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The development of new technologies and industry increases the number and variety of electromagnetic field (EMF) sources. Researcher are increasingly interested in the effects of EMF on brain health. The brain's function is largely dependent on electrical excitability, so it would be expected to be vulnerable to EMF. We therefore investigated the effects of brain development in the fetus, histopathological changes in female rats and the hippocampal level of MAPK proteins in male rats after exposed to pre and postnatal 2450 MHz continuous wave (CW) radiofrequency radiation (RFR) over four generations. Four groups; sham, irradiated female, irradiated male, irradiated male and female, with each consisting of four rats (one male and three females) were created. Rats in the exposure groups were whole-body exposed to 2450 MHz CW-RFR for 12 h/day during the experiment. Irradiation started one month before fertilization in the experimental group. On the 18th day of the gestational period, one pregnant rat from each group was decapitated under general anesthesia and the fetuses were taken. The remaining two pregnant rats completed the normal gestation period. When the offspring were two months old, four rats, one male and three female, were allocated for the second generation study. Next generation animals were also experienced the same processes as the first generation rats. This study were evaluated development of brain in fetuses and histopathological changes in brain of female rats using haematoxylin eosin staining, and the hippocampal level of MAPK proteins in brain of male rats by Western Bloting. We observed hemorrhagic areas, irregular cellular localization and vascular structures in the brain of fetal and adult female rat of exposed groups in the all generations. pERK, ptau, pJNK and pP38 were increased in the brain of adult male rat of exposed groups in the all generations (p < 0.005). Pre and postnatal 2450 MHz continuous wave radiofrequency radiation exposure may cause changes in the function of the MAPK pathway affecting cognitive processes such as learning and memory and may cause damage to both the fetus and adult brain tissue. Also, EMF may have potential to affect brain of future generations. © 2022 Elsevier B.V., TYL-2017-7110, The authors acknowledge the financial support from Erciyes University-The Scientific Research Projects of Turkey (ERUBAP); Project number: TYL-2017-7110 .

Asbestos is one of the most important occupational carcinogens. Currently, about 125 million people worldwide are exposed to asbestos in the workplace. According to global estimates, at least 107,000 people die each year from lung cancer, mesothelioma, and asbestosis as a result of occupational exposure to asbestos. The high pathogenicity of this material is currently known, being associated with the development of pulmonary diseases, of which lung cancer is the main cause of death due to exposure to this mineral. Pulmonary diseases related to asbestos are a common clinical problem and a major health concern worldwide. Extensive research has identified many important pathogenic mechanisms; however, the precise molecular mechanisms involved, and the generated genomic damage that lead to the development of these diseases, are not completely understood. The modes of action that underlie this type of disease seem to differ depending on the type of fiber, lung clearance, and genetics. This evidences the need to increase our knowledge about these effects on human health. This review focuses on the characteristics of asbestos and the cellular and genomic damage generated in humans via exposure. © 2019 Zhang et al.

Nerve injury provokes microglial activation, contributing to the sensory and emotional disorders associated with neuropathic pain that do not completely resolve with treatment. In C57BL/6J mice with neuropathic pain induced by chronic constriction of the sciatic nerve (CCI), we evaluated the effects of oltipraz, an antioxidant and anticancer compound, on (1) allodynia and hyperalgesia, (2) microglial activation and pain signaling pathways, (3) oxidative stress, and (4) depressive-like behaviors. Twenty-eight days after surgery, we assessed the effects of oltipraz on the expression of CD11b/c (a microglial marker), phosphoinositide 3-kinase (PI3K)/ phosphorylated protein kinase B (p-Akt), nuclear factor-?B (NF-?B) transcription factor, and mitogen activated protein kinases (MAPK) in the spinal cord, hippocampus, and prefrontal cortex. Our results show that oltipraz alleviates neuropathic pain by inhibiting microglial activation and PI3K/p-Akt, phosphorylated inhibitor of ?Ba (p-I?Ba), and MAPK overexpression, and by normalizing and/or enhancing the expression of antioxidant proteins, nuclear factor erythroid derived-2-related factor 2 (Nrf2), heme oxygenase 1 (HO-1), and NAD(P)H:quinone oxidoreductase-1 (NQO1) in the spinal cord. The inhibition of microglial activation and induction of the Nrf2/HO-1/NQO1 signaling pathway in the hippocampus and/or prefrontal cortex may explain the antidepressant effects of oltipraz during neuropathic pain. These data demonstrate the analgesic and antidepressant effects of oltipraz and reveal its protective and antioxidant properties during chronic pain. © 2019 by the authors. Licensee MDPI, Basel, Switzerland.

Constitutive Wnt signaling promotes intestinal cell proliferation, but signals from the tumor microenvironment are also required to support cancer development. The role that signaling proteins play to establish a tumor microenvironment has not been extensively studied. Therefore, we assessed the role of the proinflammatory Ikk-related kinase Ikkϵ in Wnt-driven tumor development. We found that Ikkϵ was activated in intestinal tumors forming upon loss of the tumor suppressor Apc. Genetic ablation of Ikkϵ in β-catenin-driven models of intestinal cancer reduced tumor incidence and consequently extended survival. Mechanistically, we attributed the tumor-promoting effects of Ikkϵ to limited TNF-dependent apoptosis in transformed intestinal epithelial cells. In addition, Ikkϵ was also required for lipopolysaccharide (LPS) and IL17A-induced activation of Akt, Mek1/2, Erk1/2, and Msk1. Accordingly, genes encoding pro-inflammatory cytokines, chemokines, and anti-microbial peptides were downregulated in Ikkϵ-deficient tissues, subsequently affecting the recruitment of tumor-associated macrophages and IL17A synthesis. Further studies revealed that IL17A synergized with commensal bacteria to trigger Ikkϵ phosphorylation in transformed intestinal epithelial cells, establishing a positive feedback loop to support tumor development. Therefore, TNF, LPS, and IL17A-dependent signaling pathways converge on Ikkϵ to promote cell survival and to establish an inflammatory tumor microenvironment in the intestine upon constitutive Wnt activation. Cancer Res; 76(9); 2587–99. ©2016 AACR.

