaptic connectivity at both the nerve inputs and the postsynaptic nAChR clusters.

Fostering Team Innovation: Why Is It Important to Combine Opposing Action Strategies?

We develop a framework that provides a general theoretical rationale for the claim made by several authors that combining opposing action strategies fosters team innovation. We distinguish between open and closed strategies and posit that these are opposing but complementary in that each fosters one of two processes necessary for team innovation: open action strategies (e.g., delegative leadership) promote knowledge generation, and closed action strategies (e.g., directive leadership) enhance knowledge integration. We argue that each pole of a pair of opposing action strategies both energizes and detracts from elements of innovation. Thus, it could be expected that combining opposing action strategies leads to an impasse, as the negative effects of each strategy might offset the positive effects of the opposite strategy. There is currently no viable explanation in the literature for why this mutual neutralization may not occur. We aim to fill this gap by explicating why and how opposing action strategies, when implemented simultaneously, do not countervail each other's positive effects, but rather yield positive synergies that fuel team innovation.

Regulation of TLR4-induced IL-6 response in bladder cancer cells by opposing actions of MAPK and PI3K signaling

Toll-like receptor 4 (TLR4) was found to be aberrantly expressed in bladder cancer, inducing some genes expression and facilitating tumor progression. Recent data suggest that tumor associated Interleukin-6 (IL-6) correlates with tumor size and grade in bladder cancer. However, the molecule mechanisms of the induction of IL-6 response in bladder cancer cells are not well elucidated. In this study, we manage to find out whether TLR4 signaling is involved in the production of IL-6 by human bladder cancer cells, and the detailed molecule mechanisms by which IL-6 is up-regulated. We selected human bladder cancer T24 cell line in the present study, and examined its expression of TLR4 and CD14 by using flow cytometry. TLR4 signaling was activated by lipopolysaccharide (LPS) and IL-6 secretion in culture supernatants was tested by using ELISA kit. The expression of p38, ERK, JNK and Akt were determined by western-blot analysis using specific antibodies. Our study demonstrated that CD14 and TLR4 were constitutively expressed in T24 cells and activation of TLR4 signaling by LPS resulted in phosphorylation of MAPK and PI3K pathways and up-regulation of IL-6 in dose- and time-dependent manner. Pretreatment of cells with SB203580 (inhibitor of p38) and PD98059 (inhibitor of ERK) attenuated LPS-induced IL-6 expression, whereas LY294002 (inhibitor of PI3K) markedly amplified the LPS-stimulated synthesis of IL-6. Our results demonstrate that activation of TLR4 signaling in bladder cancer cells induces tumor-associated IL-6 expression via activation of p38 and ERK, whereas activation of PI3K/Akt exerts an opposing action.

Estrogen Regulates Snail and Slug in the Down-Regulation of E-Cadherin and Induces Metastatic Potential of Ovarian Cancer Cells through Estrogen Receptor α

Tumorigenesis is a multistep process involving dysregulated cell growth and metastasis. Considerable evidence implicates a mitogenic action of estrogen in early ovarian carcinogenesis. In contrast, its influence in the metastatic cascade of ovarian tumor cells remains obscure. In the present study, we showed that 17beta-estradiol (E2) increased the metastatic potential of human epithelial ovarian cancer cell lines. E2 treatment led to clear morphological changes characteristic of epithelial-mesenchymal transition (EMT) and an enhanced cell migratory propensity. These morphological and functional alterations were associated with changes in the abundance of EMT-related genes. Upon E2 stimulation, expression and promoter activity of the epithelial marker E-cadherin were strikingly suppressed, whereas EMT-associated transcription factors, Snail and Slug, were significantly up-regulated. This up-regulation was attributed to the increase in gene transcription activated by E2. Depletion of endogenous Snail or Slug using small interfering RNA (siRNA) attenuated E2-mediated decrease in E-cadherin. In addition, E2-induced cell migration was also neutralized by the siRNAs, suggesting that both transcription factors are indispensable for the prometastatic actions of E2. More importantly, by using selective estrogen receptor (ER) agonists, forced expression, and siRNA approaches, we identified that E2 triggered the metastatic behaviors exclusively through an ERalpha-dependent pathway. We also showed that ERbeta had an opposing action on ERalpha because the presence of ERbeta completely inhibited the EMT and down-regulation of E-cadherin induced by ERalpha. Collectively, this study provides a compelling argument that estrogen can potentiate tumor progression by EMT induction and highlights the crucial role of ERalpha in ovarian tumorigenesis.