Orientadores: José Rodrigo Pauli, Leandro Pereira de Moura Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Aplicadas Resumo: A ação da insulina no hipotálamo é crucial para o controle da ingestão alimentar. No estado alimentado a insulina é secretada e atinge o hipotálamo fosforilando e extruindo do núcleo a proteína FoxO1 (fator de transcrição da família forkhead BOX O), diminuindo a transcrição de neuropeptídeos orexigênicos (NPY, neuropeptídeo Y e peptídeo relacionado ao gene agouti, AgRP). Ao contrário, quando há prejuízos na transdução do sinal da insulina FoxO1 permanece no núcleo das células neuronais elevando a síntese de neuripeptídeos orexigênicos e com isso há o aumento da fome. Nesse contexto, a proteína MAPK-fosfatase-3 (MKP-3) tem a habilidade de desfosforilar FoxO1, permanecendo no núcleo, induzindo aumento de NPY e AgRP no hipotálamo e consequentemente causando hiperfagia e aumento da massa corporal adiposa. Portanto, ações terapêuticas capazes de ter efeito inibitório sobre a MKP-3 em células neurônais do hipotálamo podem se revelar como importantes agentes contra a obesidade e co-morbidades. O objetivo do presente estudo foi verificar os efeitos do exercício físico sobre o conteúdo proteico de MKP-3 e sinalização da insulina em hipotálamo de camundongos obesos induzidos por dieta rica em gordura saturada. Foram utilizados camundongos Swiss distribuídos em grupos: Controle, alimentados com uma ração padrão para roedores, Obesos (OB), alimentados com dieta rica em gordura saturada e Obesos exercitados agudamente (OBEXE). Após as análises de composição corpórea e ingestão alimentar, foram identificadas as proteínas de interesse da cascata de sinalização de insulina através da técnica de Western Blot. Foram também realizados ensaios com células HEK293 e camundongos Swiss controle com e sem adminsitração de adenovírus para superexpressar MKP-3 e avaliar os efeitos sobre a ingestão alimentar e fosforilação de FoxO1. Para identificar a ação da fosfatase MKP-3 em desfosforilar FoxO1 foi utilizado células HEK293 tratadas ou não com insulina e um vetor adenoviral de MKP-3 e GFP (controle) injetado intracerebroventricular (i.c.v) em camundongos, após os tratamentos as análises moleculares foram feita através da técnica de Westen Blot. Verifica-se através dos resultados que a obesidade aumentou o conteúdo proteíco de MKP-3 no hipotálamo e isso foi acompanhado por aumento na ingestão alimentar e adiposidade corporal dos camungondos. Os experimentos com adenovíruos mostraram que a super expressão de MKP-3 causa a desfoforilação da FoxO1 e prejuízo no controle da fome estimulado por insulina. Entretanto, foi observado aumento do exercício físico sobre a proteína MKP-3 hipotalâmica. Conclui-se que a condição de obesidade está associada com aumento nos níveis de MKP-3 no hipotálamo e isso está associado com hiperfagia. O exercício físico não foi capaz de inibir a atividade desta proteína em desfoforilar o fator transcricional FoxO1 no hipotálamo. Intervenções capazes de inibir a MKP-3 podem se tornar estratégias relevantes no tratamento da obesidade Abstract: The insulin action in the hypothalamus is crucial for the food intake control. On fed state the insulin is secreted and reaches the hypothalamus which phosphorylates and extracts the FoxO1 protein (transcription factor of the forkhead BOX O family) of the nucleus, decreasing the transcription of orexigenic neuropeptides (NPY, neuropeptide Y, and peptide related to the agouti gene, AgRP). Conversely, when there is impairment in the insulin transduction signal the FoxO1 remains in the nucleus of the neuronal cells, elevating the synthesis of orexigenic neuripeptides ,taking to the increased hunger. In this context, the MAPK-phosphatase-3 (MKP-3) protein has the ability to dephosphorylate FoxO1, remaining in the nucleus, inducing increase of NPY and AgRP in the hypothalamus and consequently causing hyperphagia and increase of adipose body mass. Therefore, therapeutic actions capable of having an inhibitory effect on MKP-3 in neuronal cells of the hypothalamus can be shown as important agents against obesity and comorbidities. The objective of the present study was to verify the physical exercise effects on the MKP-3 protein content and insulin signaling in the obese mice hypothalamus induced by diet rich in saturated fat. Swiss mice were distributed in groups : control, fed with a standard ration for rodents, obese (OB), fed with high fat diet diet and obeses exercised acutely (OBEXE). After analysis of body composition and food intake, were indentified the proteins of interest of the insulin signaling cascade through the Western Blot technique. Assays were also performed with HEK293 cells in Swiss control mice with or not adenovirus administration to overexpress MKP-3 and to evaluate the effects on food intake and phosphorylation of the FoxO1. To identify the MKP-3 phosphatase action on FoxO1 dephosphorylation, we used HEK293 cells treated or not with insulin and an adenoviral vector of MKP-3 and GFP (control) injected intracerebroventricular (icv) in mice. After the treatments the molecular analyzes were done through the Westen Blot technique. It is verified through the results that obesity increased the proteic content of MKP-3 in the hypothalamus and this was accompanied by an increase of food intake and body adiposity of the camundongos. The adenoviruses experiments showed that the overexpression of MKP-3 causes FoxO1 dephosphorylation and impairment on hunger control stimulated by insulin. However, an increase in physical exercise was observed on the hypothalamic MKP-3 protein. We conclude that the obesity condition is associated with an increase in MKP-3 levels in the hypothalamus and this is associated with hyperphagia. Physical exercise was not able to inhibit the activity of this protein on desfoforilar the FoxO1 transcriptional factor in the hypothalamus. Capable Interventions of inhibiting the MKP-3 can become relevant strategies on the obesity treatment Doutorado Metabolismo e Biologia Molecular Doutora em Ciências da Nutrição e do Esporte e Metabolismo FAPESP 2013/25512-4

International audience; Brassinosteroids (BRs) are steroid hormones that coordinate fundamental developmental programs in plants. In this study we show that in addition to the well established roles of BRs in regulating cell elongation and cell division events, BRs also govern cell fate decisions during stomata development in Arabidopsis thaliana. In wild-type A. thaliana, stomatal distribution follows the one-cell spacing rule; that is, adjacent stomata are spaced by at least one intervening pavement cell. This rule is interrupted in BR-deficient and BR signaling-deficient A. thaliana mutants, resulting in clustered stomata. We demonstrate that BIN2 and its homologues, GSK3/Shaggy-like kinases involved in BR signaling, can phosphorylate the MAPK kinases MKK4 and MKK5, which are members of the MAPK module YODA-MKK4/5-MPK3/6 that controls stomata development and patterning. BIN2 phosphorylates a GSK3/Shaggy-like kinase recognition motif in MKK4, which reduces MKK4 activity against its substrate MPK6 in vitro. In vivo we show that MKK4andMKK5act downstream of BR signaling because their overexpression rescued stomata patterning defects in BR-deficient plants. A model is proposed in which GSK3-mediated phosphorylation of MKK4 and MKK5 enables for a dynamic integration of endogenous or environmental cues signaled by BRs into cell fate decisions governed by the YODA-MKK4/5-MPK3/6 module.

Biased agonism describes the ability of ligands to stabilize different conformations of a GPCR linked to distinct functional outcomes and offers the prospect of designing pathway-specific drugs that avoid on-target side effects. This mechanism is usually inferred from pharmacological data with the assumption that the confounding influences of observational (that is, assay dependent) and system (that is, cell background dependent) bias are excluded by experimental design and analysis. Here we reveal that 'kinetic context', as determined by ligand-binding kinetics and the temporal pattern of receptor-signalling processes, can have a profound influence on the apparent bias of a series of agonists for the dopamine D 2 receptor and can even lead to reversals in the direction of bias. We propose that kinetic context must be acknowledged in the design and interpretation of studies of biased agonism.

Human hepatic stellate cells (HHSCs) proliferation and migration play a key role in the pathogenesis of liver inflammation and fibrogenesis. Low density lipoprotein receptor-related protein (LRP1) is an endocytic receptor involved in intracellular signal transduction. The aim of this work was to analyse the role of low density lipoprotein receptor-related protein (LRP1) in HHSCs proliferation and migration and the mechanisms involved. Human LRP1 deficient-HHSCs were generated by nucleofecting the line HHSCs with siRNA anti-LRP1. HHSCs DNA synthesis was measured by [ 3H]-thymidine incorporation and cell cycle progression by flow cytometry after annexin V and iodure propidium staining. Cell migration was assessed using a wound repair model system. LRP1 expression and extracellular matrix-regulated kinase (ERK1,2) phosphorylation were analysed by Western blot analysis. Transforming growth factor-ß (TGF-ß) extracellular levels were analysed by ELISA. siRNA-antiLRP1 treatment almost completely inhibited LRP1 mRNA and protein expression. LRP1 deficient HHSCs showed higher proliferative response (172±19 vs. 93±8 [ 3H]-thymidine incorporation; 78.68% vs. 82.69% in G0/G1, 21.32% vs. 17.30% in G2/S) and higher migration rates than control HHSCs. LRP1 deficient cells showed higher levels of phosphorylated ERK1,2. TGF-ß extracellular levels were threefold higher in LRP1-deficient than in control HHSCs cells. These results demonstrate that LRP1 regulates HHSCs proliferation and migration through modulation of ERK1,2 phosphorylation and TGF-ß extracellular levels. These results suggest that HHSCs-LRP1 may play a key role in the modulation of factors determining hepatic fibrosis. J. Cell. Physiol. 227: 3528-3533, 2012. © 2012 Wiley Periodicals, Inc. Copyright © 2012 Wiley Periodicals, Inc.

Background & Aims: Interleukin (IL)-21, a T-cell–derived cytokine, is produced in excess in inflammatory bowel diseases (IBD). The IL-21 receptor (IL-21R) is expressed by immune and nonimmune cells, raising the possibility that IL-21 has broad effects in gut inflammation. In this study we examined whether intestinal epithelial cells express IL-21R and respond to IL-21 in IBD. Methods: IL-21R was evaluated in intestinal samples of IBD patients and controls by immunohistochemistry and Western blotting. Intestinal epithelial cells were stimulated with IL-21, and cell-free supernatants were evaluated by a protein array and enzyme-linked immunosorbent assay. The effect of IL-21–treated epithelial cell supernatants on blood lymphocyte migration was assessed using a chemotaxis assay. Finally, we evaluated the effect of a neutralizing IL-21 antibody on MIP-3α synthesis in ex vivo organ cultures of IBD mucosal explants. Results: Constitutive expression of IL-21R was seen in intestinal epithelial cells, but was higher in IBD patients than in controls. Stimulation of intestinal epithelial cells with IL-21 resulted in enhanced phosphorylation of ERK1/2 and p38 and increased synthesis of macrophage inflammatory protein-3 alpha (MIP-3α), a T-cell chemoattractant. Inhibition of ERK1/2 but not p38 suppressed IL-21–induced MIP-3α production. IL-21–treated cell culture supernatants enhanced in vitro lymphocyte migration, and this effect was inhibited by anti-MIP-3α antibody. Treatment of IBD explants with anti–IL-21 reduced MIP-3α production. Conclusions: These data show that intestinal epithelial cells are a target of IL-21 and that IL-21 is involved in the cross-talk between epithelial and immune cells in the gut.

Enhanced expression and activity of the Na+/H+ exchanger isoform 1 (NHE1) has been implicated in cardiomyocyte hypertrophy in various experimental models. The upregulation of NHE1 was correlated with an increase in osteopontin (OPN) expression in models of cardiac hypertrophy (CH), and the mechanism for this remains to be delineated. To determine whether the expression of active NHE1-induces OPN and contributes to the hypertrophic response in vitro, cardiomyocytes were infected with the active form of the NHE1 adenovirus or transfected with OPN silencing RNA (siRNA-OPN) and characterized for cardiomyocyte hypertrophy. Expression of NHE1 in cardiomyocytes resulted in a significant increase in cardiomyocyte hypertrophy markers: cell surface area, protein content, ANP mRNA and expression of phosphorylated-GATA4. NHE1 activity was also significantly increased in cardiomyocytes expressing active NHE1. Interestingly, transfection of cardiomyocytes with siRNA-OPN significantly abolished the NHE1-induced cardiomyocyte hypertrophy. siRNA-OPN also significantly reduced the activity of NHE1 in cardiomyocytes expressing NHE1 (68.5±0.24%; P

Radiocontrast medium induced nephrotoxicity is a major clinical problem. There is considerable interest in reducing the incidence of acute renal failure due to the use of radiocontrast media (RCM). Reduction of renal blood flow and direct toxic effect on renal tubular epithelial cells have been postulated as major causes of RCM nephropathy. Understanding the molecular mechanisms by which RCM cause cell damage may allow the development of pharmacological therapy to prevent their nephrotoxicity. In this work we have investigated the signaling pathways that may be affected by RCM. The incubation of human renal tubular proximal cells with sodium diatrizoate, iopromide and iomeprol caused a marked dephosphorylation of the kinase Akt on Ser473 within 5 min of incubation. RCM also caused a decrease in cell viability, which was substantially alleviated by transfecting the cells with a constitutively active form of Akt. Further downstream targets of Akt, including the Forkhead family of transcription factors FKHR and FKHRL1, were also dephosphorylated by RCM at Thr24 and Thr32, respectively. The P70S6 kinase was also dephosphorylated at Thr389 and Ser371 by RCM. However there was a more dramatic decrease in phosphorylation of the phosphorylated form of mammalian target of rapamycin (mTOR) and of the extracellular-signal regulated kinases (ERK) 1/2 caused by sodium diatrizoate than by iopromide. These results demonstrate the effect of RCM on some intracellular signaling pathways that may allow understanding of the mechanism of their toxicity and may allow the development of strategies to overcome their adverse effects.

Aim We investigated architectural, functional and molecular responses of human skeletal muscle to concentric (CON) or eccentric (ECC) resistance training (RT). Methods Twelve young males performed 10 weeks of concentric (CON) or eccentric (ECC) resistance training (RT) (n = 6 CON, 6 ECC). An additional 14 males were recruited to evaluate acute muscle fascicle behaviour and molecular signalling in biopsies collected from vastus lateralis (VL) after 30 min of single bouts of CON or ECC exercise. VL volume was measured by magnetic resonance imaging. Muscle architecture (fascicle length, Lf; pennation angle, PA) was evaluated by ultrasonography. Muscle remodelling signals to CON or ECC loading [MAPK/AKT-mammalian target of rapamycin (mTOR) signalling] and inflammatory pathway (TNFαMurf-1-MAFbx) were evaluated by immunoblotting. Results Despite the ~1.2-fold greater load of the ECC group, similar increases in muscle volume (+8% CON and +6% ECC) and in maximal voluntary isometric contraction (+9% CON and +11% ECC) were found after RT. However, increases in Lf were greater after ECC than CON (+12 vs. +5%) while increases in PA were greater in CON than ECC (+30 vs. +5%). Distinct architectural adaptations were associated with preferential growth in the distal regions of VL for ECC (+ECC +8% vs. +CON +2) and mid belly for CON (ECC +7 vs. CON +11%). While MAPK activation (p38MAPK, ERK1/2, p90RSK) was specific to ECC, neither mode affected AKT-mTOR or inflammatory signalling 30 min after exercise. Conclusion Muscle growth with CON and ECC RT occurs with different morphological adaptations reflecting distinct fibre fascicle behaviour and molecular responses.

Gentiana lutea belonging to the Gentianaceae family of flowering plants are routinely used in traditional Serbian medicine for their beneficial gastro-intestinal and anti-inflammatory properties. The aim of the study was to determine whether aqueous root extracts of Gentiana lutea consisting of gentiopicroside, gentisin, bellidifolin-8-O-glucoside, demethylbellidifolin-8-O-glucoside, isovitexin, swertiamarin and amarogentin prevents proliferation of aortic smooth muscle cells in response to PDGF-BB. Cell proliferation and cell cycle analysis were performed based on alamar blue assay and propidium iodide labeling respectively. In primary cultures of rat aortic smooth muscle cells (RASMCs), PDGF-BB (20 ng/ml) induced a two-fold increase in cell proliferation which was significantly blocked by the root extract (1 mg/ml). The root extract also prevented the S-phase entry of synchronized cells in response to PDGF. Furthermore, PDGF-BB induced ERK1/2 activation and consequent increase in cellular nitric oxide (NO) levels were also blocked by the extract. These effects of extract were due to blockade of PDGF-BB induced expression of iNOS, cyclin D1 and proliferating cell nuclear antigen (PCNA). Docking analysis of the extract components on MEK1, the upstream ERK1/2 activating kinase using AutoDock4, indicated a likely binding of isovitexin to the inhibitor binding site of MEK1. Experiments performed with purified isovitexin demonstrated that it successfully blocks PDGF-induced ERK1/2 activation and proliferation of RASMCs in cell culture. Thus, Gentiana lutea can provide novel candidates for prevention and treatment of atherosclerosis. � 2013 Kesavan et al.

Plant-derived polyphenolic compounds have beneficial health effects. In the present study, we determined the ability of ellagic acid (EA) to prevent platelet-derived growth factor-BB (PDGF-BB)-induced proliferation of primary cultures of rat aortic smooth muscle cells (RASMCs). We also determined the ability of EA to prevent atherosclerosis in streptozotocin-induced diabetic rats. Proliferation of cells was measured via Alamar Blue assay and through propidium iodide-based cell cycle analysis in flow cytometer. Reactive oxygen species (ROS) were measured via 2',7'-dichlorofluorescin diacetate and Amplex red methods. Expression of proliferation markers and activation of kinases were assessed by immunoblot analysis. Cotreatment of primary cultures of RASMCs with 25 ?mol/L of EA significantly reduced PDGF-BB (20 ng/ml)-induced proliferation by blocking S-phase entry. EA effectively blocked PDGF receptor-? (PDGFR-?) tyrosine phosphorylation, generation of intracellular ROS and downstream activation of extracellular signal-regulated kinase 1/2. It also blocked PDGF-BB-induced expression of cyclin D1. Computational molecular docking of EA with the PDGFR-?-PDGF-BB complex revealed two putative inhibitor binding sites which showed similar binding energies with the known PDGFR-? inhibitor AG1295. In diabetic rats, supplementation of diet with 2% EA significantly blocked diabetes-induced medial thickness, and lipid and collagen deposition in the arch of aorta. These were assessed through haematoxylin and eosin, Oil Red O and Masson's trichome staining, respectively. EA treatment also blocked cyclin D1 expression in medial smooth muscle cells in experimental animals. Thus, EA is effective in reducing atherosclerotic process by blocking proliferation of vascular smooth muscle cells. � 2013 Elsevier Inc.

Background: Tissue factor (TF) and its signaling mediators play a crucial role in angiogenesis. We have previously shown that TF-induced endothelial cell (EC) CCL2 release contributes to neovessel formation. Objective: In this study, we have investigated the signaling pathways involved in TF-induced EC tube formation. Methods: The human microvascular endothelial cell line (HMEC-1) cultured onto basement membrane-like gel (Matrigel) was used to study TF signaling pathways during neovessels formation. Results: Inhibition of endogenous TF expression in ECs using siRNA resulted in inhibition of a stable tube-like structure formation in three-dimensional cultures, associated with a down-regulation of Akt activation, an increased phosphorylation of Raf at Ser259 with a subsequent reduction of Raf kinase and a reduction of ERK1/2 phosphorylation ending up in Ets-1 transcription factor inhibition. Conversely, overexpression of TF resulted in an increase in tube formation and up-regulation of Akt protein. Moreover, immunoprecipitation of Akt and western blotting of the immunoprecipitates with anti-TF antibody revealed a direct interaction between TF and Akt. The effects of silencing TF were partially reversed by a PAR2 agonist that rescued tube formation, indicating that the TF-Akt pathway induces PAR2-independent effector signaling. Finally, enforced expression of Akt in TF-silenced ECs rescued tube formation in a Matrigel assay and induced Ets-1 phosphorylation. Conclusions: In EC, TF forms a complex with Akt activating Raf/ERK and Ets-1 signaling induces microvessel formation. © 2012 International Society on Thrombosis and Haemostasis.

Background One-third of breast cancers display amplifications of the ERBB2 gene encoding the HER2 kinase receptor. Trastuzumab, a humanized antibody directed against an epitope on subdomain IV of the extracellular domain of HER2 is used for therapy of HER2-overexpressing mammary tumors. However, many tumors are either natively resistant or acquire resistance against Trastuzumab. Antibodies directed to different epitopes on the extracellular domain of HER2 are promising candidates for replacement or combinatorial therapy. For example, Pertuzumab that binds to subdomain II of HER2 extracellular domain and inhibits receptor dimerization is under clinical trial. Alternative antibodies directed to novel HER2 epitopes may serve as additional tools for breast cancer therapy. Our aim was to generate novel anti-HER2 monoclonal antibodies inhibiting the growth of breast cancer cells, either alone or in combination with tumor necrosis factor-α (TNF-α). Methods Mice were immunized against SK-BR-3 cells and recombinant HER2 extracellular domain protein to produce monoclonal antibodies. Anti-HER2 antibodies were characterized with breast cancer cell lines using immunofluorescence, flow cytometry, immunoprecipitation, western blot techniques. Antibody epitopes were localized using plasmids encoding recombinant HER2 protein variants. Antibodies, either alone or in combination with TNF-α, were tested for their effects on breast cancer cell proliferation. Results We produced five new anti-HER2 monoclonal antibodies, all directed against conformational epitope or epitopes restricted to the native form of the extracellular domain. When tested alone, some antibodies inhibited modestly but significantly the growth of SK-BR-3, BT-474 and MDA-MB-361 cells displaying ERBB2 amplification. They had no detectable effect on MCF-7 and T47D cells lacking ERBB2 amplification. When tested in combination with TNF-α, antibodies acted synergistically on SK-BR-3 cells, but antagonistically on BT-474 cells. A representative anti-HER2 antibody inhibited Akt and ERK1/2 phosphorylation leading to cyclin D1 accumulation and growth arrest in SK-BR-3 cells, independently from TNF-α. Conclusions Novel antibodies against extracellular domain of HER2 may serve as potent anti-cancer bioactive molecules. Cell-dependent synergy and antagonism between anti-HER2 antibodies and TNF-α provide evidence for a complex interplay between HER2 and TNF-α signaling pathways. Such complexity may drastically affect the outcome of HER2-directed therapeutic interventions.

Pituitary adenylyl cyclase activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) augment the biosynthesis of tyrosine hydroxylase (TH). We tested whether secretin belonging to the glucagon/PACAP/VIP superfamily would increase transcription of the tyrosine hydroxylase (Th) gene and modulate catecholamine secretion. Secretin activated transcription of the endogenous Th gene and its transfected promoter (EC50 ?4.6 nM) in pheochromocytoma (PC12) cells. This was abolished by pre-treatment with a secretin receptor (SCTR) antagonist and by inhibition of protein kinase A (PKA), mitogen-activated protein kinase, or CREB (cAMP response element-binding protein). In agreement, secretin increased PKA activity and induced phosphorylation of CREB and binding to Th CRE, suggesting secretin signaling to transcription via a PKA-CREB pathway. Secretin stimulated catecholamine secretion (EC50 ?3.5 ?M) from PC12 cells, but this was inhibited by pre-treatment with VIP-preferring receptor (VPAC1)/PACAP-preferring receptor (PAC1) antagonists. Secretin-evoked secretion occurred without extracellular Ca2+ and was abolished by intracellular Ca2+ chelation. Secretin augmented phospholipase C (PLC) activity and increased inositol-1,4,5-triphosphate (IP3) levels in PC12 cells; PLC-? inhibition blocked secretin-induced catecholamine secretion, indicating the participation of intracellular Ca2+ from a phospholipase pathway in secretion. Like PACAP, secretin evoked long-lasting catecholamine secretion, even after only a transient exposure. Thus, transcription is triggered by nanomolar concentrations of the peptide through SCTR, with signaling along the cAMP-PKA and extracellular-signal-regulated kinase 1/2 pathways and through CREB. By contrast, secretion is triggered only by micromolar concentrations of peptide through PAC1/VPAC receptors and by utilizing a PLC/intracellular Ca 2+ pathway. � 2011 Springer-Verlag.

Leptin, a 16-kDa protein mainly produced by adipose tissue, has been involved in the control of energy balance through its hypothalamic receptor. However, pleiotropic effects of leptin have been identified in reproduction and pregnancy, particularly in placenta, where it was found to be expressed. In the current study, we examined the effect of cAMP in the regulation of leptin expression in trophoblastic cells. We found that dibutyryl cAMP [(Bu) 2cAMP], a cAMP analog, showed an inducing effect on endogenous leptin expression in BeWo and JEG-3 cell lines when analyzed by Western blot analysis and quantitative RT-PCR. Maximal effect was achieved at 100 μM. Leptin promoter activity was also stimulated, evaluated by transient transfection with a reporter plasmid construction. Similar results were obtained with human term placental explants, thus indicating physiological relevance. Because cAMP usually exerts its actions through activation of protein kinase A (PKA) signaling, this pathway was analyzed. We found that cAMP response element-binding protein (CREB) phosphorylation was significantly increased with (Bu)2cAMP treatment. Furthermore, cotransfection with the catalytic subunit of PKA and/or the transcription factor CREB caused a significant stimulation on leptin promoter activity. On the other hand, the cotransfection with a dominant negative mutant of the regulatory subunit of PKA inhibited leptin promoter activity. We determined that cAMP effect could be blocked by pharmacologic inhibition of PKA or adenylyl ciclase in BeWo cells and in human placental explants. Thereafter, we decided to investigate the involvement of the MAPK/ERK signaling pathway in the cAMP effect on leptin induction. We found that 50 μM PD98059, a MAPK kinase inhibitor, partially blocked leptin induction by cAMP, measured both by Western blot analysis and reporter transient transfection assay. Moreover, ERK 1/2 phosphorylation was significantly increased with (Bu)2cAMP treatment, and this effect was dose dependent. Finally, we observed that 50 μM PD98059 inhibited cAMP-dependent phosphorylation of CREB in placental explants. In summary, we provide some evidence suggesting that cAMP induces leptin expression in placental cells and that this effect seems to be mediated by a cross talk between PKA and MAPK signaling pathways. Copyright © 2010 by The Endocrine Society. Fil:Maymó, J.L. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Calvo, J.C. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Varone, C.L. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

Neutrophils are short-lived cells that rapidly undergo apoptosis. However, their survival can be regulated by signals from the environment. Flagellin, the primary component of the bacterial flagella, is known to induce neutrophil activation. In this study we examined the ability of flagellin to modulate neutrophil apoptosis. Neutrophils cultured for 12 and 24 h in the presence of flagellin from Salmonella thyphimurim at concentrations found in pathological situations underwent a marked prevention of apoptosis. In contrast, Helicobacter pylori flagellin did not affect neutrophil survival, suggesting that Salmonella flagellin exerts the antiapoptotic effect by interacting with TLR5. The delaying in apoptosis mediated by Salmonella flagellin was coupled to higher expression levels of the antiapoptotic protein Mcl-1 and lower levels of activated caspase-3. Analysis of the signaling pathways indicated that Salmonella flagellin induced the activation of the p38 and ERK1/2 MAPK pathways as well as the PI3K/Akt pathway. Furthermore, it also stimulated IBα degradation and the phosphorylation of the p65 subunit, suggesting that Salmonella flagellin also triggers NF-B activation. Moreover, the pharmacological inhibition of ERK1/2 pathway and NF-B activation partially prevented the antiapoptotic effects exerted by flagellin. Finally, the apoptotic delaying effect exerted by flagellin was also evidenced when neutrophils were cultured with whole heat-killed S. thyphimurim. Both a wild-type and an aflagellate mutant S. thyphimurim strain promoted neutrophil survival; however, when cultured in low bacteria/neutrophil ratios, the flagellate bacteria showed a higher capacity to inhibit neutrophil apoptosis, although both strains showed a similar ability to induce neutrophil activation. Taken together, our results indicate that flagellin delays neutrophil apoptosis by a mechanism partially dependent on the activation of ERK1/2 MAPK and NF-B. The ability of flagellin to delay neutrophil apoptosis could contribute to perpetuate the inflammation during infections with flagellated bacteria. © 2010 USCAP, Inc All rights reserved. Fil:Salamone, G.V. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Gabelloni, M.L. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Vermeulen, M.E. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Trevani, A.S. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

Alternative splicing of precursor mRNA is a fundamental mechanism to generate multiple proteins from a single gene. Although constitutive and alternative mRNA splicing is temporally and spatially regulated, deregulation of mRNA splicing could cause development, progression, and metastasis of tumors. Through yeast two-hybrid screening of a human breast cDNA library using estrogen receptor-α (ERα) as bait, we identified a novel nuclear receptor box containing full-length protein, nuclear protein E3-3 (NPE3-3). Our results revealed that NPE3-3 associates with not only ERα but also with splicing factors, serine/arginine-rich protein (SRp)-30c, SRp40, and splicing factor SC-35, suggesting that NPE3-3 is likely to be involved in regulation of mRNA splicing. Accordingly, transient expression of NPE3-3 in cells resulted in expected splicing of the CD44 control minigene. We also discovered that NPE3-3-overexpressing clones produced a novel, previously unrecognized, alternatively spliced variant of ERα (termed ERαV), which had a molecular size of 37 kDa composed of only exons 1, 2, 7, and 8. ERαV was expressed and sequestered in the cytoplasm in MCF-7 cells stably overexpressing NPE3-3, suggesting its involvement in nongenomic hormone signaling. NPE3-3 clones exhibited up-regulation of ERK1/2 signaling, cyclin D1, and cathepsin D and enhanced tumor cell proliferation, migration, and tumorigenicity. Moreover, direct expression of the ERαV in breast cancer cells stimulated ERK1/2 up-regulation and cyclin D1 expression. We found that ERαV physically interacted with MAPK kinase (MEK)-1/2, and thus, an ERαV and MEK1/2 complex could lead to the activation of the ERK1/2 pathway. Interestingly, NPE3-3 was up-regulated in human breast tumors. These findings revealed a role for NPE3-3 in alternative splicing and suggest that ERα is a physiological target of NPE3-3, leading to a constitutive nongenomic signaling pathway in breast cancer cells.

Extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) are members of the MAPK family and participate in the transduction of stimuli in cellular responses. Their long-term actions are accomplished by promoting the expression of specific genes whereas faster responses are achieved by direct phosphorylation of downstream effectors located throughout the cell. In this study we determined that hERK1 translocates to the mitochondria of HeLa cells upon a proliferative stimulus. In the mitochondrial environment, hERK1 physically associates with (i) at least 5 mitochondrial proteins with functions related to transport (i.e. VDAC1), signalling, and metabolism; (ii) histones H2A and H4; and (iii) other cytosolic proteins. This work indicates for the first time the presence of diverse ERK-complexes in mitochondria and thus provides a new perspective for assessing the functions of ERK1 in the regulation of cellular signalling and trafficking in HeLa cells. © 2009 Galli et al. Fil:Galli, S. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Jares-Erijman, E.A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

Leptin, the 16,000 molecular weight protein product of the obese gene, was originally considered as an adipocyte-derived signaling moleculeforthe central control of metabolism. However, leptin has been suggested to be involved in other functions during pregnancy, particularly in placenta, in which it was found to be expressed. In the present work, we have found that recombinant human chorionic gonadotropin (hCG) added to BeWo choriocarcinoma cell line showed a stimulatory effect on endogenous leptin expression, when analyzed by Western blot. This effect was time and dose dependent. Maximal effect was achieved at hCG 100 IU/ml. Moreover, hCG treatment enhanced leptin promoter activity up to 12.9 times, evaluated by transient transfection with a plasmid construction containing different promoter regions and the reporter gene luciferase. This effect was dose dependent and evidenced with all the promoter regions analyzed, regardless of length. Similar results were obtained with placental explants, thus indicating physiological relevance. Because hCG signal transduction usually involves cAMP signaling, this pathway was analyzed. Contrarily, we found that dibutyryl cAMP counteracted hCG effect on leptin expression. Furthermore, cotransfection with the catalytic subunit of PKA and/or the transcription factor cAMP response element binding protein repressed leptin expression. Thereafter we determined that hCG effect could be partially blocked by pharmacologic inhibition of MAPK pathway with 50 μM PD98059 but not by the inhibition of the phosphatidylinositol 3-kinase pathway with 0.1 μm wortmannin. Moreover, hCG treatment promoted MAPK kinase and ERK1/ERK2 phosphorylation in placental cells. Finally, cotransfection with a dominant-negative mutant of MAPK blocked the hCG-mediated activation of leptin expression. In conclusion, we provide some evidence suggesting that hCG induces leptin expression in trophoblastic cells probably involving the MAPK signal transduction pathway. Copyright © 2009. Fil:Maymó, J.L. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Calvo, J.C. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Varone, C.L. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

Different VGF peptides derived from Vgf, originally identified as a nerve growth factor responsive gene, have been detected in neurons within the central and peripheral nervous system and in various endocrine cells. In the current study, we have evaluated the ability of TLQP-21, a VGF-derived peptide, to protect, in a dose- and time-dependent manner, primary cultures of rat cerebellar granule cells (CGCs) from serum and potassium deprivation-induced cell death. We demonstrated that TLQP-21 increased survival of CGCs by decreasing the degree of apoptosis as assessed by cell viability and DNA fragmentation. Moreover, TLQP-21 significantly activated extracellular signal-regulated kinase 1/2, serine/threonine protein kinase, and c-jun N-terminal kinase phosphorylation, while decreased the extent of protein kinase C phosphorylation, as demonstrated by western blot analysis. In addition, TLQP-21 induced significant increase in intracellular calcium (as measured by fura-2AM) in about 60% of the recorded neurons. Taken together, the present results demonstrate that TLQP-21 promotes the survival of CGCs via pathways involving, within few minutes, modulation of kinases associated with CGCs survival, and by increasing intracellular calcium which can contribute to the neuroprotective effect of the peptide.

Introduction: It has been demonstrated that leukemia inhibitory factor (LIF) induces epithelium apoptosis through Stat3 activation during mouse mammary gland involution. In contrast, it has been shown that this transcription factor is commonly activated in breast cancer cells, although what causes this effect remains unknown. Here we have tested the hypothesis that locally produced LIF can be responsible for Stat3 activation in mouse mammary tumors. Methods: The studies were performed in different tumorigenic and non-tumorigenic mammary cells. The expression of LIF and LIF receptor was tested by RT-PCR analysis. In tumors, LIF and Stat3 proteins were analyzed by immunohistochemistry, whereas Stat3 and extracellular signal-regulated kinase (ERK)1/2 expression and phosphorylation were studied by Western blot analysis. A LIF-specific blocking antibody was used to determine whether this cytokine was responsible for Stat3 phosphorylation induced by conditioned medium. Specific pharmacological inhibitors (PD98059 and Stat3ip) that affect ERK1/2 and Stat3 activation were used to study their involvement in LIF-induced effects. To analyze cell survival, assays with crystal violet were performed. Results: High levels of LIF expression and activated Stat3 were found in mammary tumors growing in vivo and in their primary cultures. We found a single mouse mammary tumor cell line, LM3, that showed low levels of activated Stat3. Incidentally, these cells also showed very little expression of LIF receptor. This suggested that autocrine/paracrine LIF would be responsible for Stat3 activation in mouse mammary tumors. This hypothesis was confirmed by the ability of conditioned medium of mammary tumor primary cultures to induce Stat3 phosphorylation, activity that was prevented by pretreatment with LIF-blocking antibody. Besides, we found that LIF increased tumor cell viability. Interestingly, blocking Stat3 activation enhanced this effect in mammary tumor cells. Conclusion: LIF is overexpressed in mouse mammary tumors, where it acts as the main Stat3 activator. Interestingly, the positive LIF effect on tumor cell viability is not dependent on Stat3 activation, which inhibits tumor cell survival as it does in normal mammary epithelium. © 2007 Quaglino et al.; licensee BioMed Central Ltd. Fil:Quaglino, A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Schere-Levy, C. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Romorini, L. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Kordon, E.C. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

MAP kinases have been linked to guard cell signalling. Arabidopsis thaliana MAP Kinase 3 (MPK3) is known to be activated by abscisic acid (ABA) and hydrogen peroxide (H2O2), which also control stomatal movements. We therefore studied the possible role of MPK3 in guard cell signalling through guard cell-specific antisense inhibition of MPK3 expression. Such transgenic plants contained reduced levels of MPK3 mRNA in the guard cells and displayed partial insensitivity to ABA in inhibition of stomatal opening, but responded normally to this hormone in stomatal closure. However, ABA-induced stomatal closure was reduced compared with controls when cytoplasmic alkalinization was prevented with sodium butyrate. MPK3 antisense plants were less sensitive to exogenous H2O2, both in inhibition of stomatal opening and in promotion of stomatal closure, thus MPK3 is required for the signalling of this compound. ABA-induced H2O2 synthesis was normal in these plants, indicating that MPK3 probably acts in signalling downstream of H2O2. These results provide clear evidence for the important role of MPK3 in the perception of ABA and H 2O2 in guard cells. © The Authors (2007). Fil:Gudesblat, G.E. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Iusem, N.D. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

In the current study, we have evaluated the ability of substance P (SP) and other neurokinin 1 receptor (NK1) agonists to protect, in a dose- and time-dependent manner, primary cultures of rat cerebellar granule cells (CGCs) from serum and potassium deprivation-induced cell death (S-K5). We also established the presence of SP high affinity NK1 transcripts and the NK1 protein localization in the membrane of a sub-population of CGCs. Moreover, SP significantly and dose-dependently reduced the Akt 1/2 and Erk1/2 dephosphorylation induced by S-K5 conditions, as demonstrated by Western blot analysis. Surprisingly, in SP-treated CGCs caspase-3 activity was not inhibited, while the calpain-1 activity was moderately reduced. Corroborating this result, SP blocked calpain-mediated cleavage of tau protein, as demonstrated by the reduced appearance of a diagnostic fragment of 17 kDa by Western blot analysis. In addition, SP induced a significant reduction of the delayed rectifier K+ currents (Ik) in about 42% of the patched neurons, when these were evoked with depolarizing potential steps. Taken together, the present results demonstrate that the activation of NK1 receptors expressed in CGCs promote the neuronal survival via pathways involving Akt and Erk activation and by inhibition of Ik which can contribute to the neuroprotective effect of the peptide.

Prostaglandin F2alpha (PGF2alpha) induces cyclin D1 expression and DNA synthesis in Swiss 3T3 cells. In order to assess which signaling mechanisms are implicated in these processes, we have used both a pharmacological approach and interfering mutants. We demonstrate that PGF2alpha induces extracellular-signal-regulated kinase (ERK1-2) and p38MAPK activation, and inhibition of any of these signaling pathways completely blocks PGF2alpha-stimulated DNA synthesis. We also show that ERK1-2, but not p38MAPK activation is required to induce cyclin D1 expression, strongly suggesting that the concerted action of cyclin D1 gene expression and other events are required to induce complete phosphorylation of retinoblastoma protein and S-phase entry in response to PGF2alpha. Fil: Dekanty, Andres. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Giulianelli, Sebastian Jesus. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Coso, Omar Adrian. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina Fil: Rudland, Phillip S.. University of Liverpool; Reino Unido Fil: Jimenez de Asua, Luis Adan Felipe. University of Liverpool; Reino Unido. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina

Although transcriptional effects of thyroid hormones have substantial influence on oxidative metabolism, how thyroid sets basal metabolic rate remains obscure. Compartmental localization of nitric-oxide synthases is important for nitric oxide signaling. We therefore examined liver neuronal nitric-oxide synthase-α (nNOS) subcellular distribution as a putative mechanism for thyroid effects on rat metabolic rate. At low 3,3′,5-triiodo-L-thyronine levels, nNOS mRNA increased by 3-fold, protein expression by one-fold, and nNOS was selectively translocated to mitochondria without changes in other isoforms. In contrast, under thyroid hormone administration, mRNA level did not change and nNOS remained predominantly localized in cytosol. In hypothyroidism, nNOS translocation resulted in enhanced mitochondrial nitric-oxide synthase activity with low O2 uptake. In this context, NO utilization increased active O2 species and peroxynitrite yields and tyrosine nitration of complex I proteins that reduced complex activity. Hypothyroidism was also associated to high phospho-p38 mitogen-activated protein kinase and decreased phospho-extracellular signal-regulated kinase 1/2 and cyclin D1 levels. Similarly to thyroid hormones, but without changing thyroid status, nitric-oxide synthase inhibitor Nω-nitro-L-arginine methyl ester increased basal metabolic rate, prevented mitochondrial nitration and complex I derangement, and turned mitogen-activated protein kinase signaling and cyclin D1 expression back to control pattern. We surmise that nNOS spatial confinement in mitochondria is a significant downstream effector of thyroid hormone and hypothyroid phenotype. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Fil:Franco, M.C. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Antico Arciuch, V.G. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Galli, S. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Romorini, L. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

Several G protein-coupled receptors function within lipid rafts plasma membrane microdomains, which may be important in limiting signal transduction. Here we show that treatment of rat C6 glioma cells with the raft disruptor methyl-beta-cyclodextrin (MCD) doubles the binding efficiency (i.e. the ratio between maximum binding and dissociation constant) of type-1 cannabinoid receptors (CB1R), which belong to the rhodopsin family of G protein-coupled receptors. In parallel, activation of CB1R by the endogenous agonist anandamide (AEA) leads to approximately 3-fold higher [35S]GTPgammaS binding in MCD-treated cells than in controls, and CB1R-dependent signaling via adenylate cyclase, and p42/p44 MAPK is almost doubled by MCD. Unlike CB1R, the other AEA-binding receptor TRPV1, the AEA synthetase NAPE-PLD, and the AEA hydrolase FAAH are not modulated by MCD, whereas the activity of the AEA membrane transporter (AMT) is reduced to approximately 50% of the controls. We also show that MCD reduces dose-dependently AEA-induced apoptosis in C6 cells but not in human CHP100 neuroblastoma cells, which mirror the endocannabinoid system of C6 cells but are devoid of CB1R. MCD reduces also cytochrome c release from mitochondria of C6 cells, and this effect is CB1R-dependent and partly mediated by activation of p42/p44 MAPK. Altogether, the present data suggest that lipid rafts control CB1R binding and signaling, and that CB1R activation underlies the protective effect of MCD against apoptosis.

Uterine decidualization is characterized by stromal cell proliferation and differentiation, which are controlled by ovarian hormones estradiol and progesterone. Here we report that the proliferative response of UIII rat uterine stromal cells to a short treatment with progestins requires active progesterone receptor (PR) and estrogen receptor β (ERβ) as well as a rapid and transient activation of Erk1-2 and Akt signaling. The optimal R5020 concentration for the proliferative response as well as for activation of the signaling cascades was between 10 and 100 pM. UIII cells are negative for ERα and have low levels of ERβ and PR located mainly in the cytoplasm. Upon progestin treatment PR translocated to the cell nucleus where it colocalized with activated Erk1-2. Neither progestins nor estradiol transactivated the corresponding transfected reporter genes, suggesting that endogenous PR and ERβ are transcriptionally incompetent. A fraction of endogenous PR and ERβ form a complex as demonstrated by coimmunoprecipitation. Taken together, our results suggest that the proliferative response of uterine stromal cells to picomolar concentrations of progestins does not require direct transcriptional effects and is mediated by activation of the Erk1-2 and Akt signaling pathways via cross talk between PR and ERβ. Copyright © 2005 by The Endocrine Society. Fil:Vallejo, G. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Ballaré, C. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Barañao, J.L. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Saragüeta, P. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

In this report we describe our studies on intracellular signals that mediate neurite outgrowth and long-term survival of cerebellar granule cells. The effect of voltage-gated calcium channel activation on neurite complexity was evaluated in cultured cerebellar granule cells grown for 48 h at low density; the parameter measured was the fractal dimension of the cell. We explored the contribution of two intracellular pathways, Ca2+ calmodulin-dependent protein kinase II and mitogen-activated protein kinase kinase (MEK1), to the effects of high [K+]e under serum-free conditions. We found that 25 mM KCI (25K) induced an increase in calcium influx through L subtype channels. In neurones grown for 24-48 h under low-density conditions, the activation of these channels induced neurite outgrowth through the activation of Ca2+ calmodulin-dependent protein kinase II. This also produced an increase in long-term neuronal survival with a partial contribution from the MEK1 pathway. We also found that the addition of 25K increased the levels of the phosphorylated forms of Ca2+ calmodulin-dependent protein kinase II and of the extracellular signal-regulated kinases 1 and 2. Neuronal survival under resting conditions is supported by the MEK1 pathway. We conclude that intracellular calcium oscillations can triggered different biological effects depending on the stage of maturation of the neuronal phenotype. Ca2+ calmodulin-dependent protein kinase II activation determines the growth of neurites and the development of neuronal complexity. Fil:Borodinsky, L.N. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Coso, O.A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

Background: Shortly after weaning, a complex multi-step process that leads to massive epithelial apoptosis is triggered by tissue local factors in the mouse mammary gland. Several reports have demonstrated the relevance of mechanical stress to induce adaptive responses in different cell types. Interestingly, these signaling pathways also participate in mammary gland involution. Then, it has been suggested that cell stretching caused by milk accumulation after weaning might be the first stimulus that initiates the complete remodeling of the mammary gland. However, no previous report has demonstrated the impact of mechanical stress on mammary cell physiology. To address this issue, we have designed a new practical device that allowed us to evaluate the effects of radial stretching on mammary epithelial cells in culture. Results: We have designed and built a new device to analyze the biological consequences of applying mechanical stress to cells cultured on flexible silicone membranes. Subsequently, a geometrical model that predicted the percentage of radial strain applied to the elastic substrate was developed. By microscopic image analysis, the adjustment of these calculations to the actual strain exerted on the attached cells was verified. The studies described herein were all performed in the HC11 non-tumorigenic mammary epithelial cell line, which was originated from a pregnant BALB/c mouse. In these cells, as previously observed in other tissue types, mechanical stress induced ERK1/2 phosphorylation and c-Fos mRNA and protein expression. In addition, we found that mammary cell stretching triggered involution associated cellular events as Leukemia Inhibitory Factor (LIF) expression induction, STAT3 activation and AKT phosphorylation inhibition. Conclusion: Here, we show for the first time, that mechanical strain is able to induce weaning-associated events in cultured mammary epithelial cells. These results were obtained using a new practical and affordable device specifically designed for such a purpose. We believe that our results indicate the relevance of mechanical stress among the early post-lactation events that lead to mammary gland involution. © 2009 Quaglino et al., licensee BioMed Central Ltd. Fil:Quaglino, A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Salierno, M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Rubinstein, N. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Kordon, E.C. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

Apoptosis is the predominant process controlling cell deletion during post-lactational mammary gland remodeling. The members of the Bcl-2 protein family, whose expression levels are under the control of lactogenic hormones, internally control this mechanism. Epidermal growth factor (EGF) belongs to a family of proteins that act as survival factors for mammary epithelial cells upon binding to specific membrane tyrosine kinase receptors. Expression of EGF peaks during lactation and dramatically decreases in the involuting mammary gland. Though it was suggested that the protective effect of EGF is mediated through the phosphatidylinositol-3-kinase (PI3K) or MEK/ERK kinases activities, little is known about the downstream mechanisms involved on the anti-apoptotic effect of EGF on mammary epithelial cells; particularly the identity of target genes controlling apoptosis. Here, we focused on the effect of EGF on the survival of mammary epithelial cells. We particularly aimed at the characterization of the signaling pathways that were triggered by this growth factor, impinge upon expression of Bcl-2 family members and therefore have an impact on the regulation of cell survival. We demonstrate that EGF provokes the induction of the anti-apoptotic isoform Bcl-XL and the phosphorylation and down-regulation of the pro-apoptotic protein Bad. The activation of JNK and PI3K/AKT signaling pathways promotes the induction of Bcl-XL while AKT activation also leads to Bad phosphorylation and down-regulation. This protective effect of EGF correlates mainly with the up-regulation of Bcl-XL than with the down-regulation of Bad. In fact, HC11 cells unable to express bcl-X, die even in the presence of EGF. In this context, Bcl-XL emerges as a key anti-apoptotic molecule critical for mediating EGF cell survival. © 2008 Elsevier B.V. All rights reserved. Fil:Romorini, L. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Coso, O.A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Pecci, A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

Background The poor response to chemotherapy and the brief response to vemurafenib in metastatic melanoma patients, make the identification of new therapeutic approaches an urgent need. Interestingly the increased expression and activity of the Aurora kinase B during melanoma progression suggests it as a promising therapeutic target. Methods The efficacy of the Aurora B kinase inhibitor barasertib-HQPA was evaluated in BRAF mutated cells, sensitive and made resistant to vemurafenib after chronic exposure to the drug, and in BRAF wild type cells. The drug effectiveness has been evaluated as cell growth inhibition, cell cycle progression and cell migration. In addition, cellular effectors of drug resistance and response were investigated. Results The characterization of the effectors responsible for the resistance to vemurafenib evidenced the increased expression of MITF or the activation of Erk1/2 and p-38 kinases in the newly established cell lines with a phenotype resistant to vemurafenib. The sensitivity of cells to barasertib-HQPA was irrespective of BRAF mutational status. Barasertib-HQPA induced the mitotic catastrophe, ultimately causing apoptosis and necrosis of cells, inhibited cell migration and strongly affected the glycolytic metabolism of cells inducing the release of lactate. In association i) with vemurafenib the gain in effectiveness was found only in BRAF(V600K) cells while ii) with nab-paclitaxel, the combination was more effective than each drug alone in all cells. Conclusions These findings suggest barasertib as a new therapeutic agent and as enhancer of chemotherapy in metastatic melanoma treatment. Electronic supplementary material The online version of this article (doi:10.1186/s12967-015-0385-4) contains supplementary material, which is available to authorized users.